PROTEINS: Structure, Function, and Genetics 32:362–380 (1998)

Engineering an Mg2ϩ Site to Replace a Structurally Conserved Arginine in the Catalytic Center of Histidyl-tRNA Synthetase by Computer Experiments

John G. Arnez, Karen Flanagan, Dino Moras, and Thomas Simonson* Laboratoire de Biologie Structurale, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (Centre National de la Recherche Scientifique/Institut National de la Sante´ et de la Recherche Me´dicale/Universite´ Louis Pasteur), Strasbourg-Illkirch, France

ABSTRACT Histidyl-tRNA synthetase INTRODUCTION (HisRS) differs from other class II aminoacyl- Aminoacylation of a tRNA by its cognate amino tRNA synthetases (aaRS) in that it harbors an acid is a two-step reaction. The first step is the for- arginine at a position where the others bind a mation of a mixed anhydride, the aminoacyl-adenyl- 2ϩ catalytic Mg ion. In computer experiments, ate, from adenosine triphosphate (ATP) and amino four mutants of HisRS from Escherichia coli acid, with release of pyrophosphate. The second step were engineered by removing the arginine and is the transfer of the aminoacyl moiety to the 2’ or 3’ introducing a Mg2ϩ ion and residues from seryl- OH of the terminal ribose of the tRNA, with release tRNA synthetase (SerRS) that are involved in of adenosine monophosphate (AMP). The entire reac- Mg2ϩ binding. The mutants recreate an active tion is catalyzed by a family of enzymes, the amino- site carboxylate pair conserved in other class acyl-tRNA synthetases (aaRS). Each aaRS is specific II aaRSs, in two possible orders: Glu-Asp or for one amino acid and a set of cognate tRNAs, Asp-Glu, replacing Glu-Thr in native HisRS. known as isoacceptors. The aaRS family can be The mutants were simulated by molecular divided into two classes based on homologous se- dynamics in complex with histidyl-adenylate. quence and structural motifs,1 which lead to two As controls, the native HisRS was simulated different architectures of the active site.2,3 Here, we in complexes with histidine, histidyl-adenyl- focus on interactions that occur during the first step ate, and histidinol. The native structures of the overall reaction in histidyl-tRNA synthetase, sampled were in good agreement with ex- i.e.,, the formation of histidyl-adenylate. perimental structures and biochemical data. Histidyl-tRNA synthetase (HisRS) is a homodi- The two mutants with the Glu-Asp sequence meric class II aaRS. The monomer consists of two showed significant differences in active site structural domains.4 The C-terminal domain is an structure and Mg2ϩ coordination from SerRS. ␣-␤ structure implicated in binding the anticodon of The others were more similar to SerRS, and tRNAHis. The N-terminal domain contains the active one of them was analyzed further through site, formed of a seven-stranded antiparallel ␤-sheet simulations in complex with histidine, and surrounded by ␣-helices and containing the three HisϩATP. The latter complex sampled two Mg2ϩ class II signature motifs. The active site is a platform positions, depending on the conformation of a that binds histidine and ATP and fixes them in the loop anchoring the second carboxylate. The correct mutual orientation4,5 (Fig. 1a). The floor of lowest energy conformation led to an active the histidine-binding pocket is formed by a glycine- site geometry very similar to SerRS, with the rich portion of the ␤-sheet, and the sides are formed principal Mg2ϩ bridging the ␣- and ␤-phos- by the HisRS-conserved motifs HisA (Escherichia phates, the first carboxylate (Asp) coordinat- coli residues 257–264) and HisB (residues 284–292) ing the ion through a water molecule, and the and the conserved Glu131, which is at the carboxy second (Glu) coordinating it directly. This end of motif 2. The ATP moiety is held in place by mutant is expected to be catalytically active class II conserved residues, most notable of which and suggests a basis for the previously un- are the motif 2 arginine 113, the motif 2 loop explained conservation of the active site arginine 121, and the motif 3 arginine 311. The last Asp-Glu pair in class II aaRSs other than two interact with the ␥-phosphate of the ATP,whereas HisRS. Proteins 32:362–380, 1998. ௠ 1998 Wiley-Liss, Inc. *Correspondence to: Thomas Simonson, Institut de Ge´ne´t- Key words: molecular dynamics simulations; ique et de Biologie Mole´culaire et Cellulaire, BP 163, 67404 Strasbourg-Illkirch, France. E-mail: simonson@igbmc. mutagenesis; aminoacyl-tRNA syn- ustrasbg.fr thetase; ATP Received 27 January 1998; Accepted 26 March 1998

௠ 1998 WILEY-LISS, INC. ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 363

