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
2006; Elsevier BV; Volume: 281; Issue: 33 Linguagem: Inglês
10.1074/jbc.m602870200
ISSN1083-351X
AutoresDamien Thompson, Thomas Simonson,
Tópico(s)RNA modifications and cancer
ResumoMolecular 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·Mg32+ 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 tRNAbound 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. 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·Mg32+ 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 tRNAbound 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. Specific molecular association is fundamental to many biochemical processes and is frequently used to transfer energy or information. Aminoacyl-tRNA synthetases (aaRSs) 3The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; aa, amino acid(s); AspRS, aspartyl-tRNA synthetase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; MD, molecular dynamics; MDFE, molecular dynamics free energy; PBFE, Poisson-Boltzmann free energy; PME, particle mesh Ewald; CRF, continuum reaction field; r.m.s., root mean square. are an important class of information-processing enzymes (1Ibba M. Francklyn C. Cusack S. Aminoacyl-tRNA Synthetases. Landes Bioscience, Georgetown, TX2005Google Scholar, 2Arnez J.G. Moras D. Nagai K. Mattaj I. RNA-Protein Interactions. Oxford University Press, Oxford, UK1994: 52-81Google Scholar, 3Meinnel T. Mechulam Y. Blanquet S. Söll D. Raj Bhandary T.L. tRNA: Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 251-291Google Scholar, 4Schimmel P. Ribas de Pouplana L. Trends Biochem. Sci. 2000; 25: 207-209Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Each aaRS catalyzes the aminoacylation of a specific tRNA by a cognate amino acid, establishing the genetic code (5Pallanck L. Pak M. Schulman L.H. Söll D. Raj Bhandary T.L. tRNA: Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 371-393Google Scholar, 6Arnez J.G. Moras D. Trends Biochem. Sci. 1997; 22: 211-216Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 7Francklyn C.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9650-9652Crossref PubMed Scopus (32) Google Scholar). The amino acid (aa) and ATP react first to form an aminoacyl adenylate; in a second step, the amino acid is transferred to the tRNA. Some aaRSs have evolved a third, editing step where incorrect tRNA-aa products are hydrolyzed (8Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar, 9Fersht A. Kirkwood T. Rosenberger R. Galas D. Accuracy of Molecular Processes. Chapman and Hall, New York1986: 67-82Google Scholar, 10Soutourina J. Plateau P. Blanquet S. J. Biol. Chem. 2000; 275: 32535-32542Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Specificity for the aa and the tRNA can arise from different component steps, such as binding or release of the amino acid or binding or acylation of the tRNA. Furthermore, through their particular combination of reversible binding and irreversible reaction steps, aaRSs can use specificity in successive steps to amplify the overall effect (11Hopfield J.J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4135-4139Crossref PubMed Scopus (1103) Google Scholar). For example, amino acid specificity can be established in the aaRS·aa complex, the aaRS·aaAMP complex, or both. The 20 aaRSs form two distinct classes of 10 members each (6Arnez J.G. Moras D. Trends Biochem. Sci. 1997; 22: 211-216Abstract Full Text PDF PubMed Scopus (291) Google Scholar). Below we have focused mainly on aspartyl-tRNA synthetase (AspRS), one of the best studied aaRSs. AspRS belongs to the aaRS class II, forming a subclass IIb with AsnRS and LysRS. Although aaRSs are generally very amino acid-specific, they have a complex evolutionary history (4Schimmel P. Ribas de Pouplana L. Trends Biochem. Sci. 2000; 25: 207-209Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 12Artymiuk P.J. Rice D.W. Poirrette A.R. Willett P. Nat. Struct. Biol. 1994; 11: 758-760Crossref Scopus (72) Google Scholar), which has led to a remarkable diversity in the modern enzymes. Within class IIb, for example, LysRS is very specific in yeast; but in Escherichia coli, it is more promiscuous (13Jakubowski H. Biochemistry. 