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2001
Sauter C, Lorber B, Théobald-Dietrich A, Giege R
Crystallogenesis in tRNA aminoacylation systems: how packing accounts for crystallization drawbacks with yeast aspartyl-tRNA synthetase Article de journal
Dans: J Crystal Growth, vol. 232, no. 1-4, p. 399-408, 2001, ISBN: 10.1016/S0022-0248(01)01072-7.
Résumé | Liens | BibTeX | Étiquettes: GIEGE Crystal packing Crystal structure X-ray diffraction Growth from solutions Aspartyl-tRNA synthetase Proteins, SAUTER, Unité ARN
@article{,
title = {Crystallogenesis in tRNA aminoacylation systems: how packing accounts for crystallization drawbacks with yeast aspartyl-tRNA synthetase},
author = {C Sauter and B Lorber and A Théobald-Dietrich and R Giege},
url = {http://www.sciencedirect.com/science/article/pii/S0022024801010727},
isbn = {10.1016/S0022-0248(01)01072-7},
year = {2001},
date = {2001-01-01},
journal = {J Crystal Growth},
volume = {232},
number = {1-4},
pages = {399-408},
abstract = {Two active forms of homodimeric aspartyl-tRNAsynthetase from Saccharomyces cerevisiae differing in length at their N-terminus crystallize in the same orthorhombic space group (P41212) with identical cell parameters. Initial studies were hampered by the poor and anisotropic diffraction of the crystals of enzyme extracted from yeast cells. Isotropic diffraction at higher resolution was obtained when crystals were grown from an engineered protein deprived of its 70 N-terminal amino acids. The present work describes the packing contacts in crystals of the shortened protein whose structure was solved at 2.3 Å resolution. Each subunit of the enzyme develops two lattice interactions covering a surface of 670 Å2, about 7-fold smaller than that of the interface between monomers. The smallest lattice interaction, covering 150 Å2, brings the anticodon binding domain adjacent to the N-terminus of one monomer in contact with a loop from the active-site domain of a neighboring monomer. Modeling of the extension in the solvent channels shows that the 150 Å2 intermolecular contact is perturbed in protein molecules possessing a floppy appendix while their second and larger 520 Å2 contact area is unaffected. Altogether the packing organization explains the poor diffraction properties of the native enzyme crystals and the enhanced diffraction of the crystals of shortened synthetase.},
keywords = {GIEGE Crystal packing Crystal structure X-ray diffraction Growth from solutions Aspartyl-tRNA synthetase Proteins, SAUTER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Two active forms of homodimeric aspartyl-tRNAsynthetase from Saccharomyces cerevisiae differing in length at their N-terminus crystallize in the same orthorhombic space group (P41212) with identical cell parameters. Initial studies were hampered by the poor and anisotropic diffraction of the crystals of enzyme extracted from yeast cells. Isotropic diffraction at higher resolution was obtained when crystals were grown from an engineered protein deprived of its 70 N-terminal amino acids. The present work describes the packing contacts in crystals of the shortened protein whose structure was solved at 2.3 Å resolution. Each subunit of the enzyme develops two lattice interactions covering a surface of 670 Å2, about 7-fold smaller than that of the interface between monomers. The smallest lattice interaction, covering 150 Å2, brings the anticodon binding domain adjacent to the N-terminus of one monomer in contact with a loop from the active-site domain of a neighboring monomer. Modeling of the extension in the solvent channels shows that the 150 Å2 intermolecular contact is perturbed in protein molecules possessing a floppy appendix while their second and larger 520 Å2 contact area is unaffected. Altogether the packing organization explains the poor diffraction properties of the native enzyme crystals and the enhanced diffraction of the crystals of shortened synthetase.
