Publications

2006

• Évolution in silico des protéines monomériques et dimériques.
  
  • Noirel Josselin
  , 2006. La simulation in silico des gènes codant pour des ARN de transfert et des protéines a connu un développement considérable ces dernières années car elle permet de déduire de modèles simples des comportements inattendus qui découlent de la structure des génotypes dans l'espace des séquences et de la correspondance génotype-phénotype. Le modèle d'évolution le plus élémentaire, la théorie dite "de l'évolution neutre" conçue et défendue par Kimura principalement, donne lieu à un phénomène à présent bien documenté: les génotypes robustes aux mutations et exprimant des protéines se repliant efficacement, sont surreprésentés en comparaison des génotypes "fragiles". De nombreuses questions restent en suspens notamment en ce qui concerne l'incidence que peut avoir une modélisation plus réaliste de la fonctionnalité d'une protéine sur le schéma tracé à partir de considérations purement structurales et cinétiques. Pour cela, nous avons développé un modèle incluant une contrainte sélective imposant une dimérisation spécifique minimale de deux protéines codées par deux gènes pour qu'un individu puisse survivre. Nous démontrons que les réseaux neutres construits d'après des critères structuraux sont grandement plastiques et peuvent s'adapter à une fonction vitale sans souffrir de baisse de stabilité. La surreprésentation des génotypes robustes est maintenue, elle est même amplifiée par l'interaction épistatique existant entre les deux gènes. On observe que cela s'accompagne d'une augmentation en moyenne de la fonctionnalité résultant de l'émergence d'un *superfunnel* fonctionnel dans l'espace des séquences. Cette propriété remarquable pourrait avoir d'importantes implications dans l'explication de l'émergence de nouvelles fonctions biologiques. Une autre question concerne les simplifications impliquées par le choix des modèles protéiques. Puisque les simulations évolutives supposent un coût de calcul important, les protéines sur réseau ont eu la préférence de nombreux modélisateurs. Dans ce mémoire, nous proposons un modèle de protéine hors réseau possédant des cartes de contacts plus complexes que les protéines sur réseau. Il confirme les conclusions tirées des simulations sur les protéines sur réseau.
• The tetracycline: Mg2+ complex: a molecular mechanics force field.
  
  • Aleksandrov Alexey
  • Simonson T.
  Journal of Computational Chemistry, Wiley, 2006, 27 (13), pp.1517-33. Tetracycline (Tc) is an important antibiotic, which binds specifically to the ribosome and several proteins, in the form of a Tc-:Mg2+ complex. To model Tc:protein and Tc:RNA interactions, we have developed a molecular mechanics force field model of Tc, which is consistent with the CHARMM force field for proteins and nucleic acids. We used structures from the Cambridge Crystallographic Data Base to identify the main Tc conformations that are likely to be present in solution and in biomolecular complexes. A conformational search was also done, using the MM3 force field to perform simulated annealing of Tc. Several resulting, low-energy structures were optimized with an ab initio model and used in developing the new Tc force field. Atomic charges and Lennard-Jones parameters were derived from a supermolecule ab initio approach. We considered the ab initio energies and geometries of a probe water molecule interacting with Tc at 36 different positions. We considered both a neutral and a zwitterionic Tc form, with and without bound Mg2+. The final rms deviation between the ab initio and force field energies, averaged over all forms, was just 0.35 kcal/mol. The model also reproduces the ab initio geometry and flexibility of Tc. As further tests, we did simulations of a Tc crystal, of Tc:Mg2+ and Tc:Ca2+ complexes in aqueous solution, and of a solvated complex between Tc:Mg2+ and the Tet repressor protein (TetR). With slight, ad hoc adjustments, the model can reproduce the experimental, relative, Tc binding affinities of Mg2+ and Ca2+. It performs well for the structure and fluctuations of the Tc:Mg2+:TetR complex. The model should therefore be suitable to investigate the interactions of Tc with proteins and RNA. It provides a starting point to parameterize other compounds in the large Tc family. (10.1002/jcc.20453)
  DOI : 10.1002/jcc.20453
• Identification in archaea of a novel D-Tyr-tRNATyr deacylase.
  