1999; 38: 8088-8093Crossref PubMed Scopus (56) Google Scholar). In E. coli, AspRS discriminates strongly against Asn, but more weakly against d-Asp (10Soutourina J. Plateau P. Blanquet S. J. Biol. Chem. 2000; 275: 32535-32542Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Several aaRSs achieve a high fidelity through their editing step. An ambiguous IleRS was constructed recently by deleting the IleRS editing domain (14Pezo V. Metzgar D. Hendrickson T.L. Waas W.F. Doring V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8593-8597Crossref PubMed Scopus (63) Google Scholar); the resulting Ile/Val ambiguity actually led to a growth advantage in bacteria. As a last example, many archaebacteria lack AsnRS and produce tRNAAsn-Asn by an indirect route; tRNAAsn is aspartylated by a "nondiscriminating" AspRS (which accepts both tRNAAsp and tRNAAsn), and then the Asp moiety is amidated (1Ibba M. Francklyn C. Cusack S. Aminoacyl-tRNA Synthetases. Landes Bioscience, Georgetown, TX2005Google Scholar). The mechanism of the two enzyme reactions, amino acid adenylation and tRNA aminoacylation, is qualitatively understood in both aaRS classes (1Ibba M. Francklyn C. Cusack S. Aminoacyl-tRNA Synthetases. Landes Bioscience, Georgetown, TX2005Google Scholar). For AspRS, crystal structures are available from several organisms, encompassing the three "kingdoms" of life and the whole reaction pathway: apoenzyme, complexes with Asp alone, ATP alone, aspartyl-adenylate alone (AspAMP) (15Poterszmann A. Delarue M. Thierry J.C. Moras D. J. Mol. Biol. 1994; 244: 158-167Crossref PubMed Scopus (66) Google Scholar, 16Delarue M. Poterszman A. Nikonov S. Garber M. Moras D. Thierry J.C. EMBO J. 1994; 13: 3219-3229Crossref PubMed Scopus (91) Google Scholar, 17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubMed Scopus (125) Google Scholar, 18Eiler S. Dock-Bregeon A.C. Moulinier L. Thierry J.C. Moras D. EMBO J. 1999; 18: 6532-6541Crossref PubMed Scopus (156) Google Scholar, 19Sauter C. Lorber B. Cavarelli J. Moras D. Giege R. J. Mol. Biol. 2000; 299: 1313-1324Crossref PubMed Scopus (64) Google Scholar, 20Rees B. Webster G. Delarue M. Boeglin M. Moras D. J. Mol. Biol. 2000; 299: 1157-1164Crossref PubMed Scopus (35) Google Scholar), and complexes with tRNAAsp present (21Ruff M. Krishnaswamy S. Boeglin M. Poterszman A. Mitschler A. Podjarny A. Rees B. Thierry J.C. Moras D. Science. 1991; 252: 1682-1689Crossref PubMed Scopus (607) Google Scholar, 22Briand C. Poterszman A. Eiler S. Webster G. Thierry J.C. Moras D. J. Mol. Biol. 2000; 299: 1051-1060Crossref PubMed Scopus (44) Google Scholar, 23Moulinier L. Eiler S. Eriani G. Gangloff J. Thierry J.C. Gabriel K. McClain W.H. Moras D. EMBO J. 2001; 20: 5290-5301Crossref PubMed Scopus (88) Google Scholar). Each substrate is recognized by side chains conserved throughout AspRSs (Asp sidechain recognition) or throughout all or most of class II (ATP, Asp backbone recognition). The active site is highly preorganized to receive the Asp and ATP substrates, with the apoenzyme largely superimposable on the various complex structures (22Briand C. Poterszman A. Eiler S. Webster G. Thierry J.C. Moras D. J. Mol. Biol. 2000; 299: 1051-1060Crossref PubMed Scopus (44) Google Scholar). Each substrate, taken separately, is almost exactly superimposable on the corresponding moiety in the known AspAMP or tRNA·Asp complexes. In all of class II, ATP binds in a very unusual, completely bent conformation, with three associated Mg2+ cations (or two in a few cases; see below). The principal cation coordinates both the reactive α-phosphate and the β-phosphate and is positioned by conserved side chains (24Ador L. Jaeger S. Geslain R. Martin F. Cavarelli J. Eriani G. Biochemistry. 