2000
Sauter C, Lorber B, Cavarelli J, Moras D, Giege R
The free yeast aspartyl-tRNA synthetase differs from the tRNA(Asp)-complexed enzyme by structural changes in the catalytic site, hinge region, and anticodon-binding domain Article de journal
Dans: J Mol Biol, vol. 299, no. 5, p. 1313-1324, 2000, ISBN: 10873455, (0022-2836 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Anticodon/chemistry/genetics/*metabolism Aspartate-tRNA Ligase/*chemistry/genetics/*metabolism Binding Sites Catalytic Domain Conserved Sequence/genetics Crystallization Crystallography, Asp/chemistry/genetics/*metabolism Rotation Sequence Deletion/genetics Support, Fungal/chemistry/genetics/metabolism RNA, Molecular Molecular Sequence Data Movement Nucleic Acid Conformation Protein Structure, Non-U.S. Gov't Yeasts/*enzymology/genetics, SAUTER, Secondary RNA, Transfer, Unité ARN, X-Ray Models
@article{,
title = {The free yeast aspartyl-tRNA synthetase differs from the tRNA(Asp)-complexed enzyme by structural changes in the catalytic site, hinge region, and anticodon-binding domain},
author = {C Sauter and B Lorber and J Cavarelli and D Moras and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10873455},
isbn = {10873455},
year = {2000},
date = {2000-01-01},
journal = {J Mol Biol},
volume = {299},
number = {5},
pages = {1313-1324},
abstract = {Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp). The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module.},
note = {0022-2836
Journal Article},
keywords = {Anticodon/chemistry/genetics/*metabolism Aspartate-tRNA Ligase/*chemistry/genetics/*metabolism Binding Sites Catalytic Domain Conserved Sequence/genetics Crystallization Crystallography, Asp/chemistry/genetics/*metabolism Rotation Sequence Deletion/genetics Support, Fungal/chemistry/genetics/metabolism RNA, Molecular Molecular Sequence Data Movement Nucleic Acid Conformation Protein Structure, Non-U.S. Gov't Yeasts/*enzymology/genetics, SAUTER, Secondary RNA, Transfer, Unité ARN, X-Ray Models},
pubstate = {published},
tppubtype = {article}
}
Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp). The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module.
Giege R, Sauter C, Zhu D W, Ng J D, Lorber B
La cristallogenèse des macromolécules biologiques. Chapitre d'ouvrage
Dans: Samarut, J (Ed.): Images de la Recherche en Biologie Structurale, p. 145-151, Editions du CNRS, Paris, 2000.
Résumé | Liens | BibTeX | Étiquettes: SAUTER, Unité ARN
@inbook{,
title = {La cristallogenèse des macromolécules biologiques.},
author = {R Giege and C Sauter and D W Zhu and J D Ng and B Lorber},
editor = {J Samarut},
url = {http://cj.sauter.free.fr/xtal/abstracts.html#31},
year = {2000},
date = {2000-01-01},
booktitle = {Images de la Recherche en Biologie Structurale},
pages = {145-151},
publisher = {Editions du CNRS, Paris},
abstract = {The goal of macromolecular crystallogenesis is to understand and control the crystallisation of proteins and other macromolecular compounds. This survey presents the novel trends in the field and discusses physical methods employed to characterise crystallisation. Similarities and differences with crystal growth of small molecules are emphasised and ways to obtain crystals of higher perfection given. Crystal engineering perspectives and frontiers towards which crystallogenesis leads are outlined.},
keywords = {SAUTER, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
The goal of macromolecular crystallogenesis is to understand and control the crystallisation of proteins and other macromolecular compounds. This survey presents the novel trends in the field and discusses physical methods employed to characterise crystallisation. Similarities and differences with crystal growth of small molecules are emphasised and ways to obtain crystals of higher perfection given. Crystal engineering perspectives and frontiers towards which crystallogenesis leads are outlined.
