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2018
Dubois N, Khoo K K, Ghossein S, Seissler T, Wolff P, McKinstry W J, Mak J, Paillart J C, Marquet R, Bernacchi S
The C-terminal p6 domain of the HIV-1 Pr55Gag precursor is required for specific binding to the genomic RNA. Journal Article
In: RNA Biol, vol. 15, no. 7, pp. 923-936, 2018, ISBN: 29954247.
Abstract | Links | BibTeX | Tags: ENNIFAR, HIV-1 Pr55Gag RNA-protein binding specificity fluorescence spectroscopy genomic RNA p6 domain, MARQUET, PAILLART, Unité ARN
@article{,
title = {The C-terminal p6 domain of the HIV-1 Pr55^{Gag} precursor is required for specific binding to the genomic RNA.},
author = {N Dubois and K K Khoo and S Ghossein and T Seissler and P Wolff and W J McKinstry and J Mak and J C Paillart and R Marquet and S Bernacchi},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29954247?dopt=Abstract},
doi = {10.1080/15476286.2018.1481696},
isbn = {29954247},
year = {2018},
date = {2018-01-01},
journal = {RNA Biol},
volume = {15},
number = {7},
pages = {923-936},
abstract = {The Pr55Gag precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55Gag interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gag∆p6 is a truncated form of Pr55Gag lacking the p6 domain usually used as a default surrogate for wild type Pr55Gag for in vitro analysis. With recent advance in production of full-length recombinant Pr55Gag, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55Gag by systematically comparing binding of Pr55Gag and Gag∆p6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55Gag to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gag∆p6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55Gag and Gag∆p6 uniformly increased the Kd values of the RNA-protein complexes by ~2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55Gag-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation.},
keywords = {ENNIFAR, HIV-1 Pr55Gag RNA-protein binding specificity fluorescence spectroscopy genomic RNA p6 domain, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Pr55Gag precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55Gag interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gag∆p6 is a truncated form of Pr55Gag lacking the p6 domain usually used as a default surrogate for wild type Pr55Gag for in vitro analysis. With recent advance in production of full-length recombinant Pr55Gag, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55Gag by systematically comparing binding of Pr55Gag and Gag∆p6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55Gag to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gag∆p6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55Gag and Gag∆p6 uniformly increased the Kd values of the RNA-protein complexes by ~2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55Gag-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation.
Pellegrino S, Demeshkina N, Mancera-Martinez E, Melnikov S, Simonetti A, Myasnikov A, Yusupov M, Yusupova G, Hashem Y
Structural insights into the role of diphthamide on elongation factor 2 in messenger RNA reading frame maintenance Journal Article
In: J Mol Biol, vol. 430, no. 17, pp. 2677-2687, 2018, ISBN: 29886014.
Abstract | Links | BibTeX | Tags: ENNIFAR, Ribosome translocation cryo-EM diphthamide eEF2 reading-frame maintenance, Unité ARN
@article{,
title = {Structural insights into the role of diphthamide on elongation factor 2 in messenger RNA reading frame maintenance},
author = {S Pellegrino and N Demeshkina and E Mancera-Martinez and S Melnikov and A Simonetti and A Myasnikov and M Yusupov and G Yusupova and Y Hashem},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29886014?dopt=Abstract},
doi = {10.1016/j.jmb.2018.06.006},
isbn = {29886014},
year = {2018},
date = {2018-01-01},
journal = {J Mol Biol},
volume = {430},
number = {17},
pages = {2677-2687},
abstract = {One of the most critical steps of protein biosynthesis is the coupled movement of messenger RNA (mRNA), that encodes genetic information, with transfer RNAs (tRNAs) on the ribosome. In eukaryotes this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-EM structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights on the role of diphthamide in the mechanism of translation fidelity in eukaryotes.},
keywords = {ENNIFAR, Ribosome translocation cryo-EM diphthamide eEF2 reading-frame maintenance, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
One of the most critical steps of protein biosynthesis is the coupled movement of messenger RNA (mRNA), that encodes genetic information, with transfer RNAs (tRNAs) on the ribosome. In eukaryotes this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-EM structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights on the role of diphthamide in the mechanism of translation fidelity in eukaryotes.
2017
Leonarski F, D'Ascenzo L, Auffinger P
Mg2+ ions: do they bind to nucleobase nitrogens? Journal Article
In: Nucleic Acids Res, vol. 45, no. 2, pp. 987-1004, 2017, ISBN: 27923930.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Mg2+ ions: do they bind to nucleobase nitrogens?},
author = {F Leonarski and L D'Ascenzo and P Auffinger},
url = {https://www.ncbi.nlm.nih.gov/pubmed/27923930?dopt=Abstract},
doi = {10.1093/nar/gkw1175},
isbn = {27923930},
year = {2017},
date = {2017-01-01},
journal = {Nucleic Acids Res},
volume = {45},
number = {2},
pages = {987-1004},
abstract = {Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments.
Riml C, Lusser A, Ennifar E, Micura R
Synthesis, Thermodynamic Properties, and Crystal Structure of RNA Oligonucleotides Containing 5-Hydroxymethylcytosine. Journal Article
In: J Org Chem, vol. 82, no. 15, pp. 7939-7945, 2017, ISBN: 28707898.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Synthesis, Thermodynamic Properties, and Crystal Structure of RNA Oligonucleotides Containing 5-Hydroxymethylcytosine.},
author = {C Riml and A Lusser and E Ennifar and R Micura},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28707898?dopt=Abstract},
doi = {10.1021/acs.joc.7b01171},
isbn = {28707898},
year = {2017},
date = {2017-01-01},
journal = {J Org Chem},
volume = {82},
number = {15},
pages = {7939-7945},
abstract = {5-Hydroxymethylcytosine (hm5C) is an RNA modification that has attracted significant interest because of the finding that RNA hydroxymethylation can favor mRNA translation. For insight into the mechanistic details of hm5C function to be obtained, the availability of RNAs containing this modification at defined positions that can be used for in vitro studies is highly desirable. In this work, we present an eight-step route to 5-hydroxymethylcytidine (hm5rC) phosphoramidite for solid-phase synthesis of modified RNA oligonucleotides. Furthermore, we examined the effects of hm5rC on RNA duplex stability and its impact on structure formation using the sarcin-ricin loop (SRL) motif. Thermal denaturation experiments revealed that hm5rC increases RNA duplex stability. By contrast, when cytosine within an UNCG tetraloop motif was replaced by hm5rC, the thermodynamic stability of the corresponding hairpin fold was attenuated. Importantly, incorporation of hm5rC into the SRL motif resulted in an RNA crystal structure at 0.85 Å resolution. Besides changes in the hydration pattern at the site of modification, a slight opening of the hm5rC-G pair compared to the unmodified C-G in the native structure was revealed.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
5-Hydroxymethylcytosine (hm5C) is an RNA modification that has attracted significant interest because of the finding that RNA hydroxymethylation can favor mRNA translation. For insight into the mechanistic details of hm5C function to be obtained, the availability of RNAs containing this modification at defined positions that can be used for in vitro studies is highly desirable. In this work, we present an eight-step route to 5-hydroxymethylcytidine (hm5rC) phosphoramidite for solid-phase synthesis of modified RNA oligonucleotides. Furthermore, we examined the effects of hm5rC on RNA duplex stability and its impact on structure formation using the sarcin-ricin loop (SRL) motif. Thermal denaturation experiments revealed that hm5rC increases RNA duplex stability. By contrast, when cytosine within an UNCG tetraloop motif was replaced by hm5rC, the thermodynamic stability of the corresponding hairpin fold was attenuated. Importantly, incorporation of hm5rC into the SRL motif resulted in an RNA crystal structure at 0.85 Å resolution. Besides changes in the hydration pattern at the site of modification, a slight opening of the hm5rC-G pair compared to the unmodified C-G in the native structure was revealed.
Fernandez-Millan P, Autour A, Ennifar E, Westhof E, Ryckelynck M
Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI Journal Article
In: RNA, vol. 23, no. 12, pp. 1788-1795, 2017, ISBN: 28939697.
Abstract | Links | BibTeX | Tags: DFHBI Spinach crystal structure fluorescence fluorogenic RNA aptamer, ENNIFAR, RYCKELYNCK, Unité ARN, WESTHOF
@article{,
title = {Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI},
author = {P Fernandez-Millan and A Autour and E Ennifar and E Westhof and M Ryckelynck},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28939697?dopt=Abstract},
doi = {10.1261/rna.063008},
isbn = {28939697},
year = {2017},
date = {2017-01-01},
journal = {RNA},
volume = {23},
number = {12},
pages = {1788-1795},
abstract = {Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach comprising five mutations whom the exact role was not deciphered yet. In this work, we co-crystallized a re-engineered version of iSpinach in complex with the DFHBI and solved the x-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach allows shedding some light on the possible function of the different beneficial mutations carried by iSpinach.},
keywords = {DFHBI Spinach crystal structure fluorescence fluorogenic RNA aptamer, ENNIFAR, RYCKELYNCK, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach comprising five mutations whom the exact role was not deciphered yet. In this work, we co-crystallized a re-engineered version of iSpinach in complex with the DFHBI and solved the x-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach allows shedding some light on the possible function of the different beneficial mutations carried by iSpinach.
Auffinger P, Ennifar E
Nucleic acid nanomaterials: Silver-wired DNA Journal Article
In: Nat Chem, vol. 9, no. 10, pp. 932-934, 2017, ISBN: 28937666.
Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Nucleic acid nanomaterials: Silver-wired DNA},
author = {P Auffinger and E Ennifar},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28937666?dopt=Abstract},
doi = {10.1038/nchem.2869},
isbn = {28937666},
year = {2017},
date = {2017-01-01},
journal = {Nat Chem},
volume = {9},
number = {10},
pages = {932-934},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Wolff P, Veiga C Da, Ennifar E, Bec G, Guichard G, Burnouf D, Dumas P
Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations. Journal Article
In: J Am Soc Mass Spectrom, vol. 28, no. 2, pp. 347-357, 2017, ISBN: 27957716.
Abstract | Links | BibTeX | Tags: ARN-MS, DNA-polymerase processivity rings ESI-MS ITC Kd determination, ENNIFAR, Unité ARN
@article{,
title = {Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations.},
author = {P Wolff and C Da Veiga and E Ennifar and G Bec and G Guichard and D Burnouf and P Dumas},
url = {https://www.ncbi.nlm.nih.gov/pubmed/27957716?dopt=Abstract},
doi = {10.1007/s13361-016-1534-6},
isbn = {27957716},
year = {2017},
date = {2017-01-01},
journal = {J Am Soc Mass Spectrom},
volume = {28},
number = {2},
pages = {347-357},
abstract = {We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate Kd values (from 0.03 to 0.5 μM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to Kd(ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (Kd, ΔH) pairs greatly improved the fits and yielded a second Kd(ITC) close to Kd(ESI-MS). The striking features are: (1) ITC detected a minor binding mode (~20%) of 'low-affinity' that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (Kd, ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments. Graphical Abstract ᅟ.},
keywords = {ARN-MS, DNA-polymerase processivity rings ESI-MS ITC Kd determination, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate Kd values (from 0.03 to 0.5 μM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to Kd(ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (Kd, ΔH) pairs greatly improved the fits and yielded a second Kd(ITC) close to Kd(ESI-MS). The striking features are: (1) ITC detected a minor binding mode (~20%) of 'low-affinity' that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (Kd, ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments. Graphical Abstract ᅟ.
Mancera-Martinez E, Querido J Brito, Valasek L S, Simonetti A, Hashem Y
ABCE1: A special factor that orchestrates translation at the crossroad between recycling and initiation. Journal Article
In: RNA Biol, vol. 14, no. 10, pp. 1279-1285, 2017, ISBN: 28498001.