2004; 43: 7028-7037Crossref PubMed Scopus (12) Google Scholar). The other two cations coordinate the β- and γ-phosphates on either side of the ATP. In AspRS, the adenylation reaction can occur in the absence of tRNA. Once Asp and ATP are in place, the Asp backbone reacts with the α-phosphate through an in-line mechanism and a pentacoordinate transition state. Inversion of the α-phosphate is clearly seen when crystals of AspRS·ATP and AspRS·AspAMP are compared (19Sauter C. Lorber B. Cavarelli J. Moras D. Giege R. J. Mol. Biol. 2000; 299: 1313-1324Crossref PubMed Scopus (64) Google Scholar). As with most enzyme reactions, the exact role of each surrounding group is hard to establish. It is presumed that the principal Mg2+ cation helps activate the α-phosphate by withdrawing electrons and pulling its oxygens into a pentacoordinate geometry, helping to stabilize the transition state through electrostatic interactions. The other two cations neutralize the leaving pyrophosphate product and may also contribute to transition state stabilization. We have focused here on the role of Mg2+ in Asp/Asn discrimination by AspRS from E. coli. AspRS specificity is a complex problem. Although the enzyme is preorganized, Asp binding does induce structural reorganization in two important regions: (i) a so-called histidine loop (residues 436-449 in E. coli) shifts and becomes more ordered, with His-448 making a hydrogen bond to Asp; (ii) a flexible "flipping loop" (residues 167-173 in E. coli) closes over the aa binding site, bringing the negative Glu-171 close to the Asp ligand. The flipping loop is conserved in eukaryal, eubacterial, and archaebacterial AspRS (17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubMed Scopus (125) Google Scholar, 18Eiler S. Dock-Bregeon A.C. Moulinier L. Thierry J.C. Moras D. EMBO J. 1999; 18: 6532-6541Crossref PubMed Scopus (156) Google Scholar, 23Moulinier L. Eiler S. Eriani G. Gangloff J. Thierry J.C. Gabriel K. McClain W.H. Moras D. EMBO J. 2001; 20: 5290-5301Crossref PubMed Scopus (88) Google Scholar), and a corresponding mobile loop is found in AsnRS (27Berthet-Colominas C. Seignovert L. Hartlein M. Grotli M. Cusack S. Leberman R. EMBO J. 1998; 17: 2947-2960Crossref PubMed Scopus (88) Google Scholar) and LysRS (28Desogus D. Tadone F. Brick P. Onesti S. Biochemistry. 2000; 39: 8418-8425Crossref PubMed Scopus (76) Google Scholar). The exact populations of the open and closed loop states, with and without bound Asp, are unknown. Another difficulty is that the substrates Asp and ATP are both charged. Therefore, both short and long range electrostatic interactions are expected to play a role in both binding and specificity. Amino acid binding might couple to proton binding or release by His-448 or His-449 and to Mg2+ binding or release by ATP. AspRS specificity has been analyzed with a powerful combination of crystallography, site-directed mutagenesis, kinetic and thermodynamic experiments, and phylogenetic analyses. These methods have limitations, however. Conserved residues may contribute to binding, binding specificity, catalysis, or all three. Experimental assays based on catalytic activity (29Fersht A.R. Structure and Mechanism in Protein Science. W.H. Freeman & Company, New York1999Google Scholar, 30Blanquet S. Fayat G. Waller J.P. Eur. J. Biochem. 