  • Ferri-Fioni Maria-Laura
  • Fromant Michel
  • Bouin Anne-Pascale
  • Aubard Caroline
  • Lazennec Christine
  • Plateau Pierre
  • Blanquet Sylvain
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2006, 281 (37), pp.27575-85. Most bacteria and eukarya contain an enzyme capable of specifically hydrolyzing D-aminoacyl-tRNA. Here, the archaea Sulfolobus solfataricus is shown to also contain an enzyme activity capable of recycling misaminoacylated D-Tyr-tRNATyr. N-terminal sequencing of this enzyme identifies open reading frame SS02234 (dtd2), the product of which does not present any sequence homology with the known D-Tyr-tRNATyr deacylases of bacteria or eukaryotes. On the other hand, homologs of dtd2 occur in archaea and plants. The Pyrococcus abyssi dtd2 ortholog (PAB2349) was isolated. It rescues the sensitivity to D-tyrosine of a mutant Escherichia coli strain lacking dtd, the gene of its endogeneous D-Tyr-tRNATyr deacylase. Moreover, in vitro, the PAB2349 product, which behaves as a monomer and carries 2 mol of zinc/mol of protein, catalyzes the cleavage of D-Tyr-tRNATyr. The three-dimensional structure of the product of the Archaeoglobus fulgidus dtd2 ortholog has been recently solved by others through a structural genomics approach (Protein Data Bank code 1YQE). This structure does not resemble that of Escherichia coli D-Tyr-tRNATyr deacylase. Instead, it displays homology with that of a bacterial peptidyl-tRNA hydrolase. We show, however, that the archaeal PAB2349 enzyme does not act against diacetyl-Lys-tRNALys, a model substrate of peptidyl-tRNA hydrolase. Based on the Protein Data Bank 1YQE structure, site-directed mutagenesis experiments were undertaken to remove zinc from the PAB2349 enzyme. Several residues involved in zinc binding and supporting the activity of the deacylase were identified. Taken together, these observations suggest evolutionary links between the various hydrolases in charge of the recycling of metabolically inactive tRNAs during translation. (10.1074/jbc.M605860200)
  DOI : 10.1074/jbc.M605860200
• Molecular dynamics simulations show that bound Mg2+ contributes to amino acid and aminoacyl adenylate binding specificity in aspartyl-tRNA synthetase through long range electrostatic interactions.
  
  • Thompson Damien
  • Simonson T.
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2006, 281 (33), pp.23792-803. Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry. The Mg2+ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP x Mg3(2+) complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg2+ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNA-bound AspRS, the single remaining Mg2+ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg2+ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states. (10.1074/jbc.M602870200)
  DOI : 10.1074/jbc.M602870200
• Structural basis of RNA-dependent recruitment of glutamine to the genetic code.
  
  • Oshikane Hiroyuki
  • Sheppard Kelly
  • Fukai Shuya
  • Nakamura Yuko
  • Ishitani Ryuichiro
  • Numata Tomoyuki
  • Sherrer R Lynn
  • Feng Liang
  • Schmitt Emmanuelle
  • Panvert Michel
  • Blanquet Sylvain
  • Mechulam Yves
  • Söll Dieter
  • Nureki Osamu
  Science, American Association for the Advancement of Science (AAAS), 2006, 312 (5782), pp.1950-4. Glutaminyl-transfer RNA (Gln-tRNA(Gln)) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNA(Gln) by the heterodimeric Glu-tRNA(Gln) amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNA(Gln) at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNA(Gln) mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom-long channel for ammonia transport connects the active sites in GatD and GatE. tRNA(Gln) recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code. (10.1126/science.1128470)
  DOI : 10.1126/science.1128470
• Cys(x)His(y)-Zn2+ interactions: possibilities and limitations of a simple pairwise force field.
  
  • Calimet N.
  • Simonson T.
  Journal of Molecular Graphics and Modelling, Elsevier, 2006, 24 (5), pp.404-11. In zinc proteins, the Zn2+ cation frequently binds with a tetrahedral coordination to cysteine and histidine side chains. We examine the possibilities and limitations of a classical, pairwise force field for molecular dynamics of such systems. Hartree Fock and density functional calculations are used to obtain geometries, charge distributions, and association energies of side chain analogues bound to Zn2+. Both ionized and neutral cysteines are considered. Two parameterizations are obtained, then tested and compared through molecular dynamics simulations of two small, homologous proteins in explicit solvent: Protein Kinase C and the Cysteine Rich Domain (CRD) of Raf, which have two Cys3His-Zn2+ groups each. The lack of explicit polarizability and charge transfer in the force field leads to poor accuracy for the association energies, and to parameters--including the zinc charge, that depend on the number of bound cysteines and their protonation state. Nevertheless, the structures sampled with the best parameterization are in good overall agreement with experiment, and have zinc coordination geometries compatible with related structures in the Cambridge Structural Database and the Protein Data Bank. Non-optimized parameters lead to poorer structures. This suggests that while a simple force field is not appropriate for processes involving exchange between water and amino acids in the zinc coordination sphere (e.g. protein unfolding), it can be useful for equilibrium simulations of stable Cys3His zinc fingers. (10.1016/j.jmgm.2005.10.006)
  DOI : 10.1016/j.jmgm.2005.10.006
• Active sites by computational protein design
  