Abstract | Links | BibTeX | Tags: ABCE1 cryo-EM recycling ribosome translation, ENNIFAR, Unité ARN
@article{,
title = {ABCE1: A special factor that orchestrates translation at the crossroad between recycling and initiation.},
author = {E Mancera-Martinez and J Brito Querido and L S Valasek and A Simonetti and Y Hashem},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28498001?dopt=Abstract},
doi = {10.1080/15476286.2016.1269993},
isbn = {28498001},
year = {2017},
date = {2017-01-01},
journal = {RNA Biol},
volume = {14},
number = {10},
pages = {1279-1285},
abstract = {For many years initiation and termination of mRNA translation have been studied separately. However, a direct link between these two isolated stages has been suggested by the fact that some initiation factors also control termination and can even promote ribosome recycling; i.e. the last stage where post-terminating 80 S ribosomes are split to start a new round of initiation. Notably, it is now firmly established that, among other factors, ribosomal recycling critically requires the NTPase ABCE1. However, several earlier reports have proposed that ABCE1 also somehow participates in the initiation complex assembly. Based on an extended analysis of our recently published late-stage 48 S initiation complex from rabbit, here we provide new mechanistic insights into this putative role of ABCE1 in initiation. This point of view represents the first structural evidence in which the regulatory role of the recycling factor ABCE1 in initiation is discussed and establishes a corner stone for elucidating the interplay between ABCE1 and several initiation factors during the transit from ribosomal recycling to formation of the elongation competent 80 S initiation complex.},
keywords = {ABCE1 cryo-EM recycling ribosome translation, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
For many years initiation and termination of mRNA translation have been studied separately. However, a direct link between these two isolated stages has been suggested by the fact that some initiation factors also control termination and can even promote ribosome recycling; i.e. the last stage where post-terminating 80 S ribosomes are split to start a new round of initiation. Notably, it is now firmly established that, among other factors, ribosomal recycling critically requires the NTPase ABCE1. However, several earlier reports have proposed that ABCE1 also somehow participates in the initiation complex assembly. Based on an extended analysis of our recently published late-stage 48 S initiation complex from rabbit, here we provide new mechanistic insights into this putative role of ABCE1 in initiation. This point of view represents the first structural evidence in which the regulatory role of the recycling factor ABCE1 in initiation is discussed and establishes a corner stone for elucidating the interplay between ABCE1 and several initiation factors during the transit from ribosomal recycling to formation of the elongation competent 80 S initiation complex.
D'Ascenzo L, Leonarski F, Vicens Q, Auffinger P
Revisiting GNRA and UNCG folds: U-turns versus Z-turns in RNA hairpin loops. Journal Article
In: RNA, vol. 23, no. 3, pp. 259-269, 2017, ISBN: 27999116.
Abstract | Links | BibTeX | Tags: ENNIFAR, RNA folding RNA motif structure prediction tRNA anticodon tetraloop, Unité ARN
@article{,
title = {Revisiting GNRA and UNCG folds: U-turns versus Z-turns in RNA hairpin loops.},
author = {L D'Ascenzo and F Leonarski and Q Vicens and P Auffinger},
url = {https://www.ncbi.nlm.nih.gov/pubmed/27999116?dopt=Abstract},
doi = {10.1261/rna.059097.116},
isbn = {27999116},
year = {2017},
date = {2017-01-01},
journal = {RNA},
volume = {23},
number = {3},
pages = {259-269},
abstract = {When thinking about RNA three-dimensional structures, coming across GNRA and UNCG tetraloops is perceived as a boon since their folds have been extensively described. Nevertheless, analyzing loop conformations within RNA and RNP structures led us to uncover several instances of GNRA and UNCG loops that do not fold as expected. We noticed that when a GNRA does not assume its "natural" fold, it adopts the one we typically associate with a UNCG sequence. The same folding interconversion may occur for loops with UNCG sequences, for instance within tRNA anticodon loops. Hence, we show that some structured tetranucleotide sequences starting with G or U can adopt either of these folds. The underlying structural basis that defines these two fold types is the mutually exclusive stacking of a backbone oxygen on either the first (in GNRA) or the last nucleobase (in UNCG), generating an oxygen-π contact. We thereby propose to refrain from using sequences to distinguish between loop conformations. Instead, we suggest using descriptors such as U-turn (for "GNRA-type" folds) and a newly described Z-turn (for "UNCG-type" folds). Because tetraloops adopt for the largest part only two (inter)convertible turns, we are better able to interpret from a structural perspective loop interchangeability occurring in ribosomes and viral RNA. In this respect, we propose a general view on the inclination for a given sequence to adopt (or not) a specific fold. We also suggest how long-noncoding RNAs may adopt discrete but transient structures, which are therefore hard to predict.},
keywords = {ENNIFAR, RNA folding RNA motif structure prediction tRNA anticodon tetraloop, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
When thinking about RNA three-dimensional structures, coming across GNRA and UNCG tetraloops is perceived as a boon since their folds have been extensively described. Nevertheless, analyzing loop conformations within RNA and RNP structures led us to uncover several instances of GNRA and UNCG loops that do not fold as expected. We noticed that when a GNRA does not assume its "natural" fold, it adopts the one we typically associate with a UNCG sequence. The same folding interconversion may occur for loops with UNCG sequences, for instance within tRNA anticodon loops. Hence, we show that some structured tetranucleotide sequences starting with G or U can adopt either of these folds. The underlying structural basis that defines these two fold types is the mutually exclusive stacking of a backbone oxygen on either the first (in GNRA) or the last nucleobase (in UNCG), generating an oxygen-π contact. We thereby propose to refrain from using sequences to distinguish between loop conformations. Instead, we suggest using descriptors such as U-turn (for "GNRA-type" folds) and a newly described Z-turn (for "UNCG-type" folds). Because tetraloops adopt for the largest part only two (inter)convertible turns, we are better able to interpret from a structural perspective loop interchangeability occurring in ribosomes and viral RNA. In this respect, we propose a general view on the inclination for a given sequence to adopt (or not) a specific fold. We also suggest how long-noncoding RNAs may adopt discrete but transient structures, which are therefore hard to predict.
Khusainov I, Vicens Q, Ayupov R, Usachev K, Myasnikov A, Simonetti A, Validov S, Kieffer B, Yusupova G, Yusupov M, Hashem Y
Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF. Journal Article
In: EMBO J, vol. 36, no. 14, pp. 2073-2087, 2017, ISBN: 28645916.
Abstract | Links | BibTeX | Tags: cryo-electron microscopy hibernation pathogen ribosome, ENNIFAR, Unité ARN
@article{,
title = {Structures and dynamics of hibernating ribosomes from \textit{Staphylococcus aureus} mediated by intermolecular interactions of HPF.},
author = {I Khusainov and Q Vicens and R Ayupov and K Usachev and A Myasnikov and A Simonetti and S Validov and B Kieffer and G Yusupova and M Yusupov and Y Hashem},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28645916?dopt=Abstract},
doi = {10.15252/embj.201696105},
isbn = {28645916},
year = {2017},
date = {2017-01-01},
journal = {EMBO J},
volume = {36},
number = {14},
pages = {2073-2087},
abstract = {In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation-promoting factor (SaHPF) that we solved using cryo-electron microscopy. Our reconstructions reveal that the N-terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C-terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD-dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.},
keywords = {cryo-electron microscopy hibernation pathogen ribosome, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation-promoting factor (SaHPF) that we solved using cryo-electron microscopy. Our reconstructions reveal that the N-terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C-terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD-dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.
2016
Auffinger P, D'Ascenzo L, Ennifar E
Sodium and Potassium Interactions with Nucleic Acids Book Chapter
In: Sigel, A; Sigel, H; Sigel, R (Ed.): The Alkali Metal Ions: Their Role for Life, vol. 16, pp. 167-201, Springer, 2016, ISBN: 26860302.
Abstract | Links | BibTeX | Tags: DNA Hydration K+ Metal binding Molecular dynamics simulations Monovalent ions Na+·NMR Potassium RNA Sodium Solvation X-ray crystallography, ENNIFAR, Unité ARN
@inbook{,
title = {Sodium and Potassium Interactions with Nucleic Acids},
author = {P Auffinger and L D'Ascenzo and E Ennifar},
editor = {A Sigel and H Sigel and R Sigel},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26860302?dopt=Abstract},
doi = {10.1007/978-3-319-21756-7_6},
isbn = {26860302},
year = {2016},
date = {2016-01-01},
booktitle = {The Alkali Metal Ions: Their Role for Life},
volume = {16},
pages = {167-201},
publisher = {Springer},
series = {Metal Ions in Life Sciences},
abstract = {Metal ions are essential cofactors for the structure and functions of nucleic acids. Yet, the early discovery in the 70s of the crucial role of Mg(2+) in stabilizing tRNA structures has occulted for a long time the importance of monovalent cations. Renewed interest in these ions was brought in the late 90s by the discovery of specific potassium metal ions in the core of a group I intron. Their importance in nucleic acid folding and catalytic activity is now well established. However, detection of K(+) and Na(+) ions is notoriously problematic and the question about their specificity is recurrent. Here we review the different methods that can be used to detect K(+) and Na(+) ions in nucleic acid structures such as X-ray crystallography, nuclear magnetic resonance or molecular dynamics simulations. We also discuss specific versus non-specific binding to different structures through various examples.},
keywords = {DNA Hydration K+ Metal binding Molecular dynamics simulations Monovalent ions Na+·NMR Potassium RNA Sodium Solvation X-ray crystallography, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Metal ions are essential cofactors for the structure and functions of nucleic acids. Yet, the early discovery in the 70s of the crucial role of Mg(2+) in stabilizing tRNA structures has occulted for a long time the importance of monovalent cations. Renewed interest in these ions was brought in the late 90s by the discovery of specific potassium metal ions in the core of a group I intron. Their importance in nucleic acid folding and catalytic activity is now well established. However, detection of K(+) and Na(+) ions is notoriously problematic and the question about their specificity is recurrent. Here we review the different methods that can be used to detect K(+) and Na(+) ions in nucleic acid structures such as X-ray crystallography, nuclear magnetic resonance or molecular dynamics simulations. We also discuss specific versus non-specific binding to different structures through various examples.
Urzhumtsev A, Urzhumtseva L, Baumann U
Helical Symmetry of Nucleic Acids: Obstacle or Help in Structure Solution? Book Chapter
In: Ennifar, E (Ed.): Nucleic Acid Crystallography: Methods and Protocols, vol. 1320, pp. 259-267, Humana Press, NY, 2016, ISBN: 26227048.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@inbook{,
title = {Helical Symmetry of Nucleic Acids: Obstacle or Help in Structure Solution?},
author = {A Urzhumtsev and L Urzhumtseva and U Baumann},
editor = {E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26227048?dopt=Abstract},
doi = {10.1007/978-1-4939-2763-0_16},
isbn = {26227048},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
booktitle = {Nucleic Acid Crystallography: Methods and Protocols},
volume = {1320},
pages = {259-267},
publisher = {Humana Press},
address = {NY},
series = {Methods in Molecular Biology},
abstract = {Crystallographic molecular replacement method is the key tool to define an atomic structure of nucleic acids. Frequently nucleic acids are packed forming continuous helices in the crystal. This arrangement of individual molecules in "infinite" pseudo helical structures in crystal may be the reason why the molecular replacement fails to find a unique position of the search atomic model as the method requires. The Patterson function, calculated as a Fourier series with diffraction intensities, has auxiliary peaks for such a molecular packing. Those near the origin peak indicate the orientation of the helices. The coordinates of other peaks are related to the molecular position and the rotation angle between two such "infinite" helices. Thus, the peak analysis allows getting molecular position even without a search model. An intelligent selecting and averaging of the phase sets corresponding to multiple probable positions of the search model again result in a unique solution but in the form of a Fourier synthesis and not a model. This synthesis can be used then to build an atomic model as it is the case for usual phasing methods.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Crystallographic molecular replacement method is the key tool to define an atomic structure of nucleic acids. Frequently nucleic acids are packed forming continuous helices in the crystal. This arrangement of individual molecules in "infinite" pseudo helical structures in crystal may be the reason why the molecular replacement fails to find a unique position of the search atomic model as the method requires. The Patterson function, calculated as a Fourier series with diffraction intensities, has auxiliary peaks for such a molecular packing. Those near the origin peak indicate the orientation of the helices. The coordinates of other peaks are related to the molecular position and the rotation angle between two such "infinite" helices. Thus, the peak analysis allows getting molecular position even without a search model. An intelligent selecting and averaging of the phase sets corresponding to multiple probable positions of the search model again result in a unique solution but in the form of a Fourier synthesis and not a model. This synthesis can be used then to build an atomic model as it is the case for usual phasing methods.