1974; 44: 343-351Crossref PubMed Scopus (71) Google Scholar) usually become infeasible once a single essential residue is mutated. The strength of electrostatic interactions is very difficult to infer from crystal structures, because the complex dielectric environment within a solvated protein causes large deviations from a simple Coulomb's law (31Simonson T. Rep. Prog. Phys. 2003; 66: 737-787Crossref Scopus (199) Google Scholar). Crystallography does not reveal the ionization states of acidic and basic residues, and pKa measurements are difficult for AspRS, which is a homodimer of 1180 residues. Most importantly, weakly populated states are difficult to study experimentally. One extensive mutation study explored changes in the apparent Asp and ATP dissociation constants spanning only 2 orders of magnitude, corresponding to a free energy span of less than 3 kcal/mol (26Cavarelli J. Eriani B. Rees B. Ruff M. Boeglin M. Mitschler A. Martin F. Gangloff J. Thierry J.C. Moras D. EMBO J. 1994; 13: 327-337Crossref PubMed Scopus (220) Google Scholar). Non-cognate complexes like AspRS·Asn could not be analyzed. Weakly populated states are invisible in a crystal structure. Thus, from the AspRS·ATP crystal structures, ATP binds preferentially with three associated Mg2+ cations (17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubMed Scopus (125) Google Scholar). However, the crystallographic data do not reveal the exact occupancies of the three magnesium sites. A complex with ATP and only two cations, present in the crystal with a population of 10 or 20%, for example, would not have an appreciable effect on the electron density maps and would be impossible to infer from the crystallographic data. Theoretical methods represent a valuable complementary tool that can resolve these difficulties (32Fothergill M. Goodman M.F. Petruska J. Warshel A. J. Am. Chem. Soc. 1995; 117: 11619-11627Crossref Scopus (93) Google Scholar, 33Archontis G. Simonson T. Moras D. Karplus M. J. Mol. Biol. 1998; 275: 823-846Crossref PubMed Scopus (76) Google Scholar, 34Archontis G. Simonson T. J. Am. Chem. Soc. 2001; 123: 11047-11056Crossref PubMed Scopus (51) Google Scholar, 35Archontis G. Simonson T. Karplus M. J. Mol. Biol. 2001; 306: 307-327Crossref PubMed Scopus (99) Google Scholar, 36Thompson D. Plateau P. Simonson T. ChemBioChem. 2006; 7: 337-344Crossref PubMed Scopus (34) Google Scholar, 37Simonson T. J. Phys. Chem. B. 2000; 104: 6509-6513Crossref Scopus (38) Google Scholar). The specificity of Asp binding to AspRS is governed by the binding free energy difference between the cognate Asp and competitor ligands. This difference can be obtained from molecular dynamics free energy simulations (MDFE), which have matured enormously in recent years and have been used to study several aaRSs. Extensive studies (33Archontis G. Simonson T. Moras D. Karplus M. J. Mol. Biol. 1998; 275: 823-846Crossref PubMed Scopus (76) Google Scholar, 34Archontis G. Simonson T. J. Am. Chem. Soc. 2001; 123: 11047-11056Crossref PubMed Scopus (51) Google Scholar, 35Archontis G. Simonson T. Karplus M. J. Mol. Biol. 2001; 306: 307-327Crossref PubMed Scopus (99) Google Scholar) show that when the AspRS binding pocket is in the "open" state (open flipping loop), there is an enormous preference for Asp over Asn, thanks to a network of electrostatic interactions in the active site. A thermodynamic cycle (see below) was used to obtain binding free energy differences, and a group decomposition of the free energy was used to identity the residues determining amino acid binding specificity. When the flipping loop closes (17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubMed Scopus (125) Google Scholar, 23Moulinier L. Eiler S. Eriani G. Gangloff J. Thierry J.C. Gabriel K. McClain W.H. Moras D. EMBO J. 2001; 20: 5290-5301Crossref PubMed Scopus (88) Google Scholar), the negative Glu-171 is brought close to the Asp ligand site. We recently predicted computationally (36Thompson D. Plateau P. Simonson T. ChemBioChem. 