pancy, which interacts with the ␣-phosphate. The other two bridge the ␤- and ␥-phosphates (Fig. 1b) and were recently observed in HisRS as well.5 In addition to their role in binding ATP, these ions, particularly the principal ion when present, are presumed to play a role in aaRS catalysis by stabiliz- ing the pentavalent and highly charged ␣-phosphate in the transition state. A catalytic role for Mg2ϩ ions in the exonuclease mechanism of DNA polymerase I has been suggested and supported by mutagenesis experiments as well as detailed simulations.7,8 In HisRS, the Mg2ϩ proxy Arg259 was shown to contrib- ute to catalysis by mutation experiments that re- moved this side chain. For example, the R259H mutation slows the adenylation reaction by three orders of magnitude.5 The HisRS active site is unusual in yet another aspect. Most class II aaRSs, such as AspRS9,10 and SerRS,6 have two conserved carboxylates adjacent to the ␤-strand of motif 3. The first carboxylate, an Asp, extends from the ␤-strand distal to the motif 3 strand. The second carboxylate, a Glu, is on a loop preceding the ␤-strand proximal to the motif 3 strand (Fig. 1b). This Glu coordinates the principal metal ion directly, whereas the Asp interacts with the ion via a water molecule. In HisRS, the first carboxylate is Glu270, but the second carboxylate is replaced by a threonine, Thr281 (Fig. 1a). The most obvious role of Glu270 in HisRS appears to be positioning Arg259.5 The present study suggests that the identity of the more common Asp-Glu pair is important for correct positioning of the principal metal ion. Another feature of the HisRS active site that is shared by all class II aaRSs, but is unique with respect to all other ATP- or GTP-binding proteins, is the completely bent geometry of the bound ATP. A Fig. 1. Ribbon diagram of the active site of (a) HisRS5 and (b) highly bent geometry is also observed in three ATP SerRS6, complexed with ATP. The HisRS site also contains an structures in the Cambridge Structural Database in analog of histidine, histidinol. Shown are the two class II con- 11 served arginines (a, Arg 113 and Arg 311; b, Arg 256 and Arg 386) the presence of sodium counterions (entries adentp and the nearly invariant arginine (a, Arg 121; b, Arg 271). The and citwux1012). Two ATP structures in the presence metal ions are shown in light blue; there are two in HisRS (a) and of divalent Mg2ϩ or Mn2ϩ ions are found in the data three in SerRS (b). Arg 259 in (a) takes the place of the metal ion base (entries cicrah1013 and decdiy14), in which the bridging the ␣- and ␤-phosphates in (b); it is replaced by Gly in the mutants studied here. Also shown are the residues in HisRS (a) ATP is less bent and coordinates the ion with all that are replaced by similarly positioned residues in SerRS (b). three phosphates. The ATP environment in the pro- This and the other structure figures were generated with the tein is very different. The analysis of a mutant programs MOLSCRIPT43 and RASTER3D44,45. HisRS:His:ATP complex below is expected to provide insights into this protein-ATP interaction mode. Arg113 interacts with the ␣-phosphate. In addition To understand these unique features of HisRS, we to these arginines, HisRS is unique among class II have examined through computer simulations the aaRSs in that it possesses a fourth arginine, Arg259, possibility of engineering an Mg2ϩ site into HisRS which is part of the conserved HisA motif (Fig. 1a). that would functionally replace Arg259. This exer- This arginine interacts with the ␣-phosphate; its cise is of general interest, since the engineering of guanidino group replaces a metal ion found in SerRS5 metal-binding sites in enzymes makes it possible to and thought to exist in all other class II aaRSs. fine-tune catalysis and has potential applications in Three metal sites were observed in SerRS by anoma- industry and medicine. Ab initio engineering of lous dispersion of Mn2ϩ ions and are presumed to metal-binding sites is a difficult task, for it requires correspond to actual Mg2ϩ sites.6 Arg259 replaces the concerted changes not only at the site of metal principal site, i.e., the one with the highest occu- binding but also in other parts of the target mol- 364 J.G. ARNEZ ET AL. ecule.15–18In this study, we are not designing a site ab 1kmn). The structures of the mutant complexes were initio, but transferring a site from a similar frame- built as described below. work, already optimized through evolution. Related Atomic charges, van der Waals, and stereochemi- engineering exercises have been carried out experi- cal force field parameters for protein and ligand, mentally: a manganese-binding site was trans- with the exception of a portion of the adenylate, were planted from manganese peroxidase into cytochrome taken from the CHARMM22 all-atom force field.22 c oxidase by mutagenesis,19 and a magnesium- Parameters for the central part