Latrick C M, Marek M, Ouararhni K, Papin C, Stoll I, Ignatyeva M, Obri A, Ennifar E, Dimitrov S, Romier C, Hamiche A
Molecular basis and specificity of H2A.Z-H2B recognition and deposition by the histone chaperone YL1. Journal Article
In: Nat Struct Mol Biol, vol. 23, no. 4, pp. 309-316, 2016, ISBN: 26974126.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Molecular basis and specificity of H2A.Z-H2B recognition and deposition by the histone chaperone YL1.},
author = {C M Latrick and M Marek and K Ouararhni and C Papin and I Stoll and M Ignatyeva and A Obri and E Ennifar and S Dimitrov and C Romier and A Hamiche},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26974126?dopt=Abstract},
doi = {10.1038/nsmb.3189},
isbn = {26974126},
year = {2016},
date = {2016-01-01},
journal = {Nat Struct Mol Biol},
volume = {23},
number = {4},
pages = {309-316},
abstract = {H2A.Z, a widely conserved histone variant, is evicted from chromatin by the histone chaperone ANP32E. However, to date, no deposition chaperone for H2A.Z is known in metazoans. Here, we identify YL1 as a specific H2A.Z-deposition chaperone. The 2.7-Å-resolution crystal structure of the human YL1-H2A.Z-H2B complex shows that YL1 binding, similarly to ANP32E binding, triggers an extension of the H2A.Z αC helix. The interaction with YL1 is, however, more extensive and includes both the extended acidic patch and the entire DNA-binding surface of H2A.Z-H2B. Substitution of only four amino acid residues of H2A is sufficient for the formation of an H2A.Z-like interface specifically recognized by YL1. Collectively, our data reveal the molecular basis of H2A.Z-specific recognition by YL1 and shed light on the mechanism of H2A.Z transfer to the nucleosome by the ATP-dependent chromatin-remodeling complexes SRCAP and P400-TIP60.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
H2A.Z, a widely conserved histone variant, is evicted from chromatin by the histone chaperone ANP32E. However, to date, no deposition chaperone for H2A.Z is known in metazoans. Here, we identify YL1 as a specific H2A.Z-deposition chaperone. The 2.7-Å-resolution crystal structure of the human YL1-H2A.Z-H2B complex shows that YL1 binding, similarly to ANP32E binding, triggers an extension of the H2A.Z αC helix. The interaction with YL1 is, however, more extensive and includes both the extended acidic patch and the entire DNA-binding surface of H2A.Z-H2B. Substitution of only four amino acid residues of H2A is sufficient for the formation of an H2A.Z-like interface specifically recognized by YL1. Collectively, our data reveal the molecular basis of H2A.Z-specific recognition by YL1 and shed light on the mechanism of H2A.Z transfer to the nucleosome by the ATP-dependent chromatin-remodeling complexes SRCAP and P400-TIP60.
Guedich S, Puffer-Enders B, Baltzinger M, Hoffmann G, Veiga C Da, Jossinet F, Thore S, Bec G, Ennifar E, Burnouf D, Dumas P
Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches. Journal Article
In: RNA Biol, vol. 13, no. 4, pp. 373-390, 2016, ISBN: 26932506.
Abstract | Links | BibTeX | Tags: ENNIFAR, Kinetic Regulation kinITC pyrophosphate riboswitches thiamine, LESCURE, Unité ARN
@article{,
title = {Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.},
author = {S Guedich and B Puffer-Enders and M Baltzinger and G Hoffmann and C Da Veiga and F Jossinet and S Thore and G Bec and E Ennifar and D Burnouf and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26932506?dopt=Abstract},
doi = {10.1080/15476286.2016.1142040},
isbn = {26932506},
year = {2016},
date = {2016-01-01},
journal = {RNA Biol},
volume = {13},
number = {4},
pages = {373-390},
abstract = {Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.},
keywords = {ENNIFAR, Kinetic Regulation kinITC pyrophosphate riboswitches thiamine, LESCURE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.
Ennifar E (Ed.)
Nucleic Acid Crystallography: Methods and Protocols Book
Humana Press, New York, NY, 2016.
Abstract | BibTeX | Tags: ENNIFAR, Unité ARN
@book{,
title = {Nucleic Acid Crystallography: Methods and Protocols},
editor = {E Ennifar},
year = {2016},
date = {2016-01-01},
volume = {1320},
publisher = {Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {This volume includes comprehensive and up-to-date coverage of all nucleic acid-specific steps used in X-ray crystallography, from macromolecule production to structure determination. Chapters dedicated to RNA preparation and crystallogenesis will be of interest to beginners, while chapters focused on data collection, phasing and refinement will be particularly useful to researchers with a higher level of expertise. Several functional case studies are also presented in the last part of the book. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and thorough, Nucleic Acid Crystallography: Methods and Protocols presents protocols that are aimed at both researchers and students who are interested in the structural biology of DNA or RNA, alone or in complex with proteins or ligands.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {book}
}
This volume includes comprehensive and up-to-date coverage of all nucleic acid-specific steps used in X-ray crystallography, from macromolecule production to structure determination. Chapters dedicated to RNA preparation and crystallogenesis will be of interest to beginners, while chapters focused on data collection, phasing and refinement will be particularly useful to researchers with a higher level of expertise. Several functional case studies are also presented in the last part of the book. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and thorough, Nucleic Acid Crystallography: Methods and Protocols presents protocols that are aimed at both researchers and students who are interested in the structural biology of DNA or RNA, alone or in complex with proteins or ligands.
Authoritative and thorough, Nucleic Acid Crystallography: Methods and Protocols presents protocols that are aimed at both researchers and students who are interested in the structural biology of DNA or RNA, alone or in complex with proteins or ligands.
Dumas P, Ennifar E, Veiga C Da, Bec G, Palau W, Primo C Di, Piñeiro A, Sabin J, Muñoz E, Rial J
Extending ITC to Kinetics with kinITC. Journal Article
In: Methods Enzymol, vol. 567, pp. 157-180, 2016, ISBN: 26794354.
Abstract | Links | BibTeX | Tags: Biological calorimetry Equilibration time curve ITC Kinetics Microcalorimetry kinITC, ENNIFAR, Unité ARN
@article{,
title = {Extending ITC to Kinetics with kinITC.},
author = {P Dumas and E Ennifar and C Da Veiga and G Bec and W Palau and C Di Primo and A Piñeiro and J Sabin and E Muñoz and J Rial},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26794354?dopt=Abstract},
doi = {10.1016/bs.mie.2015.08.026},
isbn = {26794354},
year = {2016},
date = {2016-01-01},
journal = {Methods Enzymol},
volume = {567},
pages = {157-180},
abstract = {Isothermal titration calorimetry (ITC) has long been used for kinetic studies in chemistry, but this remained confined to enzymatic studies in the biological field. In fact, the biological community has long had the tendency of ignoring the kinetic possibilities of ITC considering it solely as a thermodynamic technique, whereas surface plasmon resonance is seen as the kinetic technique par excellence. However, the primary signal recorded by ITC is a heat power which is directly related to the kinetics of the reaction. Here, it is shown how this kinetic signal can be recovered by using kinITC, the kinetic extension of ITC. The theoretical basis of kinITC is detailed for the most common situation of a second-order reaction A+B Ω C characterized by kinetic parameters kon, koff. A simplified kinITC-ETC method based upon the determination of an "Equilibration Time Curve" (ETC) is presented. The ETC is obtained by automatic determination of the "effective end" of each injection. The method is illustrated with experimental results with a comparison to Surface Plasmon Resonance (SPR) data. kon values were obtained in a wide range, from 10(3) to 0.5×10(6)M(-1)s(-1). All procedures were implemented in the program AFFINImeter (https://www.affinimeter.com/).},
keywords = {Biological calorimetry Equilibration time curve ITC Kinetics Microcalorimetry kinITC, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Isothermal titration calorimetry (ITC) has long been used for kinetic studies in chemistry, but this remained confined to enzymatic studies in the biological field. In fact, the biological community has long had the tendency of ignoring the kinetic possibilities of ITC considering it solely as a thermodynamic technique, whereas surface plasmon resonance is seen as the kinetic technique par excellence. However, the primary signal recorded by ITC is a heat power which is directly related to the kinetics of the reaction. Here, it is shown how this kinetic signal can be recovered by using kinITC, the kinetic extension of ITC. The theoretical basis of kinITC is detailed for the most common situation of a second-order reaction A+B Ω C characterized by kinetic parameters kon, koff. A simplified kinITC-ETC method based upon the determination of an "Equilibration Time Curve" (ETC) is presented. The ETC is obtained by automatic determination of the "effective end" of each injection. The method is illustrated with experimental results with a comparison to Surface Plasmon Resonance (SPR) data. kon values were obtained in a wide range, from 10(3) to 0.5×10(6)M(-1)s(-1). All procedures were implemented in the program AFFINImeter (https://www.affinimeter.com/).
Veiga C Da, Mezher J, Dumas P, Ennifar E
Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes Book Chapter
In: Ennifar, E (Ed.): Nucleic Acid Crystallography: Methods and Protocols, vol. 1320, pp. 127-143, Humana Press, NY, 2016, ISBN: 26227041.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@inbook{,
title = {Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes},
author = {C Da Veiga and J Mezher and P Dumas and E Ennifar},
editor = {E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26227041?dopt=Abstract},
doi = {10.1007/978-1-4939-2763-0_9},
isbn = {26227041},
year = {2016},
date = {2016-01-01},
booktitle = {Nucleic Acid Crystallography: Methods and Protocols},
volume = {1320},
pages = {127-143},
publisher = {Humana Press},
address = {NY},
series = {Methods in Molecular Biology},
abstract = {The success rate of nucleic acids/ligands co-crystallization can be significantly improved by performing preliminary biophysical analyses. Among suitable biophysical approaches, isothermal titration calorimetry (ITC) is certainly a method of choice. ITC can be used in a wide range of experimental conditions to monitor in real time the formation of the RNA- or DNA-ligand complex, with the advantage of providing in addition the complete binding profile of the interaction. Following the ITC experiment, the complex is ready to be concentrated for crystallization trials. This chapter describes a detailed experimental protocol for using ITC as a tool for monitoring RNA/small molecule binding, followed by co-crystallization.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
The success rate of nucleic acids/ligands co-crystallization can be significantly improved by performing preliminary biophysical analyses. Among suitable biophysical approaches, isothermal titration calorimetry (ITC) is certainly a method of choice. ITC can be used in a wide range of experimental conditions to monitor in real time the formation of the RNA- or DNA-ligand complex, with the advantage of providing in addition the complete binding profile of the interaction. Following the ITC experiment, the complex is ready to be concentrated for crystallization trials. This chapter describes a detailed experimental protocol for using ITC as a tool for monitoring RNA/small molecule binding, followed by co-crystallization.
Challier L, Miranda-Castro R, Barbe B, Fave C, Limoges B, Peyrin E, Ravelet C, Fiore E, Labbé P, Coche-Guérente L, Ennifar E, Bec G, Dumas P, Mavré F, Noël V
In: Anal Chem, vol. 88, no. 23, pp. 11963-11971, 2016, ISBN: 27934108.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3'- versus 5'-End Sequence Modification.},
author = {L Challier and R Miranda-Castro and B Barbe and C Fave and B Limoges and E Peyrin and C Ravelet and E Fiore and P Labbé and L Coche-Guérente and E Ennifar and G Bec and P Dumas and F Mavré and V Noël},
url = {https://www.ncbi.nlm.nih.gov/pubmed/27934108?dopt=Abstract},
doi = {10.1021/acs.analchem.6b04046},
isbn = {27934108},
year = {2016},
date = {2016-01-01},
journal = {Anal Chem},
volume = {88},
number = {23},
pages = {11963-11971},
abstract = {Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.