2006; 7: 337-344Crossref PubMed Scopus (34) Google Scholar) that this conformational change induces proton binding by the nearby His-448. The His-448 positive charge then accounts for most of the large, computed Asp/Asn discrimination. In another long range electrostatic effect, a substrate-assisted specificity was observed: co-binding of ATP increases the Asp/Asn discrimination further. In eukaryotic AspRSs, His-448 is absent, being replaced by an Arg that is more distant from the ligand site. In these organisms, the role of ATP as a mobile discriminator is therefore important, protecting against Asn binding. In the same study, the computational model was tested and validated by experimental measurements of Asp-stimulated pyrophosphate exchange and its inhibition by Asn (36Thompson D. Plateau P. Simonson T. ChemBioChem. 2006; 7: 337-344Crossref PubMed Scopus (34) Google Scholar). The present article focuses on the precise stability of the ATP-associated Mg2+ cations in the AspRS structure and their role in the thermodynamics of Asp and Asn binding. We consider AspRS from both E. coli and the archaebacterium Pyrococcus kodakaraensis. We report free energy simulations that compare Asp and Asn binding to AspRS in the presence of either bound ATP·Mg22+ or bound ATP·Mg32+. The level of Asp/Asn discrimination in each case was computed using two distinct, largely independent methods for the free energy changes. The first method, MDFE, alchemically transforms Asp into Asn during a series of molecular simulations with an explicit solvent representation (33Archontis G. Simonson T. Moras D. Karplus M. J. Mol. Biol. 1998; 275: 823-846Crossref PubMed Scopus (76) Google Scholar). The second, Poisson-Boltzmann free energies (PBFE), models the ligand binding reactions using a continuum dielectric model of both protein and solvent (35Archontis G. Simonson T. Karplus M. J. Mol. Biol. 2001; 306: 307-327Crossref PubMed Scopus (99) Google Scholar). MDFE was then used to compute the relative affinities of AspRS for ATP·Mg22+ and ATP·Mg32+ and to demonstrate that the ATP·Mg22+ complex has a negligible occupancy and does not play any role in the specificity. In the closely homologous E. coli LysRS, the ATP 3-cation complex is again strongly favored. Binding of the positively charged Lys substrate, however, is not affected by cation binding. We also considered the post-adenylation, AspRS·tRNA·AspAMP complex. Our simulations predict a strongly bound Mg2+ cation that aids AspAMP recognition. We show that specificity is maintained in the post-adenylation state (11Hopfield J.J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4135-4139Crossref PubMed Scopus (1103) Google Scholar), with AspAMP binding more strongly than AsnAMP in the presence of one Mg2+ cation and tRNAAsp. The Mg2+ cation boosts AspAMP binding specificity, in a long range electrostatic effect similar to that of ATP·Mg32+ in the preadenylation complex. Thus, the introduction of negative charge into AspRS, by conformational change (36Thompson D. Plateau P. Simonson T. ChemBioChem. 2006; 7: 337-344Crossref PubMed Scopus (34) Google Scholar) or tRNA binding, is compensated by histidine protonation (36Thompson D. Plateau P. Simonson T. ChemBioChem. 2006; 7: 337-344Crossref PubMed Scopus (34) Google Scholar) and/or cation binding to preserve Asp recognition. Finally, in the supplemental material we have reported a survey of 238 x-ray structures of protein complexes with ATP or GTP taken from the Protein Data Bank, which sheds additional light on the role of the Mg2+ cations and supports the predicted 3-cation AspRS state. Indeed, we find that ATP binding in a completely bent conformation with three associated Mg2+ cations is a characteristic property of class II aaRSs. Starting structures for AspRS with bound Asp and ATP were generated from a 2.6-Å resolution crystal structure of E. coli AspRS with bound