  • Tortosa Pablo
  • Jaramillo Alfonso
  , 2006, 851, pp.96-101. We have developed an automated method to design active sites into protein scaffolds using computational protein design techniques. We search through the amino acid sequence and conformation spaces by optimising protein stability and ligand binding. We use an all-atom force field, a high- resolution protein structure and a rotamer library to model a protein's unfolded and folded states. We enlarge a rotamer library by using a minimization procedure that optimizes rotamers to maximize intermolecular h-bonds. We validate our methodology by re-designing SH3-domain proteins to bind a set of 64 peptides. © 2006 American Institute of Physics. (10.1063/1.2345625)
  DOI : 10.1063/1.2345625
• Free-energy simulations and experiments reveal long-range electrostatic interactions and substrate-assisted specificity in an aminoacyl-tRNA synthetase.
  
  • Thompson Damien
  • Plateau Pierre
  • Simonson T.
  ChemBioChem, Wiley-VCH Verlag, 2006, 7 (2), pp.337-44. Specific recognition of their cognate amino acid substrates by the aminoacyl-tRNA synthetase enzymes is essential for the correct translation of the genetic code. For aspartyl-tRNA synthetase (AspRS), electrostatic interactions are expected to play an important role, since its three substrates (aspartate, ATP, tRNA) are all electrically charged. We used molecular-dynamics free-energy simulations and experiments to compare the binding of the substrate Asp and its electrically neutral analogue Asn to AspRS. The preference for Asp is found to be very strong, with good agreement between simulations and experiment. The simulations reveal long-range interactions that electrostatically couple the amino acid ligand, ATP, and its associated Mg2+ cations, a histidine side chain (His448) next to the amino acid ligand and a flexible loop that closes over the active site in response to amino acid binding. Closing this loop brings a negatively charged glutamate into the active site; this causes His448 to recruit a labile proton, which interacts favorably with Asp and accounts for most of the Asp/Asn discrimination. Cobinding of the second substrate, ATP, increases specificity for Asp further and makes the system robust towards removal of His448, which is mutated to a neutral amino acid in many organisms. Thus, AspRS specificity is assisted by a labile proton and a cosubstrate, and ATP acts as a mobile discriminator for specific Asp binding to AspRS. In asparaginyl-tRNA synthetase, a close homologue of AspRS, a few binding-pocket differences modify the charge balance so that asparagine binding predominates. (10.1002/cbic.200500364)
  DOI : 10.1002/cbic.200500364
• Structural switch of the gamma subunit in an archaeal aIF2 alpha gamma heterodimer.
  
  • Yatime Laure
  • Mechulam Yves
  • Blanquet Sylvain
  • Schmitt Emmanuelle
  Structure / Struct Fold Des; Structure (Camb ), 2006, 14 (1), pp.119-28. Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (alphabetagamma) supplying the small subunit of the ribosome with methionylated initiator tRNA. This study reports the crystallographic structure of an aIF2alphagamma heterodimer from Sulfolobus solfataricus bound to Gpp(NH)p-Mg(2+). aIF2gamma is in a closed conformation with the G domain packed on domains II and III. The C-terminal domain of aIF2alpha interacts with domain II of aIF2gamma. Conformations of the two switch regions involved in GTP binding are similar to those encountered in an EF1A:GTP:Phe-tRNA(Phe) complex. Comparison with the EF1A structure suggests that only the gamma subunit of the aIF2alphagamma heterodimer contacts tRNA. Because the alpha subunit markedly reinforces the affinity of tRNA for the gamma subunit, a contribution of the alpha subunit to the switch movements observed in the gamma structure is considered. (10.1016/j.str.2005.09.020)
  DOI : 10.1016/j.str.2005.09.020