1999
Wolfson A D, Khvorova A M, Sauter C, Florentz C, Giege R
Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase Article de journal
Dans: Biochemistry, vol. 38, no. 37, p. 11926-11932, 1999, ISBN: 10508395, (0006-2960 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Acylation Aspartate-tRNA Ligase/*chemistry/metabolism Base Sequence Catalysis Cloning, Asp/*chemistry/genetics/metabolism Saccharomyces cerevisiae Support, FLORENTZ, Molecular Enzyme Activation/genetics Genetic Engineering Molecular Mimicry Molecular Sequence Data Mutagenesis, Non-U.S. Gov't, SAUTER, Site-Directed Plasmids/chemical synthesis RNA, Transfer, Unité ARN
@article{,
title = {Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase},
author = {A D Wolfson and A M Khvorova and C Sauter and C Florentz and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10508395},
isbn = {10508395},
year = {1999},
date = {1999-01-01},
journal = {Biochemistry},
volume = {38},
number = {37},
pages = {11926-11932},
abstract = {Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm. The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.},
note = {0006-2960
Journal Article},
keywords = {Acylation Aspartate-tRNA Ligase/*chemistry/metabolism Base Sequence Catalysis Cloning, Asp/*chemistry/genetics/metabolism Saccharomyces cerevisiae Support, FLORENTZ, Molecular Enzyme Activation/genetics Genetic Engineering Molecular Mimicry Molecular Sequence Data Mutagenesis, Non-U.S. Gov't, SAUTER, Site-Directed Plasmids/chemical synthesis RNA, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm. The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.
Sauter C, Ng J D, Lorber B, Keith G, Brion P, Hosseini D W, Lehn J M, Giege R
Additives for the crystallization of proteins and nucleic acids. Article de journal
Dans: J Crystal Growth, vol. 196, no. 2-4, p. 365-376, 1999.
Résumé | Liens | BibTeX | Étiquettes: Crystallogenesis Additives Polyamines, SAUTER, Unité ARN
@article{,
title = {Additives for the crystallization of proteins and nucleic acids.},
author = {C Sauter and J D Ng and B Lorber and G Keith and P Brion and D W Hosseini and J M Lehn and R Giege},
url = {http://www.sciencedirect.com/science/article/pii/S0022024898008525},
year = {1999},
date = {1999-01-01},
journal = {J Crystal Growth},
volume = {196},
number = {2-4},
pages = {365-376},
abstract = {Numerous molecules have been described in literature as additives that were indispensable either for nucleation or
growth of macromolecular crystals. In some cases, such additives were shown to improve the quality of the X-ray
di¤raction and to extend di¤raction limits. We have investigated the e¤ects of more than Þfty compounds, belonging to
several chemical families, on the crystallization of four model proteins (hen and turkey egg-white lysozymes, thaumatin,
and aspartyl-tRNA synthetase from ¹hermus thermophilus). In addition, we have studied the crystallization of a ribonucleic
acid from yeast, the transfer RNA speciÞc for phenylalanine in the presence of synthetic polyamines. Crystals grown
in the presence of the additives were optically evaluated and X-ray di¤raction analyses were performed on selective
crystals to compare their space group, cell parameters, and di¤raction limit with those of controls. Whereas no changes in
space group nor cell parameters were observed for the model proteins, signiÞcant improvements in di¤raction limit were
found when the transfer RNA was crystallized with certain synthetic polyamines. (1999 Elsevier Science B.V. All
rights reserved.},
keywords = {Crystallogenesis Additives Polyamines, SAUTER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Numerous molecules have been described in literature as additives that were indispensable either for nucleation or
growth of macromolecular crystals. In some cases, such additives were shown to improve the quality of the X-ray
di¤raction and to extend di¤raction limits. We have investigated the e¤ects of more than Þfty compounds, belonging to
several chemical families, on the crystallization of four model proteins (hen and turkey egg-white lysozymes, thaumatin,
and aspartyl-tRNA synthetase from ¹hermus thermophilus). In addition, we have studied the crystallization of a ribonucleic
acid from yeast, the transfer RNA speciÞc for phenylalanine in the presence of synthetic polyamines. Crystals grown
in the presence of the additives were optically evaluated and X-ray di¤raction analyses were performed on selective
crystals to compare their space group, cell parameters, and di¤raction limit with those of controls. Whereas no changes in
space group nor cell parameters were observed for the model proteins, signiÞcant improvements in di¤raction limit were
found when the transfer RNA was crystallized with certain synthetic polyamines. (1999 Elsevier Science B.V. All
rights reserved.