aspartyl-adenylate, AspAMP (Protein Data Bank entry 1IL2) (23Moulinier L. Eiler S. Eriani G. Gangloff J. Thierry J.C. Gabriel K. McClain W.H. Moras D. EMBO J. 2001; 20: 5290-5301Crossref PubMed Scopus (88) Google Scholar). The two tRNA ligands were removed. We considered protein residues within a 24-Å sphere centered on the γ-carbon of the adenylate ligand of one monomer of the 1IL2 dimer. We overlaid either Asp or Asn on the adenylate molecule and deleted the original ligand. ATP and its associated cations were positioned by taking ATP·Mg32+ from the 1.9-Å resolution P. kodakaraensis AspRS·ATP complex (Protein Data Bank entry 1B8A) (17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubMed Scopus (125) Google Scholar) and building it into the 1IL2 structure so as to overlap with the AMP moiety of the original AspAMP ligand. Hydrogens were constructed with ideal stereochemistry. Protonation states of histidines were assigned by visual inspection, except for His-448 and His-449 in the active site, which were assigned earlier through extensive simulations (36Thompson D. Plateau P. Simonson T. ChemBioChem. 2006; 7: 337-344Crossref PubMed Scopus (34) Google Scholar). Orientations of His, Asn, and Gln side chains in the active site were taken from the crystal structure and verified by inspection (33Archontis G. Simonson T. Moras D. Karplus M. J. Mol. Biol. 1998; 275: 823-846Crossref PubMed Scopus (76) Google Scholar). In addition to crystal waters, a 73-Å cubic box of water was overlaid, and waters overlapping the protein were removed. The final model contained the amino acid and ATP ligands, 357 protein residues, and around 10,000 waters, including 113 crystal waters. Periodic boundary conditions were assumed; i.e. the entire 73-Å box was replicated periodically in all directions. We also generated AspRS·Asp·ATP complexes from the P. kodakaraensis AspRS·ATP complex (17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubMed Scopus (125) Google Scholar). We built in Asp and Asn ligands by alignment with the E. coli structure (Protein Data Bank entry 1IL2; see above) and generated solvated 24-Å spheres centered on the ligand. We then used pKa calculations (see below) to determine the protonation state of His-223, a histidine residue oriented into the ATP binding pocket. LysRS structures were generated from a 2.1-Å resolution E. coli LysRS·Lys·ATP·Mg32+ crystal structure (28Desogus D. Tadone F. Brick P. Onesti S. Biochemistry. 2000; 39: 8418-8425Crossref PubMed Scopus (76) Google Scholar). We took a 24-Å sphere centered on the γ-carbon of the Lys ligand, replaced the Mn2+ cations with Mg2+, and then solvated the system. Histidine protonation states were assigned by visual inspection except for His-270, which points into the LysRS ATP-binding pocket. In both AspRS and LysRS, the protonation state of the histidine oriented into the ATP pocket is coupled to cation binding. Structures for AspRS with bound AspAMP and tRNA were generated from the 2.4-Å resolution E. coli crystal structure 1C0A (18Eiler S. Dock-Bregeon A.C. Moulinier L. Thierry J.C. Moras D. EMBO J. 1999; 18: 6532-6541Crossref PubMed Scopus (156) Google Scholar). Again, we considered a 24-Å sphere centered on the ligand γ-carbon and solvated the system. pKa calculations were used to determine the predominant state of His-448, a histidine residue close to AspAMP. The cation associated with the AspAMP ligand phosphate group (15Poterszmann A. Delarue M. Thierry J.C. Moras D. J. Mol. Biol. 1994; 244: 158-167Crossref PubMed Scopus (66) Google Scholar) was placed by structural alignment with the principal cation in the Asp·ATP·Mg32+ state (17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubMed Scopus (125) Google Scholar). The cation occupies the space assigned to water molecule 1073 in the x-ray structure (18Eiler S. Dock-Bregeon