D'Ascenzo L, Leonarski F, Vicens Q, Auffinger P
'Z-DNA like' fragments in RNA: a recurring structural motif with implications for folding, RNA/protein recognition and immune response. Journal Article
In: Nucleic Acids Res, vol. 44, no. 12, pp. 5944-5956, 2016, ISBN: 27151194.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {'Z-DNA like' fragments in RNA: a recurring structural motif with implications for folding, RNA/protein recognition and immune response.},
author = {L D'Ascenzo and F Leonarski and Q Vicens and P Auffinger},
url = {http://www.ncbi.nlm.nih.gov/pubmed/27151194?dopt=Abstract},
doi = {10.1093/nar/gkw388},
isbn = {27151194},
year = {2016},
date = {2016-01-01},
journal = {Nucleic Acids Res},
volume = {44},
number = {12},
pages = {5944-5956},
abstract = {Since the work of Alexander Rich, who solved the first Z-DNA crystal structure, we have known that d(CpG) steps can adopt a particular structure that leads to forming left-handed helices. However, it is still largely unrecognized that other sequences can adopt 'left-handed' conformations in DNA and RNA, in double as well as single stranded contexts. These 'Z-like' steps involve the coexistence of several rare structural features: a C2'-endo puckering, a syn nucleotide and a lone pair-π stacking between a ribose O4' atom and a nucleobase. This particular arrangement induces a conformational stress in the RNA backbone, which limits the occurrence of Z-like steps to ≈0.1% of all dinucleotide steps in the PDB. Here, we report over 600 instances of Z-like steps, which are located within r(UNCG) tetraloops but also in small and large RNAs including riboswitches, ribozymes and ribosomes. Given their complexity, Z-like steps are probably associated with slow folding kinetics and once formed could lock a fold through the formation of unique long-range contacts. Proteins involved in immunologic response also specifically recognize/induce these peculiar folds. Thus, characterizing the conformational features of these motifs could be a key to understanding the immune response at a structural level.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Since the work of Alexander Rich, who solved the first Z-DNA crystal structure, we have known that d(CpG) steps can adopt a particular structure that leads to forming left-handed helices. However, it is still largely unrecognized that other sequences can adopt 'left-handed' conformations in DNA and RNA, in double as well as single stranded contexts. These 'Z-like' steps involve the coexistence of several rare structural features: a C2'-endo puckering, a syn nucleotide and a lone pair-π stacking between a ribose O4' atom and a nucleobase. This particular arrangement induces a conformational stress in the RNA backbone, which limits the occurrence of Z-like steps to ≈0.1% of all dinucleotide steps in the PDB. Here, we report over 600 instances of Z-like steps, which are located within r(UNCG) tetraloops but also in small and large RNAs including riboswitches, ribozymes and ribosomes. Given their complexity, Z-like steps are probably associated with slow folding kinetics and once formed could lock a fold through the formation of unique long-range contacts. Proteins involved in immunologic response also specifically recognize/induce these peculiar folds. Thus, characterizing the conformational features of these motifs could be a key to understanding the immune response at a structural level.
2015
Ramirez J, Recht R, Charbonnier S, Ennifar E, Atkinson R A, Travé G, Nominé Y, Kieffer B
Disorder-to-order transition of MAGI-1 PDZ1 C-terminal extension upon peptide binding: thermodynamic and dynamic insights. Journal Article
In: Biochemistry, vol. 54, no. 6, pp. 1327-1337, 2015, ISBN: 25590897.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Disorder-to-order transition of MAGI-1 PDZ1 C-terminal extension upon peptide binding: thermodynamic and dynamic insights.},
author = {J Ramirez and R Recht and S Charbonnier and E Ennifar and R A Atkinson and G Travé and Y Nominé and B Kieffer},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25590897?dopt=Abstract},
doi = {10.1021/bi500845j},
isbn = {25590897},
year = {2015},
date = {2015-01-01},
journal = {Biochemistry},
volume = {54},
number = {6},
pages = {1327-1337},
abstract = {PDZ domains are highly abundant protein-protein interaction modules commonly found in multidomain scaffold proteins. The PDZ1 domain of MAGI-1, a protein present at cellular tight junctions that contains 6 PDZ domains, is targeted by the E6 oncoprotein of the high-risk Human Papilloma Virus. Thermodynamic and dynamic studies using complementary ITC and NMR 15N heteronuclear relaxation measurements were conducted at different temperatures to decipher the molecular mechanism of this interaction. The binding of E6 peptides to the MAGI-1 PDZ1 is accompanied by an unusually large and negative change in heat capacity ΔCp that is attributed to a disorder-to-order transition of the C-terminal extension of the PDZ1 domain, observed upon E6 binding. Analysis of the temperature dependent thermodynamic parameters and 15N NMR relaxation data of a PDZ1 mutant for which this disorder-to-order transition was abolished allows the unusual thermodynamic signature of the E6 binding to be correlated to the local folding of PDZ1 C-terminal extension. The comparison of exchange contributions observed for wild-type and mutant proteins explains how the variation in solvent exposed area may compensate for the loss of conformational entropy and further designates a distinct set of few residues that mediate this local folding phenomenon.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
PDZ domains are highly abundant protein-protein interaction modules commonly found in multidomain scaffold proteins. The PDZ1 domain of MAGI-1, a protein present at cellular tight junctions that contains 6 PDZ domains, is targeted by the E6 oncoprotein of the high-risk Human Papilloma Virus. Thermodynamic and dynamic studies using complementary ITC and NMR 15N heteronuclear relaxation measurements were conducted at different temperatures to decipher the molecular mechanism of this interaction. The binding of E6 peptides to the MAGI-1 PDZ1 is accompanied by an unusually large and negative change in heat capacity ΔCp that is attributed to a disorder-to-order transition of the C-terminal extension of the PDZ1 domain, observed upon E6 binding. Analysis of the temperature dependent thermodynamic parameters and 15N NMR relaxation data of a PDZ1 mutant for which this disorder-to-order transition was abolished allows the unusual thermodynamic signature of the E6 binding to be correlated to the local folding of PDZ1 C-terminal extension. The comparison of exchange contributions observed for wild-type and mutant proteins explains how the variation in solvent exposed area may compensate for the loss of conformational entropy and further designates a distinct set of few residues that mediate this local folding phenomenon.
Koch M, Flür S, Kreutz C, Ennifar E, Micura R, Polacek N
Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered GTP hydrolysis. Journal Article
In: Proc Natl Acad Sci U S A, vol. 112, no. 20, pp. E2561-2568, 2015, ISBN: 25941362.
Abstract | Links | BibTeX | Tags: ENNIFAR, RNA solid-phase synthesis methylphosphonate modification protein biosynthesis tRNA translocation, Unité ARN
@article{,
title = {Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered GTP hydrolysis.},
author = {M Koch and S Flür and C Kreutz and E Ennifar and R Micura and N Polacek},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25941362?dopt=Abstract},
doi = {10.1073/pnas.1505231112},
isbn = {25941362},
year = {2015},
date = {2015-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {112},
number = {20},
pages = {E2561-2568},
abstract = {Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.},
keywords = {ENNIFAR, RNA solid-phase synthesis methylphosphonate modification protein biosynthesis tRNA translocation, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.
Sydow D, Leonarski F, D'Ascenzo L, Auffinger P
105 Stabilization effects induced by modified nucleotides in tRNA T-loop motifs. Journal Article
In: J Biomol Struct Dyn, vol. 33, no. Suppl 1, pp. 66, 2015, ISBN: 26103316.
Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {105 Stabilization effects induced by modified nucleotides in tRNA T-loop motifs.},
author = {D Sydow and F Leonarski and L D'Ascenzo and P Auffinger},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26103316?dopt=Abstract},
doi = {10.1080/07391102.2015.1032667},
isbn = {26103316},
year = {2015},
date = {2015-01-01},
journal = {J Biomol Struct Dyn},
volume = {33},
number = {Suppl 1},
pages = {66},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
2014
Mundigala H, Michaux J B, Feig A L, Ennifar E, Rueda D
HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway. Journal Article
In: Nucleic Acids Res, vol. 42, no. 11, pp. 7281-7289, 2014, ISBN: 24813449.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway.},
author = {H Mundigala and J B Michaux and A L Feig and E Ennifar and D Rueda},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24813449?dopt=Abstract},
doi = {10.1093/nar/gku332},
isbn = {24813449},
year = {2014},
date = {2014-01-01},
journal = {Nucleic Acids Res},
volume = {42},
number = {11},
pages = {7281-7289},
abstract = {The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5'-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a 'kissing complex' between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg2+, but not by K+ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg2+ ions.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5'-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a 'kissing complex' between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg2+, but not by K+ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg2+ ions.
Wolff P, Amal I, Oliéric V, Chaloin O, Gygli G, Ennifar E, Lorber B, Guichard G, Wagner J E, Dejaegere A, Burnouf D Y
Differential Modes of Peptide Binding onto Replicative Sliding Clamps from Various Bacterial Origins. Journal Article
In: J Med Chem, vol. 57, no. 18, pp. 7565-7576, 2014, ISBN: 25170813.
Abstract | Links | BibTeX | Tags: ENNIFAR, FRUGIER, Unité ARN
@article{,
title = {Differential Modes of Peptide Binding onto Replicative Sliding Clamps from Various Bacterial Origins.},
author = {P Wolff and I Amal and V Oliéric and O Chaloin and G Gygli and E Ennifar and B Lorber and G Guichard and J E Wagner and A Dejaegere and D Y Burnouf},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25170813?dopt=Abstract},
doi = {10.1021/jm500467a},
isbn = {25170813},
year = {2014},
date = {2014-01-01},
journal = {J Med Chem},
volume = {57},
number = {18},
pages = {7565-7576},
abstract = {Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interacts efficiently with our designed peptides and assembles the Escherichia coli and related orthologs clamps, while group II poorly interact with the same peptides and includes Bacillus subtilis and other Gram+ clamps. These studies also suggest that the peptide binding process could occur via different mechanisms depending on which type of clamp it binds to.},
keywords = {ENNIFAR, FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interacts efficiently with our designed peptides and assembles the Escherichia coli and related orthologs clamps, while group II poorly interact with the same peptides and includes Bacillus subtilis and other Gram+ clamps. These studies also suggest that the peptide binding process could occur via different mechanisms depending on which type of clamp it binds to.
Blond A, Ennifar E, Tisné C, Micouin L
The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study. Journal Article
In: ChemMedChem, vol. 9, no. 9, pp. 1982-96, 2014, ISBN: 25100137.
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, RNA recognition antiviral agents drug design human immunodeficiency virus new targets, Unité ARN
@article{,
title = {The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study.},
author = {A Blond and E Ennifar and C Tisné and L Micouin},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25100137?dopt=Abstract},
doi = {10.1002/cmdc.201402259},
isbn = {25100137},
year = {2014},
date = {2014-01-01},
journal = {ChemMedChem},
volume = {9},
number = {9},
pages = {1982-96},
abstract = {The human immunodeficiency virus 1 (HIV-1) replication cycle is finely tuned with many important steps involving RNA-RNA or protein-RNA interactions, all of them being potential targets for the development of new antiviral compounds. This cycle can also be considered as a good benchmark for the evaluation of early-stage strategies aiming at designing drugs that bind to RNA, with the possibility to correlate in vitro activities with antiviral properties. In this review, we highlight different approaches developed to interfere with four important steps of the HIV-1 replication cycle: the early stage of reverse transcription, the transactivation of viral transcription, the nuclear export of partially spliced transcripts and the dimerization step.},
keywords = {DUMAS, ENNIFAR, RNA recognition antiviral agents drug design human immunodeficiency virus new targets, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The human immunodeficiency virus 1 (HIV-1) replication cycle is finely tuned with many important steps involving RNA-RNA or protein-RNA interactions, all of them being potential targets for the development of new antiviral compounds. This cycle can also be considered as a good benchmark for the evaluation of early-stage strategies aiming at designing drugs that bind to RNA, with the possibility to correlate in vitro activities with antiviral properties. In this review, we highlight different approaches developed to interfere with four important steps of the HIV-1 replication cycle: the early stage of reverse transcription, the transactivation of viral transcription, the nuclear export of partially spliced transcripts and the dimerization step.
Košutić M, Jud L, Veiga C Da, Frener M, Fauster K, Kreutz C, Ennifar E, Micura R
Surprising base pairing and structural properties of 2'-trifluoromethylthio-modified ribonucleic acids. Journal Article
In: J Am Chem Soc, vol. 136, no. 18, pp. 6656-63, 2014, ISBN: 24766131.
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Surprising base pairing and structural properties of 2'-trifluoromethylthio-modified ribonucleic acids.},
author = {M Košutić and L Jud and C Da Veiga and M Frener and K Fauster and C Kreutz and E Ennifar and R Micura},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24766131?dopt=Abstract},
doi = {10.1021/ja5005637},
isbn = {24766131},
year = {2014},
date = {2014-01-01},
journal = {J Am Chem Soc},
volume = {136},
number = {18},
pages = {6656-63},
abstract = {The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2'-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2'-endo conformation of the 2'-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858-2865) that estimated the extent of destabilization caused by a single C2'-endo nucleotide within a native RNA duplex to amount to 6 kcal mol(-1) because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2'-endo conformation of 2'-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2'-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2'-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson-Crick base pair, adopted the same C3'-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2'-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2'-SCF3 nucleoside modification and correlate them to base pairing properties.},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2'-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2'-endo conformation of the 2'-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858-2865) that estimated the extent of destabilization caused by a single C2'-endo nucleotide within a native RNA duplex to amount to 6 kcal mol(-1) because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2'-endo conformation of 2'-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2'-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2'-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson-Crick base pair, adopted the same C3'-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2'-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2'-SCF3 nucleoside modification and correlate them to base pairing properties.