growth of macromolecular crystals. In some cases, such additives were shown to improve the quality of the X-ray
di¤raction and to extend di¤raction limits. We have investigated the e¤ects of more than Þfty compounds, belonging to
several chemical families, on the crystallization of four model proteins (hen and turkey egg-white lysozymes, thaumatin,
and aspartyl-tRNA synthetase from ¹hermus thermophilus). In addition, we have studied the crystallization of a ribonucleic
acid from yeast, the transfer RNA speciÞc for phenylalanine in the presence of synthetic polyamines. Crystals grown
in the presence of the additives were optically evaluated and X-ray di¤raction analyses were performed on selective
crystals to compare their space group, cell parameters, and di¤raction limit with those of controls. Whereas no changes in
space group nor cell parameters were observed for the model proteins, signiÞcant improvements in di¤raction limit were
found when the transfer RNA was crystallized with certain synthetic polyamines. (1999 Elsevier Science B.V. All
rights reserved.
Sauter C, Lorber B, Kern D, Cavarelli J, Moras D, Giege R
Crystallogenesis studies on yeast aspartyl-tRNA synthetase: use of phase diagram to improve crystal quality Article de journal
Dans: Acta Crystallogr D Biol Crystallogr, vol. 55, no. Pt 1, p. 149-156, 1999, (0907-4449 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Aspartate-tRNA Ligase/*chemistry/genetics/*isolation & purification Crystallization Crystallography, Fungal Saccharomyces cerevisiae/*enzymology/genetics Sequence Deletion Solutions Support, Non-U.S. Gov't, SAUTER, Unité ARN, X-Ray Genes
@article{,
title = {Crystallogenesis studies on yeast aspartyl-tRNA synthetase: use of phase diagram to improve crystal quality},
author = {C Sauter and B Lorber and D Kern and J Cavarelli and D Moras and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10089405},
doi = {10089405},
year = {1999},
date = {1999-01-01},
journal = {Acta Crystallogr D Biol Crystallogr},
volume = {55},
number = {Pt 1},
pages = {149-156},
abstract = {Aspartyl-tRNA synthetase (AspRS) extracted from yeast is heterogeneous owing to proteolysis of its positively charged N-terminus; its crystals are of poor quality. To overcome this drawback, a rational strategy was developed to grow crystals of sufficient quality for structure determination. The strategy is based on improvement of the protein homogeneity and optimization of crystallization, taking advantage of predictions from crystal-growth theories. An active mutant lacking the first 70 residues was produced and initial crystallization conditions searched. The shape and habit of initial crystals were improved by establishing a phase diagram of protein versus crystallizing-agent concentrations. Growth of large well faceted crystals takes place at low supersaturations near the isochronic supersolubility curve. Further refinement led to reproducible growth of two crystalline forms of bipyramidal (I) or prismatic (II) habit. Both diffract X-rays better than crystals previously obtained with native AspRS. Complete data sets were collected at 3 A resolution for form I (space group P41212) and form II (space group P3221) and molecular-replacement solutions were found in both space groups.},
note = {0907-4449
Journal Article},
keywords = {Aspartate-tRNA Ligase/*chemistry/genetics/*isolation & purification Crystallization Crystallography, Fungal Saccharomyces cerevisiae/*enzymology/genetics Sequence Deletion Solutions Support, Non-U.S. Gov't, SAUTER, Unité ARN, X-Ray Genes},
pubstate = {published},
tppubtype = {article}
}
Aspartyl-tRNA synthetase (AspRS) extracted from yeast is heterogeneous owing to proteolysis of its positively charged N-terminus; its crystals are of poor quality. To overcome this drawback, a rational strategy was developed to grow crystals of sufficient quality for structure determination. The strategy is based on improvement of the protein homogeneity and optimization of crystallization, taking advantage of predictions from crystal-growth theories. An active mutant lacking the first 70 residues was produced and initial crystallization conditions searched. The shape and habit of initial crystals were improved by establishing a phase diagram of protein versus crystallizing-agent concentrations. Growth of large well faceted crystals takes place at low supersaturations near the isochronic supersolubility curve. Further refinement led to reproducible growth of two crystalline forms of bipyramidal (I) or prismatic (II) habit. Both diffract X-rays better than crystals previously obtained with native AspRS. Complete data sets were collected at 3 A resolution for form I (space group P41212) and form II (space group P3221) and molecular-replacement solutions were found in both space groups.