A.C. Moulinier L. Thierry J.C. Moras D. EMBO J. 1999; 18: 6532-6541Crossref PubMed Scopus (156) Google Scholar) and remains in the same octahedral binding mode throughout the simulations, coordinating the Glu-482 and Asp-475 carboxylates and two or three water molecules and remaining 4-5 Å away from the AspAMP phosphate group. For both AspRS and LysRS, if the entire aaRS protein were included in the model, rather than a spherical subset, a far greater number of solvating waters would be needed. To reduce artifacts due to the protein truncation, protein groups between 20 and 24 Å from the center were harmonically restrained to their positions in the crystal structure. In this way, protein regions beyond 24 Å are accounted for structurally. They will also be accounted for thermodynamically, in a separate step (see below), where the free energy to reintroduce the missing protein groups is computed from a continuum electrostatic model. This hybrid, atomic/continuum approach is accurate, because earlier work on this system showed that both structures and free energies obtained with spherical subsets of 20-, 24-, or 28-Å radii were all similar (37Simonson T. J. Phys. Chem. B. 2000; 104: 6509-6513Crossref Scopus (38) Google Scholar). All long range electrostatic interactions were computed efficiently by the particle mesh Ewald (PME) method. Four sodium counterions were included to reduce the formal charge of the system. One nanosecond of unrestrained molecular dynamics was performed (for each complex) at constant room temperature and pressure with a Nosé-Hoover algorithm following 200 ps of thermalization. The complexes modeled are E. coli (23Moulinier L. Eiler S. Eriani G. Gangloff J. Thierry J.C. Gabriel K. McClain W.H. Moras D. EMBO J. 2001; 20: 5290-5301Crossref PubMed Scopus (88) Google Scholar) AspRS·Asp·ATP·Mg32+, AspRS·Asp·ATP·Mg22+, AspRS·Asn·ATP·Mg32+, and AspRS·AsnATP·Mg22+. 500-ps trajectories were also produced for each amino acid ligand in solution, Asp or Asn, solvated at the center of a box of water molecules, with the ligand γ-carbon weakly restrained at the origin throughout the dynamics. Additional simulations were performed with a less expensive, spherical, continuum reaction field (CRF) (37Simonson T. J. Phys. Chem. B. 2000; 104: 6509-6513Crossref Scopus (38) Google Scholar, 38Beglov D. Roux B. J. Chem. Phys. 1994; 100: 9050-9063Crossref Scopus (813) Google Scholar) model. It included the same protein residues as mentioned above, along with the water molecules inside the 24-Å sphere (about 560 waters). Water and protein outside the 24-Å sphere were treated as a single, homogeneous, dielectric medium with a dielectric constant of 80 (38Beglov D. Roux B. J. Chem. Phys. 1994; 100: 9050-9063Crossref Scopus (813) Google Scholar). Electrostatic interactions between atoms within the sphere were computed without any cutoff, using an efficient multipole approximation for distant groups (39Stote R.H. States D.J. Karplus M. J. Chim. Phys. 1991; 88: 2419-2433Crossref Google Scholar). A multipolar expansion with 20 terms was used to approximate the reaction field due to the surrounding continuum (37Simonson T. J. Phys. Chem. B. 2000; 104: 6509-6513Crossref Scopus (38) Google Scholar, 38Beglov D. Roux B. J. Chem. Phys. 1994; 100: 9050-9063Crossref Scopus (813) Google Scholar). Newtonian dynamics were used for the inner 20 Å of the sphere and Langevin dynamics for the outer region (20-24 Å), with a bath temperature of 293 K. The same four complexes as above were simulated for 3 ns each, as well as the following 10 complexes (3 ns each): AspRS·Asp (or Asn)·ATP·Mg32+ (or Mg22+) complexes from P. kodakaraensis (17Schmitt E. Moulinier L. Fujiwara S. Imanaka T. Thierry J.C. Moras D. EMBO J. 1998; 17: 5227-5237Crossref PubM
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