2013
Neuenfeldt A, Lorber B, Ennifar E, Gaudry A, Sauter C, Sissler M, Florentz C
Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture Journal Article
In: Nucleic Acids Research, vol. 41, no. 4, pp. 2698-708, 2013.
Abstract | Links | BibTeX | Tags: ENNIFAR, FRUGIER, Unité ARN
@article{Neuenfeldt2013,
title = {Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture},
author = {A Neuenfeldt and B Lorber and E Ennifar and A Gaudry and C Sauter and M Sissler and C Florentz
},
url = {https://pubmed.ncbi.nlm.nih.gov/23275545/},
doi = { 10.1093/nar/gks1322 },
year = {2013},
date = {2013-02-04},
journal = {Nucleic Acids Research},
volume = {41},
number = {4},
pages = {2698-708},
abstract = {In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs. },
keywords = {ENNIFAR, FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs.
Bec G, Meyer B, Gerard M A, Steger J, Fauster K, Wolff P, Burnouf D, Micura R, Dumas P, Ennifar E
Thermodynamics of HIV-1 Reverse Transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors. Journal Article
In: J Am Chem Soc, vol. 135, no. 26, pp. 9743-9752, 2013, ISBN: 23742167.
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Thermodynamics of HIV-1 Reverse Transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors.},
author = {G Bec and B Meyer and M A Gerard and J Steger and K Fauster and P Wolff and D Burnouf and R Micura and P Dumas and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/pubmed/23742167},
isbn = {23742167},
year = {2013},
date = {2013-01-01},
journal = {J Am Chem Soc},
volume = {135},
number = {26},
pages = {9743-9752},
abstract = {HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermody-namic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a re-interpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation, or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermody-namic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a re-interpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation, or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.
Ennifar E
X-ray crystallography as a tool for mechanism-of-action studies and drug discovery Journal Article
In: Curr Pharm Biotechnol, vol. 14, no. 5, pp. 537-550, 2013, ISBN: ISBN/1389-2010 (Print) 1873-4316 (Online).
Abstract | Links | BibTeX | Tags: Drug design Ligand Molecular mechanism Protein RNA Structural biology X-ray crystallography, DUMAS, ENNIFAR, Unité ARN
@article{,
title = {X-ray crystallography as a tool for mechanism-of-action studies and drug discovery},
author = {E Ennifar},
url = {http://www.ingentaconnect.com/content/ben/cpb/2013/00000014/00000005/art00007?crawler=true},
isbn = {ISBN/1389-2010 (Print)
1873-4316 (Online)},
year = {2013},
date = {2013-01-01},
journal = {Curr Pharm Biotechnol},
volume = {14},
number = {5},
pages = {537-550},
abstract = {Knowledge of three-dimensional structures of biological macromolecules is essential for a complete understanding of many biological processes. X-ray crystallography is the most widely used technique in structural biology and can provide highly detailed structures of proteins, nucleic acids or macromolecular complexes without any size limit. In the past decade, several recent advances in biological crystallography and automation of data collection and structure solution allowed extraordinary progresses and now more than 58 000 crystal structures have been deposited into the Protein Data Bank. This wealth of structural data significantly helped the elucidation of many biological processes and led to the development of new drugs. In this review we will show how of X-ray crystallography can provide insights into the mechanism of action of biological processes and can contribute to the rationale development of ligands through structure-based drug design.},
keywords = {Drug design Ligand Molecular mechanism Protein RNA Structural biology X-ray crystallography, DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Knowledge of three-dimensional structures of biological macromolecules is essential for a complete understanding of many biological processes. X-ray crystallography is the most widely used technique in structural biology and can provide highly detailed structures of proteins, nucleic acids or macromolecular complexes without any size limit. In the past decade, several recent advances in biological crystallography and automation of data collection and structure solution allowed extraordinary progresses and now more than 58 000 crystal structures have been deposited into the Protein Data Bank. This wealth of structural data significantly helped the elucidation of many biological processes and led to the development of new drugs. In this review we will show how of X-ray crystallography can provide insights into the mechanism of action of biological processes and can contribute to the rationale development of ligands through structure-based drug design.
Ennifar E, Aslam M W, Strasser P, Hoffmann G, Dumas P, van Delft F L
Structure-guided Discovery of a Novel Aminoglycoside Conjugate Targeting HIV-1 RNA Viral Genome. Journal Article
In: ACS Chem Biol, vol. 8, no. 11, pp. 2509-17, 2013, ISBN: 24015986.
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Structure-guided Discovery of a Novel Aminoglycoside Conjugate Targeting HIV-1 RNA Viral Genome.},
author = {E Ennifar and M W Aslam and P Strasser and G Hoffmann and P Dumas and F L van Delft},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24015986?dopt=Abstract},
doi = {10.1021/cb400498n},
isbn = {24015986},
year = {2013},
date = {2013-01-01},
journal = {ACS Chem Biol},
volume = {8},
number = {11},
pages = {2509-17},
abstract = {The Dimerization Initiation Site (DIS) of the HIV-1 genomic RNA is a conserved stem-loop that promotes viral genome dimerization by forming a loop-loop complex. The DIS constitutes a potentially interesting target since it is crucial for several key steps of the viral replication. In this work we describe the synthesis of a rationally designed aminoglycoside conjugate that binds the HIV-1 DIS viral RNA with high specificity, as shown by an extensive in vitro binding characterization. We propose a three-dimensional model of the drug/RNA interaction that perfectly fits with binding data. Our results show the feasibility of targeting the HIV DIS viral RNA dimer and open the way to the rationale design of a new class of antiviral drugs. In addition, due to similarities between the HIV-1 DIS RNA and the bacterial aminoacyl decoding site (A site) RNA, we show that this novel aminoglycoside conjugate also binds the bacterial A site with a similar affinity than natural aminoglycoside antibiotics.},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Dimerization Initiation Site (DIS) of the HIV-1 genomic RNA is a conserved stem-loop that promotes viral genome dimerization by forming a loop-loop complex. The DIS constitutes a potentially interesting target since it is crucial for several key steps of the viral replication. In this work we describe the synthesis of a rationally designed aminoglycoside conjugate that binds the HIV-1 DIS viral RNA with high specificity, as shown by an extensive in vitro binding characterization. We propose a three-dimensional model of the drug/RNA interaction that perfectly fits with binding data. Our results show the feasibility of targeting the HIV DIS viral RNA dimer and open the way to the rationale design of a new class of antiviral drugs. In addition, due to similarities between the HIV-1 DIS RNA and the bacterial aminoacyl decoding site (A site) RNA, we show that this novel aminoglycoside conjugate also binds the bacterial A site with a similar affinity than natural aminoglycoside antibiotics.
Pendem N, Nelli Y R, Douat C, Fischer L, Laguerre M, Ennifar E, Kauffmann B, Guichard G
Controlling Helix Formation in the γ-Peptide Superfamily: Heterogeneous Foldamers with Urea/Amide and Urea/Carbamate Backbones Journal Article
In: Angew Chem Int Ed Engl, vol. 52, no. 15, pp. 4147-51, 2013, ISBN: 23460200.
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Controlling Helix Formation in the γ-Peptide Superfamily: Heterogeneous Foldamers with Urea/Amide and Urea/Carbamate Backbones},
author = {N Pendem and Y R Nelli and C Douat and L Fischer and M Laguerre and E Ennifar and B Kauffmann and G Guichard},
url = {http://www.ncbi.nlm.nih.gov/pubmed/23460200},
doi = {10.1002/anie.201209838},
isbn = {23460200},
year = {2013},
date = {2013-01-01},
journal = {Angew Chem Int Ed Engl},
volume = {52},
number = {15},
pages = {4147-51},
abstract = {One fold to rule them all: New heterogeneous aliphatic backbone foldamers belonging to the γ-peptide superfamily and containing various combinations of urea/amide (U/A) and urea/carbamate (U/C) units are reported. Structural studies at atomic resolution reveal hydrogen-bonded helical structures akin to that formed by cognate Un homooligomers.},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
One fold to rule them all: New heterogeneous aliphatic backbone foldamers belonging to the γ-peptide superfamily and containing various combinations of urea/amide (U/A) and urea/carbamate (U/C) units are reported. Structural studies at atomic resolution reveal hydrogen-bonded helical structures akin to that formed by cognate Un homooligomers.
Pendem N, Douat C, Claudon P, Laguerre M, Castano S, Desbat B, Cavagnat D, Ennifar E, Kauffmann B, Guichard G
Helix-forming propensity of aliphatic urea oligomers incorporating noncanonical residue substitution patterns Journal Article
In: J Am Chem Soc, vol. 135, no. 12, pp. 4884-92, 2013, ISBN: 23445529, (1520-5126 (Electronic) 0002-7863 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Biomolecular Peptidomimetics/*chemistry Protein Structure, Circular Dichroism Crystallography, ENNIFAR, Fourier Transform Infrared Urea/*analogs & derivatives, Molecular Molecular Conformation Nuclear Magnetic Resonance, Secondary Spectroscopy, Unité ARN, X-Ray Cyclohexylamines/chemistry Models
@article{,
title = {Helix-forming propensity of aliphatic urea oligomers incorporating noncanonical residue substitution patterns},
author = {N Pendem and C Douat and P Claudon and M Laguerre and S Castano and B Desbat and D Cavagnat and E Ennifar and B Kauffmann and G Guichard},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=23445529},
doi = {10.1021/ja401151v},
isbn = {23445529},
year = {2013},
date = {2013-01-01},
journal = {J Am Chem Soc},
volume = {135},
number = {12},
pages = {4884-92},
abstract = {Aliphatic N,N'-linked oligoureas are peptidomimetic foldamers that adopt a well-defined helical secondary structure stabilized by a collection of remote three-center H-bonds closing 12- and 14-membered pseudorings. Delineating the rules that govern helix formation depending on the nature of constituent units is of practical utility if one aims to utilize this helical fold to place side chains in a given arrangement and elaborate functional helices. In this work, we tested whether the helix geometry is compatible with alternative substitution patterns. The central -NH-CH(R)-CH2-NH-CO- residue in a model oligourea pentamer sequence was replaced by guest units bearing various substitution patterns [e.g., -NH-CH2-CH2-NH-CO-, -NH-CH2-CH(R)-NH-CO-, and -NH-CH(R(1))-CH(R(2))-NH-CO-], levels of preorganization (cyclic vs acyclic residues), and stereochemistries, and the helix formation was systematically assessed. The extent of helix perturbation or stabilization was primarily monitored in solution by Fourier transform IR, NMR, and electronic circular dichroism spectroscopies. Our results indicate that although three new substitution patterns were accommodated in the 2.5-helix, the helical urea backbone in short oligomers is particularly sensitive to variations in the residue substitution pattern (position and stereochemistry). For example, the trans-1,2-diaminocyclohexane unit was experimentally found to break the helix nucleation, but the corresponding cis unit did not. Theoretical calculations helped to rationalize these results. The conformational preferences in this series of oligoureas were also studied at high resolution by X-ray structure analyses of a representative set of modified oligomers.},
note = {1520-5126 (Electronic)
0002-7863 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Biomolecular Peptidomimetics/*chemistry Protein Structure, Circular Dichroism Crystallography, ENNIFAR, Fourier Transform Infrared Urea/*analogs & derivatives, Molecular Molecular Conformation Nuclear Magnetic Resonance, Secondary Spectroscopy, Unité ARN, X-Ray Cyclohexylamines/chemistry Models},
pubstate = {published},
tppubtype = {article}
}
Aliphatic N,N'-linked oligoureas are peptidomimetic foldamers that adopt a well-defined helical secondary structure stabilized by a collection of remote three-center H-bonds closing 12- and 14-membered pseudorings. Delineating the rules that govern helix formation depending on the nature of constituent units is of practical utility if one aims to utilize this helical fold to place side chains in a given arrangement and elaborate functional helices. In this work, we tested whether the helix geometry is compatible with alternative substitution patterns. The central -NH-CH(R)-CH2-NH-CO- residue in a model oligourea pentamer sequence was replaced by guest units bearing various substitution patterns [e.g., -NH-CH2-CH2-NH-CO-, -NH-CH2-CH(R)-NH-CO-, and -NH-CH(R(1))-CH(R(2))-NH-CO-], levels of preorganization (cyclic vs acyclic residues), and stereochemistries, and the helix formation was systematically assessed. The extent of helix perturbation or stabilization was primarily monitored in solution by Fourier transform IR, NMR, and electronic circular dichroism spectroscopies. Our results indicate that although three new substitution patterns were accommodated in the 2.5-helix, the helical urea backbone in short oligomers is particularly sensitive to variations in the residue substitution pattern (position and stereochemistry). For example, the trans-1,2-diaminocyclohexane unit was experimentally found to break the helix nucleation, but the corresponding cis unit did not. Theoretical calculations helped to rationalize these results. The conformational preferences in this series of oligoureas were also studied at high resolution by X-ray structure analyses of a representative set of modified oligomers.