of the adenylate binding site was transplanted from oncomodulin were derived from quantum chemical calculations, into parvalbumin.20 Comparing the E. coli HisRS as described in the Appendix. Water interactions active site with that of SerRS, we construct a series were described by a modified TIP3P model,23,24 which of targeted mutations of some critical residues in has a dielectric constant of 82.25 Water molecules HisRS. The simulations then address the question of were confined within the 18 A˚ sphere by the stochas- whether the modified HisRS has an active site that is tic boundary method.26 Protein atoms within the stable and likely to be catalytically active. We first outer 3 A˚ buffer shell, i.e., more than 15 A˚ from the remove the side chain of Arg259 by mutating it to a center of the simulation sphere, were harmonically Gly. Arg259 is in a loop that is shorter in SerRS, but restrained to their initial positions. Unpaired charged we retain the length of the HisRS loop so that the side chains (i.e., those not involved in salt bridges) in remaining residues of the conserved HisA motif will the buffer region had their charges reduced by a not be perturbed. Next, we replace the guanidino factor of 40, to account for the absence of bulk group of the arginine with an Mg2ϩ site, by trans- solvent.27 Protein and water heavy atoms located planting four combinations of some key residues more than 15 A˚ from the center of the sphere were from SerRS into HisRS. The resulting mutated pro- subjected to Langevin dynamics with frictional and teins, complexed with histidyl-adenylate (HisAMP), random forces that mimic a thermal bath at a chosen are subjected to molecular dynamics simulation. The temperature.26 The internal geometry of the water metal sites thus constituted are compared with the molecules and bond lengths to protein hydrogen strongest Mn2ϩ site in the SerRS active site. The atoms were constrained with the SHAKE algo- most promising mutant, i.e., the one that most rithm.28 Initial water positions were obtained by closely recapitulates the strongest site in SerRS, overlaying a large box of bulk water on the protein undergoes further molecular dynamics simulations, structure and deleting waters that overlap with in complex with histidine, and with histidine and protein. Each structure was then optimized through ATP. The resulting structures present a metal coordi- Powell minimization,29 gradually heated to room nation similar to SerRS and are expected to be temperature by molecular dynamics over a period of catalytically active. 30 ps, and then subjected to several hundred picosec- Simulations of the native HisRS were carried out onds of molecular dynamics at 293 K. An additional as controls, in complexes with His, HisAMP, and the water overlay was done after the first few picosec- inhibitor histidinol (HisOH). Experimental struc- onds of equilibration, to account for electrostriction tures are known for native E. coli HisRS complexed of the water around charged protein groups. The with HisOH and ATP,and with HisAMP.The HisAMP coordinates were stored every 20 or 40 fs for further simulations can be compared directly with the latter analysis. Energy minimization and molecular dynam- structure. In addition, the structure of HisRS from ics calculations were done with the programs Thermus thermophilus complexed with His21 became X-PLOR30 and CHARMM.31 available in the final stage of this work. The simula- All simulations but one were done with a 16 A˚ tions with His and HisOH can be compared with cutoff for electrostatic interactions (using a shifting both the latter experimental structure and the function31). The use of even a relatively large cutoff, HisOHϩATP complex, providing insights into the as here, is a significant approximation. To test its ordering of the active site prior to ATP binding. effects, one simulation of the native HisAMP com- plex was done without any cutoff, i.e., including all MATERIALS AND METHODS electrostatic interactions. This is done efficiently in The simulation model consisted of a spherical the CHARMM program by approximating small region of 18 A˚ radius, centered on the position of the groups of atoms by sets of electric multipoles when adenylate phosphorus, which includes the active site computing interactions beyond 13 A˚ .31,32 Because of region of HisRS, one ligand molecule (HisAMP, His, the good agreement between the HisAMP simula- HisOH, or HisϩATP), and about 325 explicit water tions with and without cutoff (see below), all the molecules solvating the active site. The histidine other simulations were done with a cutoff. moiety is neutral, with the N⑀, which interacts with The crystal structure of HisRS from T. thermophi- Glu131, protonated and the N␦, which interacts with lus (HisRStt) is more complete than that from E. coli, Tyr264, deprotonated. The initial protein and ligand since it includes the insertion domain,21 which is coordinates for the native complexes were taken disordered in the structure of the enzyme from from the crystal structure of the E. coli enzyme: E. coli (residues 188–223). However, its coordinates HisAMP complex4,5 (PDB accession codes 1kmm and (released Aug. 20, 1997) only became available after ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 365