2012
Fauster K, Hartl M, Santner T, Aigner M, Kreutz C, Bister K, Ennifar E, Micura R
2'-Azido RNA, a versatile tool for chemical biology: synthesis, X-ray structure, siRNA applications, click labeling. Journal Article
In: ACS Chem Biol, vol. 7, no. 3, pp. 581-589, 2012, ISBN: 22273279, (Online: January 24, 2012).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {2'-Azido RNA, a versatile tool for chemical biology: synthesis, X-ray structure, siRNA applications, click labeling.},
author = {K Fauster and M Hartl and T Santner and M Aigner and C Kreutz and K Bister and E Ennifar and R Micura},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22273279},
doi = {10.1021/cb200510k},
isbn = {22273279},
year = {2012},
date = {2012-01-01},
journal = {ACS Chem Biol},
volume = {7},
number = {3},
pages = {581-589},
abstract = {Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2'-azido cytidine and 2'-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2'-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2'-azido uridine and 2'-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2'-azido group supports the C3'-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2'-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2'-azido modifications are compatible with 2'-fluoro and/or 2'-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2'-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2'-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.},
note = {Online: January 24, 2012},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2'-azido cytidine and 2'-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2'-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2'-azido uridine and 2'-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2'-azido group supports the C3'-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2'-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2'-azido modifications are compatible with 2'-fluoro and/or 2'-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2'-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2'-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.
Burnouf D Y, Ennifar E, Guedich S, Puffer B, Hoffmann G, Bec G, Disdier F, Baltzinger M, Dumas P
kinITC: a new method for obtaining joint thermodynamic and kinetic data by Isothermal Titration Calorimetry. Journal Article
In: J Am Chem Soc, vol. 134, no. 1, pp. 559-565, 2012, ISBN: 22126339, (Publication Date (Web): November 29, 2011).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {kinITC: a new method for obtaining joint thermodynamic and kinetic data by Isothermal Titration Calorimetry.},
author = {D Y Burnouf and E Ennifar and S Guedich and B Puffer and G Hoffmann and G Bec and F Disdier and M Baltzinger and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22126339},
doi = {10.1021/ja209057d},
isbn = {22126339},
year = {2012},
date = {2012-01-01},
journal = {J Am Chem Soc},
volume = {134},
number = {1},
pages = {559-565},
abstract = {Isothermal Titration Calorimetry is the method of choice for obtaining thermodynamic data on a great variety of systems. Here we show that modern ITC apparatus and new processing methods also allow obtaining a complete kinetic description on more diverse systems than usually thought, ranging from simple ligand binding to complex RNA folding. We illustrate these new features with a simple case (HIV-1 reverse transcriptase/inhibitor interaction), but also with the more complex case of the folding of a riboswitch triggered by Thiamine PyrophosPhate binding. The originality of the new kinITC method appears clearly by its ability of dissecting, both thermodynamically and kinetically, the two components: primary ligand binding and subsequent RNA folding. We are not aware of another single method yielding in a simple way such a deep insight into a composite process. Our study also rationalizes common observations from daily ITC use.},
note = {Publication Date (Web): November 29, 2011},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Isothermal Titration Calorimetry is the method of choice for obtaining thermodynamic data on a great variety of systems. Here we show that modern ITC apparatus and new processing methods also allow obtaining a complete kinetic description on more diverse systems than usually thought, ranging from simple ligand binding to complex RNA folding. We illustrate these new features with a simple case (HIV-1 reverse transcriptase/inhibitor interaction), but also with the more complex case of the folding of a riboswitch triggered by Thiamine PyrophosPhate binding. The originality of the new kinITC method appears clearly by its ability of dissecting, both thermodynamically and kinetically, the two components: primary ligand binding and subsequent RNA folding. We are not aware of another single method yielding in a simple way such a deep insight into a composite process. Our study also rationalizes common observations from daily ITC use.
Freisz S, Mezher J, Hafirassou L, Wolff P, Nomine Y, Romier C, Dumas P, Ennifar E
Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein. Journal Article
In: RNA Biol, vol. 9, no. 7, pp. 966 - 977, 2012, ISBN: 22767258.
Abstract | Links | BibTeX | Tags: ENNIFAR, HIV Biophysics RNA-protein interactions Microcalorimetry, Unité ARN
@article{,
title = {Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein.},
author = {S Freisz and J Mezher and L Hafirassou and P Wolff and Y Nomine and C Romier and P Dumas and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22767258?dopt=Abstract},
isbn = {22767258},
year = {2012},
date = {2012-01-01},
journal = {RNA Biol},
volume = {9},
number = {7},
pages = {966 - 977},
abstract = {The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.},
keywords = {ENNIFAR, HIV Biophysics RNA-protein interactions Microcalorimetry, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.
2011
Wolff P, Olieric V, Briand J P, Chaloin O, Dejaegere A, Dumas P, Ennifar E, Guichard G, Wagner J, Burnouf D Y
Structure-based design of short peptide ligands binding onto the E. coli processivity ring Journal Article
In: J Med Chem, vol. 54, no. 13, pp. 4627-4637, 2011, ISSN: 1520-4804 (Electronic) 0022-2623 (Linking), (Wolff, Philippe Olieric, Vincent Briand, Jean Paul Chaloin, Olivier Dejaegere, Annick Dumas, Philippe Ennifar, Eric Guichard, Gilles Wagner, Jerome Burnouf, Dominique Y J Med Chem. 2011 Jul 14;54(13):4627-37. Epub 2011 Jun 9.).
Abstract | Links | BibTeX | Tags: Crystallography, ENNIFAR, Molecular Oligopeptides/*chemical synthesis/chemistry Protein Binding Structure-Activity Relationship Thermodynamics, Unité ARN, X-Ray DNA Polymerase III/*chemistry DNA Polymerase beta/*chemistry Drug Design Escherichia coli/*chemistry Escherichia coli Proteins/*chemistry Ligands Models
@article{,
title = {Structure-based design of short peptide ligands binding onto the E. coli processivity ring},
author = {P Wolff and V Olieric and J P Briand and O Chaloin and A Dejaegere and P Dumas and E Ennifar and G Guichard and J Wagner and D Y Burnouf},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21619076},
doi = {10.1021/jm200311m},
issn = {1520-4804 (Electronic) 0022-2623 (Linking)},
year = {2011},
date = {2011-01-01},
journal = {J Med Chem},
volume = {54},
number = {13},
pages = {4627-4637},
abstract = {The multimeric DNA sliding clamps confer high processivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10(-8) M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand.},
note = {Wolff, Philippe
Olieric, Vincent
Briand, Jean Paul
Chaloin, Olivier
Dejaegere, Annick
Dumas, Philippe
Ennifar, Eric
Guichard, Gilles
Wagner, Jerome
Burnouf, Dominique Y
J Med Chem. 2011 Jul 14;54(13):4627-37. Epub 2011 Jun 9.},
keywords = {Crystallography, ENNIFAR, Molecular Oligopeptides/*chemical synthesis/chemistry Protein Binding Structure-Activity Relationship Thermodynamics, Unité ARN, X-Ray DNA Polymerase III/*chemistry DNA Polymerase beta/*chemistry Drug Design Escherichia coli/*chemistry Escherichia coli Proteins/*chemistry Ligands Models},
pubstate = {published},
tppubtype = {article}
}
The multimeric DNA sliding clamps confer high processivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10(-8) M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand.
Simonetti A, Marzi S, Myasnikov A, Menetret J F, Klaholz B P
Insights into translation initiation and termination complexes and into the polysome architecture Book Chapter
In: Rodnina, M; Wintermeyer, W; Green, R (Ed.): Ribosomes Structures, Function, and Dynamics, pp. 113-128, Springer, Wien, 2011.
Abstract | Links | BibTeX | Tags: ENNIFAR, ROMBY, Unité ARN
@inbook{,
title = {Insights into translation initiation and termination complexes and into the polysome architecture},
author = {A Simonetti and S Marzi and A Myasnikov and J F Menetret and B P Klaholz},
editor = {M Rodnina and W Wintermeyer and R Green},
url = {http://www.springerlink.com/content/j5022n7218036w14},
doi = {10.1007/978-3-7091-0215-2_10},
year = {2011},
date = {2011-01-01},
booktitle = {Ribosomes Structures, Function, and Dynamics},
pages = {113-128},
publisher = {Springer},
address = {Wien},
abstract = {Translation initiation is the most strongly regulated phase of protein synthesis during which the synthesis of a given protein is decided on. Initiation is the least conserved step of translation, since bacteria, archaea and eukarya have distinct and very different ways to initiate translation, and many different trans-acting factors are involved in the process. In bacteria, translation initiation comprises the consecutive formation of three major intermediary initiation complexes that are assembled via a multi-step process and that differ in composition and in conformation. At the end of the initiation process, an active 70S ribosomal initiation complex (70S IC) has formed which can enter peptide bond formation.},
keywords = {ENNIFAR, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Translation initiation is the most strongly regulated phase of protein synthesis during which the synthesis of a given protein is decided on. Initiation is the least conserved step of translation, since bacteria, archaea and eukarya have distinct and very different ways to initiate translation, and many different trans-acting factors are involved in the process. In bacteria, translation initiation comprises the consecutive formation of three major intermediary initiation complexes that are assembled via a multi-step process and that differ in composition and in conformation. At the end of the initiation process, an active 70S ribosomal initiation complex (70S IC) has formed which can enter peptide bond formation.
Heppell B, Blouin S, Dussault A M, Mulhbacher J, Ennifar E, Penedo J C, Lafontaine D A
Molecular insights into the ligand-controlled organization of the SAM-I riboswitch Journal Article
In: Nat Chem Biol, vol. 7, no. 6, pp. 384-92, 2011, ISBN: ISBN/1552-4469 (Electronic) 1552-4450 (Linking), (Published online 01 May 2011).
Abstract | Links | BibTeX | Tags: Aptamers, Bacterial/*chemistry *Riboswitch S-Adenosylmethionine/*chemistry, DUMAS, ENNIFAR, Nucleotide/chemistry Bacillus subtilis/genetics Binding Sites Crystallography, Unité ARN, X-Ray Ligands Metals Nucleic Acid Conformation RNA
@article{,
title = {Molecular insights into the ligand-controlled organization of the SAM-I riboswitch},
author = {B Heppell and S Blouin and A M Dussault and J Mulhbacher and E Ennifar and J C Penedo and D A Lafontaine},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21532599},
doi = {10.1038/nchembio.563},
isbn = {ISBN/1552-4469 (Electronic)
1552-4450 (Linking)},
year = {2011},
date = {2011-01-01},
journal = {Nat Chem Biol},
volume = {7},
number = {6},
pages = {384-92},
abstract = {S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.},
note = {Published online 01 May 2011},
keywords = {Aptamers, Bacterial/*chemistry *Riboswitch S-Adenosylmethionine/*chemistry, DUMAS, ENNIFAR, Nucleotide/chemistry Bacillus subtilis/genetics Binding Sites Crystallography, Unité ARN, X-Ray Ligands Metals Nucleic Acid Conformation RNA},
pubstate = {published},
tppubtype = {article}
}
S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.
2010
Baussanne I, Bussiere A, Halder S, Ganem-Elbaz C, Ouberai M, Riou M, Paris J M, Ennifar E, Mingeot-Leclercq M P, Decout J L
Synthesis and antimicrobial evaluation of amphiphilic neamine derivatives Journal Article
In: J Med Chem, vol. 53, no. 1, pp. 119-27, 2010, ISBN: 20000576, (1520-4804 (Electronic) 0022-2623 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Synthesis and antimicrobial evaluation of amphiphilic neamine derivatives},
author = {I Baussanne and A Bussiere and S Halder and C Ganem-Elbaz and M Ouberai and M Riou and J M Paris and E Ennifar and M P Mingeot-Leclercq and J L Decout},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20000576},
isbn = {20000576},
year = {2010},
date = {2010-01-01},
journal = {J Med Chem},
volume = {53},
number = {1},
pages = {119-27},
abstract = {The aminoglycoside antibiotics bind to the 16S bacterial rRNA and disturb the protein synthesis. One to four hydroxyl functions of the small aminoglycoside neamine were capped with phenyl, naphthyl, pyridyl, or quinolyl rings. The 3',4'- (6), 3',6- (7a), and the 3',4',6- (10a) 2-naphthylmethylene derivatives appeared to be active against sensitive and resistant Staphylococcus aureus strains. 10a also showed marked antibacterial activities against Gram (-) bacteria, including strains expressing enzymes modifying aminoglycosides, efflux pumps, or rRNA methylases. 7a and 10a revealed a weak and aspecific binding to a model bacterial 16S rRNA. Moreover, as compared to neomycin B, 10a showed a lower ability to decrease (3)H leucine incorporation into proteins in Pseudomonas aeruginosa. All together, our results suggest that the 3',4',6-tri-2-naphthylmethylene neamine derivative 10a should act against Gram (-) bacteria through a mechanism different from inhibition of protein synthesis, probably by membrane destabilization.},
note = {1520-4804 (Electronic)
0022-2623 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The aminoglycoside antibiotics bind to the 16S bacterial rRNA and disturb the protein synthesis. One to four hydroxyl functions of the small aminoglycoside neamine were capped with phenyl, naphthyl, pyridyl, or quinolyl rings. The 3',4'- (6), 3',6- (7a), and the 3',4',6- (10a) 2-naphthylmethylene derivatives appeared to be active against sensitive and resistant Staphylococcus aureus strains. 10a also showed marked antibacterial activities against Gram (-) bacteria, including strains expressing enzymes modifying aminoglycosides, efflux pumps, or rRNA methylases. 7a and 10a revealed a weak and aspecific binding to a model bacterial 16S rRNA. Moreover, as compared to neomycin B, 10a showed a lower ability to decrease (3)H leucine incorporation into proteins in Pseudomonas aeruginosa. All together, our results suggest that the 3',4',6-tri-2-naphthylmethylene neamine derivative 10a should act against Gram (-) bacteria through a mechanism different from inhibition of protein synthesis, probably by membrane destabilization.
Ennifar E, Walter P, Dumas P
Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site Journal Article
In: Nucleic Acids Res, vol. 38, no. 17, pp. 5807-5811, 2010, ISBN: 20460458, (1362-4962 (Electronic) 0305-1048 (Linking) Journal article).
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site},
author = {E Ennifar and P Walter and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20460458},
isbn = {20460458},
year = {2010},
date = {2010-01-01},
journal = {Nucleic Acids Res},
volume = {38},
number = {17},
pages = {5807-5811},
abstract = {The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3'-phosphate and 5'-hydroxyl termini. A crystallographic analysis showed that Ba(2+), Mn(2+), Co(2+) and Zn(2+) bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an 'in-line' geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a 'Trojan horse' mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a 'harmless' species is further transformed in situ into an 'aggressive' species carrying a hydroxide anion.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal article},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3'-phosphate and 5'-hydroxyl termini. A crystallographic analysis showed that Ba(2+), Mn(2+), Co(2+) and Zn(2+) bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an 'in-line' geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a 'Trojan horse' mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a 'harmless' species is further transformed in situ into an 'aggressive' species carrying a hydroxide anion.
Fischer L, Claudon P, Pendem N, Miclet E, Didierjean C, Ennifar E, Guichard G
The canonical helix of urea oligomers at atomic resolution: insights into folding-induced axial organization Journal Article
In: Angew Chem Int Ed Engl, vol. 49, no. 6, pp. 1067-70, 2010, ISBN: 20039243, (1521-3773 (Electronic) 1433-7851 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {The canonical helix of urea oligomers at atomic resolution: insights into folding-induced axial organization},
author = {L Fischer and P Claudon and N Pendem and E Miclet and C Didierjean and E Ennifar and G Guichard},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20039243},
isbn = {20039243},
year = {2010},
date = {2010-01-01},
journal = {Angew Chem Int Ed Engl},
volume = {49},
number = {6},
pages = {1067-70},
note = {1521-3773 (Electronic)
1433-7851 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Freisz S, Bec G, Radi M, Wolff P, Crespan E, Angeli L, Dumas P, Maga G, Botta M, Ennifar E
Crystal structure of HIV-1 reverse transcriptase bound to a non-nucleoside inhibitor with a novel mechanism of action Journal Article
In: Angew Chem Int Ed Engl, vol. 49, no. 10, pp. 1805-8, 2010, ISBN: 20135654, (1521-3773 (Electronic) 1433-7851 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Links | BibTeX | Tags: 3-Ring/*chemistry Humans Indoles/*chemistry *Models, Anti-HIV Agents/*chemistry/therapeutic use Crystallography, Biological *Models, DUMAS, ENNIFAR, Molecular Molecular Structure Nitriles/*chemistry Pyridones/*chemistry Pyrimidines/*chemistry Reverse Transcriptase Inhibitors/*chemistry/therapeutic use Vinyl Compounds/*chemistry, Unité ARN, X-Ray HIV Protease Inhibitors/*chemistry/metabolism/pharmacology HIV Reverse Transcriptase/*chemistry/metabolism Heterocyclic Compounds
@article{,
title = {Crystal structure of HIV-1 reverse transcriptase bound to a non-nucleoside inhibitor with a novel mechanism of action},
author = {S Freisz and G Bec and M Radi and P Wolff and E Crespan and L Angeli and P Dumas and G Maga and M Botta and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20135654},
isbn = {20135654},
year = {2010},
date = {2010-01-01},
journal = {Angew Chem Int Ed Engl},
volume = {49},
number = {10},
pages = {1805-8},
note = {1521-3773 (Electronic)
1433-7851 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {3-Ring/*chemistry Humans Indoles/*chemistry *Models, Anti-HIV Agents/*chemistry/therapeutic use Crystallography, Biological *Models, DUMAS, ENNIFAR, Molecular Molecular Structure Nitriles/*chemistry Pyridones/*chemistry Pyrimidines/*chemistry Reverse Transcriptase Inhibitors/*chemistry/therapeutic use Vinyl Compounds/*chemistry, Unité ARN, X-Ray HIV Protease Inhibitors/*chemistry/metabolism/pharmacology HIV Reverse Transcriptase/*chemistry/metabolism Heterocyclic Compounds},
pubstate = {published},
tppubtype = {article}
}
Sato Y, Ramalanjaona N, Huet T, Potier N, Osz J, Antony P, Peluso-Iltis C, Poussin-Courmontagne P, Ennifar E, Mely Y, Dejaegere A, Moras D, Rochel N
The "Phantom Effect" of the Rexinoid LG100754: structural and functional insights Journal Article
In: PLoS One, vol. 5, no. 11, pp. e15119, 2010, ISBN: ISBN/1932-6203 (Electronic) 1932-6203 (Linking).
Abstract | Links | BibTeX | Tags: Animals Binding Sites Binding, Competitive Fluorescence Polarization Humans Ligands Mice Models, DUMAS, ENNIFAR, Molecular Protein Binding Protein Multimerization Protein Structure, Retinoic Acid/*chemistry/genetics/metabolism Recombinant Proteins/chemistry/metabolism Retinoid X Receptors/*chemistry/genetics/metabolism Retinoids/*chemistry/metabolism Scattering, Small Angle Tetrahydronaphthalenes/*chemistry/metabolism Tretinoin/*chemistry/metabolism X-Ray Diffraction, Tertiary Receptor Cross-Talk Receptors, Unité ARN
@article{,
title = {The "Phantom Effect" of the Rexinoid LG100754: structural and functional insights},
author = {Y Sato and N Ramalanjaona and T Huet and N Potier and J Osz and P Antony and C Peluso-Iltis and P Poussin-Courmontagne and E Ennifar and Y Mely and A Dejaegere and D Moras and N Rochel},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21152046},
doi = {10.1371/journal.pone.0015119},
isbn = {ISBN/1932-6203 (Electronic)
1932-6203 (Linking)},
year = {2010},
date = {2010-01-01},
journal = {PLoS One},
volume = {5},
number = {11},
pages = {e15119},
abstract = {Retinoic acid receptors (RARs) and Retinoid X nuclear receptors (RXRs) are ligand-dependent transcriptional modulators that execute their biological action through the generation of functional heterodimers. RXR acts as an obligate dimer partner in many signalling pathways, gene regulation by rexinoids depending on the liganded state of the specific heterodimeric partner. To address the question of the effect of rexinoid antagonists on RAR/RXR function, we solved the crystal structure of the heterodimer formed by the ligand binding domain (LBD) of the RARalpha bound to its natural agonist ligand (all-trans retinoic acid, atRA) and RXRalpha bound to a rexinoid antagonist (LG100754). We observed that RARalpha exhibits the canonical agonist conformation and RXRalpha an antagonist one with the C-terminal H12 flipping out to the solvent. Examination of the protein-LG100754 interactions reveals that its propoxy group sterically prevents the H12 associating with the LBD, without affecting the dimerization or the active conformation of RAR. Although LG100754 has been reported to act as a 'phantom ligand' activating RAR in a cellular context, our structural data and biochemical assays demonstrate that LG100754 mediates its effect as a full RXR antagonist. Finally we show that the 'phantom ligand effect' of the LG100754 is due to a direct binding of the ligand to RAR that stabilizes coactivator interactions thus accounting for the observed transcriptional activation of RAR/RXR.},
keywords = {Animals Binding Sites Binding, Competitive Fluorescence Polarization Humans Ligands Mice Models, DUMAS, ENNIFAR, Molecular Protein Binding Protein Multimerization Protein Structure, Retinoic Acid/*chemistry/genetics/metabolism Recombinant Proteins/chemistry/metabolism Retinoid X Receptors/*chemistry/genetics/metabolism Retinoids/*chemistry/metabolism Scattering, Small Angle Tetrahydronaphthalenes/*chemistry/metabolism Tretinoin/*chemistry/metabolism X-Ray Diffraction, Tertiary Receptor Cross-Talk Receptors, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Retinoic acid receptors (RARs) and Retinoid X nuclear receptors (RXRs) are ligand-dependent transcriptional modulators that execute their biological action through the generation of functional heterodimers. RXR acts as an obligate dimer partner in many signalling pathways, gene regulation by rexinoids depending on the liganded state of the specific heterodimeric partner. To address the question of the effect of rexinoid antagonists on RAR/RXR function, we solved the crystal structure of the heterodimer formed by the ligand binding domain (LBD) of the RARalpha bound to its natural agonist ligand (all-trans retinoic acid, atRA) and RXRalpha bound to a rexinoid antagonist (LG100754). We observed that RARalpha exhibits the canonical agonist conformation and RXRalpha an antagonist one with the C-terminal H12 flipping out to the solvent. Examination of the protein-LG100754 interactions reveals that its propoxy group sterically prevents the H12 associating with the LBD, without affecting the dimerization or the active conformation of RAR. Although LG100754 has been reported to act as a 'phantom ligand' activating RAR in a cellular context, our structural data and biochemical assays demonstrate that LG100754 mediates its effect as a full RXR antagonist. Finally we show that the 'phantom ligand effect' of the LG100754 is due to a direct binding of the ligand to RAR that stabilizes coactivator interactions thus accounting for the observed transcriptional activation of RAR/RXR.
2009
Radi M, Maga G, Alongi M, Angeli L, Samuele A, Zanoli S, Bellucci L, Tafi A, Casaluce G, Giorgi G, Armand-Ugon M, Gonzalez E, Este J A, Baltzinger M, Bec G, Dumas P, Ennifar E, Botta M
In: J Med Chem, vol. 52, no. 3, pp. 840-851, 2009, ISBN: 19140683, (1520-4804 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Discovery of chiral cyclopropyl dihydro-alkylthio-benzyl-oxopyrimidine (S-DABO) derivatives as potent HIV-1 reverse transcriptase inhibitors with high activity against clinically relevant mutants},
author = {M Radi and G Maga and M Alongi and L Angeli and A Samuele and S Zanoli and L Bellucci and A Tafi and G Casaluce and G Giorgi and M Armand-Ugon and E Gonzalez and J A Este and M Baltzinger and G Bec and P Dumas and E Ennifar and M Botta},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19140683},
isbn = {19140683},
year = {2009},
date = {2009-01-01},
journal = {J Med Chem},
volume = {52},
number = {3},
pages = {840-851},
abstract = {The role played by stereochemistry in the C2-substituent (left part) on the S-DABO scaffold for anti-HIV-1 activity has been investigated for the first time. A series of S-DABO analogues, where the double bond in the C2-substituent is replaced by an enantiopure isosteric cyclopropyl moiety, has been synthesized, leading to the identification of a potent lead compound endowed with picomolar activity against RT (wt) and nanomolar activity against selected drug-resistant mutants. Molecular modeling calculation, enzymatic studies, and surface plasmon resonance experiments allowed us to rationalize the biological behavior of the synthesized compounds, which act as mixed-type inhibitors of HIV-1 RT K103N, with a preferential association to the enzyme-substrate complex. Taken together, our data show that the right combination of stereochemistry on the left and right parts (C6-substituent) of the S-DABO scaffold plays a key role in the inhibition of both wild-type and drug-resistant enzymes, especially the K103N mutant.},
note = {1520-4804 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The role played by stereochemistry in the C2-substituent (left part) on the S-DABO scaffold for anti-HIV-1 activity has been investigated for the first time. A series of S-DABO analogues, where the double bond in the C2-substituent is replaced by an enantiopure isosteric cyclopropyl moiety, has been synthesized, leading to the identification of a potent lead compound endowed with picomolar activity against RT (wt) and nanomolar activity against selected drug-resistant mutants. Molecular modeling calculation, enzymatic studies, and surface plasmon resonance experiments allowed us to rationalize the biological behavior of the synthesized compounds, which act as mixed-type inhibitors of HIV-1 RT K103N, with a preferential association to the enzyme-substrate complex. Taken together, our data show that the right combination of stereochemistry on the left and right parts (C6-substituent) of the S-DABO scaffold plays a key role in the inhibition of both wild-type and drug-resistant enzymes, especially the K103N mutant.
Olieric V, Rieder U, Lang K, Serganov A, Schulze-Briese C, Micura R, Dumas P, Ennifar E
A fast selenium derivatization strategy for crystallization and phasing of RNA structures Journal Article
In: RNA, vol. 15, no. 4, pp. 707-715, 2009, ISBN: 19228585, (1469-9001 (Electronic) Journal Article Research Support, Non-U.S. Gov't Validation Studies).
Abstract | Links | BibTeX | Tags: Base Sequence Crystallography, ENNIFAR, Unité ARN, X-Ray/*methods HIV-1/chemistry/genetics Nucleic Acid Conformation RNA/*chemistry Selenium/*metabolism Staining and Labeling/*methods
@article{,
title = {A fast selenium derivatization strategy for crystallization and phasing of RNA structures},
author = {V Olieric and U Rieder and K Lang and A Serganov and C Schulze-Briese and R Micura and P Dumas and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19228585},
isbn = {19228585},
year = {2009},
date = {2009-01-01},
journal = {RNA},
volume = {15},
number = {4},
pages = {707-715},
abstract = {Site-specific 2'-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2'-OCH(3)-modified RNA sequences, prior to the replacement of 2'-OCH(3) groups with their 2'-SeCH(3) counterparts. The method exploits the similar crystallization properties of 2'-OCH(3)- and 2'-SeCH(3)-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2'-SeCH(3)-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality.},
note = {1469-9001 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't
Validation Studies},
keywords = {Base Sequence Crystallography, ENNIFAR, Unité ARN, X-Ray/*methods HIV-1/chemistry/genetics Nucleic Acid Conformation RNA/*chemistry Selenium/*metabolism Staining and Labeling/*methods},
pubstate = {published},
tppubtype = {article}
}
Site-specific 2'-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2'-OCH(3)-modified RNA sequences, prior to the replacement of 2'-OCH(3) groups with their 2'-SeCH(3) counterparts. The method exploits the similar crystallization properties of 2'-OCH(3)- and 2'-SeCH(3)-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2'-SeCH(3)-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality.
Olieric V, Wolff P, Takeuchi A, Bec G, Birck C, Vitorino M, Kieffer B, Beniaminov A, Cavigiolio G, Theil E, Allmang C, Krol A, Dumas P
SECIS-binding protein 2, a key player in selenoprotein synthesis, is an intrinsically disordered protein Journal Article
In: Biochimie, vol. 91, no. 8, pp. 1003-1009, 2009, ISBN: 19467292, (1638-6183 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: ENNIFAR, ERIANI, Unité ARN
@article{,
title = {SECIS-binding protein 2, a key player in selenoprotein synthesis, is an intrinsically disordered protein},
author = {V Olieric and P Wolff and A Takeuchi and G Bec and C Birck and M Vitorino and B Kieffer and A Beniaminov and G Cavigiolio and E Theil and C Allmang and A Krol and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19467292},
isbn = {19467292},
year = {2009},
date = {2009-01-01},
journal = {Biochimie},
volume = {91},
number = {8},
pages = {1003-1009},
abstract = {Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.},
note = {1638-6183 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {ENNIFAR, ERIANI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.
Wagner J, Etienne H, Fuchs R P, Cordonnier A, Burnouf D
Distinct beta-clamp interactions govern the activities of the Y family PolIV DNA polymerase Journal Article
In: Mol Microbiol, vol. 74, no. 5, pp. 1143-1151, 2009, ISBN: 19843218, (1365-2958 (Electronic) 0950-382X (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Distinct beta-clamp interactions govern the activities of the Y family PolIV DNA polymerase},
author = {J Wagner and H Etienne and R P Fuchs and A Cordonnier and D Burnouf},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19843218},
isbn = {19843218},
year = {2009},
date = {2009-01-01},
journal = {Mol Microbiol},
volume = {74},
number = {5},
pages = {1143-1151},
abstract = {The prototypic Y family DNA polymerase IV (PolIV) of Escherichia coli is involved in multiple replication-associated processes including spontaneous mutagenesis, translesion synthesis (TLS), cell fitness, survival under stressful conditions and checkpoint like functions. It interacts physically and functionally with the replisome's beta processivity clamp through the canonical PolIV C-terminal peptide (CTP). A second interaction that involves a portion of the little finger (LF) domain of PolIV has been structurally described. Here we show that the LF-beta interaction stabilizes the clamp-polymerase complex in vitro and is necessary for the access of PolIV to ongoing replication forks in vivo. However, in contrast to the CTP-beta, the LF-beta interaction is dispensable for the role of the polymerase in TLS. This discloses two independent modes of action for PolIV and, in turn, uncovers a novel way by which the cell may regulate the potentially deleterious effect of such low fidelity polymerases during replication.},
note = {1365-2958 (Electronic)
0950-382X (Linking)
Journal Article},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The prototypic Y family DNA polymerase IV (PolIV) of Escherichia coli is involved in multiple replication-associated processes including spontaneous mutagenesis, translesion synthesis (TLS), cell fitness, survival under stressful conditions and checkpoint like functions. It interacts physically and functionally with the replisome's beta processivity clamp through the canonical PolIV C-terminal peptide (CTP). A second interaction that involves a portion of the little finger (LF) domain of PolIV has been structurally described. Here we show that the LF-beta interaction stabilizes the clamp-polymerase complex in vitro and is necessary for the access of PolIV to ongoing replication forks in vivo. However, in contrast to the CTP-beta, the LF-beta interaction is dispensable for the role of the polymerase in TLS. This discloses two independent modes of action for PolIV and, in turn, uncovers a novel way by which the cell may regulate the potentially deleterious effect of such low fidelity polymerases during replication.
Burnouf D Y, Wagner J E
Kinetics of deoxy-CTP incorporation opposite a dG-C8-N-2-aminofluorene adduct by a high-fidelity DNA polymerase Journal Article
In: J Mol Biol, vol. 386, no. 4, pp. 951-961, 2009, ISBN: 19150355, (1089-8638 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Kinetics of deoxy-CTP incorporation opposite a dG-C8-N-2-aminofluorene adduct by a high-fidelity DNA polymerase},
author = {D Y Burnouf and J E Wagner},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19150355},
isbn = {19150355},
year = {2009},
date = {2009-01-01},
journal = {J Mol Biol},
volume = {386},
number = {4},
pages = {951-961},
abstract = {The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2'-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2'-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.},
note = {1089-8638 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2'-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2'-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.
2008
Lemay J, Maidou-Peindara P, Cancio R, Ennifar E, Coadou G, Maga G, Rain J C, Benarous R, Liu L X
AKAP149 binds to HIV-1 reverse transcriptase and is involved in the reverse transcription Journal Article
In: J Mol Biol, vol. 383, no. 4, pp. 783-796, 2008, ISBN: 18786546, (1089-8638 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: DUMAS A Kinase Anchor Proteins/chemistry/genetics/*metabolism Amino Acid Sequence Animals Binding Sites Cell Line HIV Reverse Transcriptase/chemistry/genetics/*metabolism HIV-1/enzymology/genetics Humans Models, ENNIFAR, Molecular Molecular Sequence Data Mutagenesis Protein Binding Protein Structure, Tertiary RNA Interference Recombinant Fusion Proteins/genetics/metabolism Reverse Transcription/*physiology Two-Hybrid System Techniques Virion/metabolism Virus Replication/physiology, Unité ARN
@article{,
title = {AKAP149 binds to HIV-1 reverse transcriptase and is involved in the reverse transcription},
author = {J Lemay and P Maidou-Peindara and R Cancio and E Ennifar and G Coadou and G Maga and J C Rain and R Benarous and L X Liu},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18786546},
isbn = {18786546},
year = {2008},
date = {2008-01-01},
journal = {J Mol Biol},
volume = {383},
number = {4},
pages = {783-796},
abstract = {Like all retroviruses, human immunodeficiency virus type 1 (HIV-1) undergoes reverse transcription during its replication cycle. The cellular cofactors potentially involved in this process still remain to be identified. We show here that A-kinase anchoring protein 149 (AKAP149) interacts with HIV-1 reverse transcriptase (RT) in both the yeast two-hybrid system and human cells. The AKAP149 binding site has been mapped to the RNase H domain of HIV-1 RT. AKAP149 silencing by RNA interference in HIV-1-infected cells inhibited viral replication at the reverse transcription step. We selected single-point mutants of RT defective for AKAP149 binding and demonstrated that mutant G462R, despite retaining significant intrinsic RT activity in vitro, failed to carry out HIV-1 reverse transcription correctly in infected cells. This suggests that the interaction between RT and AKAP149 in infected cells may play an important role in HIV-1 reverse transcription.},
note = {1089-8638 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {DUMAS A Kinase Anchor Proteins/chemistry/genetics/*metabolism Amino Acid Sequence Animals Binding Sites Cell Line HIV Reverse Transcriptase/chemistry/genetics/*metabolism HIV-1/enzymology/genetics Humans Models, ENNIFAR, Molecular Molecular Sequence Data Mutagenesis Protein Binding Protein Structure, Tertiary RNA Interference Recombinant Fusion Proteins/genetics/metabolism Reverse Transcription/*physiology Two-Hybrid System Techniques Virion/metabolism Virus Replication/physiology, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Like all retroviruses, human immunodeficiency virus type 1 (HIV-1) undergoes reverse transcription during its replication cycle. The cellular cofactors potentially involved in this process still remain to be identified. We show here that A-kinase anchoring protein 149 (AKAP149) interacts with HIV-1 reverse transcriptase (RT) in both the yeast two-hybrid system and human cells. The AKAP149 binding site has been mapped to the RNase H domain of HIV-1 RT. AKAP149 silencing by RNA interference in HIV-1-infected cells inhibited viral replication at the reverse transcription step. We selected single-point mutants of RT defective for AKAP149 binding and demonstrated that mutant G462R, despite retaining significant intrinsic RT activity in vitro, failed to carry out HIV-1 reverse transcription correctly in infected cells. This suggests that the interaction between RT and AKAP149 in infected cells may play an important role in HIV-1 reverse transcription.
Lemay J, Maidou-Peindara P, Bader T, Ennifar E, Rain J C, Benarous R, Liu L X