TABLE I. Residues Mutated in HisRS conformation, residue 281 is closer to the principal Mg2ϩ ion, and in SerRS, the Glu at this position Mutant coordinates both the principal and the second Mg2ϩ Wild type 1234SerRS ions. Exchange between the two conformations is Ile168 — Lys — Lys Lys327 slow on the time scale of the present simulations Arg259 Gly Gly Gly Gly — (200–600 ps). Therefore, both conformations were Val268 Gln Gln Gln Gln Gln330 simulated individually. In the structure of HisRS Glu270 — — Asp Asp Asp332 from T. thermophilus,21 which became available af- Thr281 Asp Asp Glu Glu Glu345 Ala284 Ser Ser Ser Ser Ser348 ter the simulations were finished, the ‘‘minor’’ confor- mation of the loop predominates. Mutants 1–4 were simulated complexed with HisAMP,with the 273–281 loop in its major conforma- the present simulations had been performed. An a tion. Mutants 1 and 3 were also simulated with the posteriori examination of the HisRStt structure 273–281 loop in its minor conformation. Mutant 3, showed that the ␣-helix containing residues 200–210 which gave the best results in terms of metal coordi- is within our 18 A˚ sphere, occupying space that is nation, was examined through three additional simu- filled with solvent in our simulations. The side chain lations. The adenylate was replaced with His alone extremities of four residues of the helix extend into in one simulation and with HisϩATP in two. Addi- the inner 15 A˚ sphere: the guanidino group of Arg204 tional space made available to solvent in the His (10 A˚ from P␣), the carboxylate of Asp207 (9.8 A˚ from complex was filled by overlaying a sphere of water P␣), the ammonium of Lys209 (11.9 A˚ from P␣), and molecules on the system and retaining 12 waters the guanidino of Arg197 (14.7 A˚ from P␣). The first that fit into the available space. In the HisϩATP three are fully solvated, so that their charges are cases, six waters were deleted to avoid overlap with largely shielded from groups in the active site. the ATP. The two HisϩATP simulations were done Arg197 forms a salt bridge to Asp59 (in HisRStt, with the 273–281 loop in its major and minor confor- equivalent to Asp61 in HisRS from E. coli) and is mations, respectively. Each complex was then mini- sufficiently far away from the phosphate and magne- mized and underwent 200–300 ps of Langevin dy- sium to be neglected. Furthermore, all these resi- namics in most cases, or 600 ps in the HisϩATP dues are expected to be relatively disordered based cases. on the low electron density observed experimentally. Hence, including these groups would not be expected RESULTS to affect the behavior of the active site significantly Native Simulations during the molecular dynamics simulations. Four sets of mutations were introduced into the The native HisAMP complex was simulated with wild-type E. coli HisRS structure. They are summa- and without an electrostatic cutoff (see Materials rized in Table I. In all four cases, Arg259 was and Methods). The structures sampled in the two changed to Gly, Val268 to Gln, and Ala284 to Ser. simulations were in good agreement with each other Thr281 was changed to a carboxylate, i.e., to Asp in and with the crystal structure (Table II). The three the first two constructs and to Glu in the other two. structures are compared in Figure 2. Glu270 was changed to Asp in the latter two in- The root-mean-square (rms) deviation between stances. Ile168 was changed to Lys once for each the two simulation structures (averaged over the carboxylate combination. A Lys is present at the last 5 ps of simulation) was only 0.4 A˚ for backbone, corresponding position in SerRS (Fig. 1). The coordi- 0.7 A˚ for side chains, and 0.5 A˚ for the adenylate. The nates of the mutated residues were built into the rms deviations between the simulation and experi- native E. coli HisRS:HisAMP complex by superimpos- mental structures were 0.7 A˚ and 0.5 A˚ with and ing atoms shared with the native residues and without cutoff (backbone atoms), or 1.2 A˚ (side chain assuming an ideal stereochemistry for the others. atoms, with or without cutoff). These are roughly The native complex was taken from the native comparable to the rms deviations between mono- simulations described above. Only minimal energy mers in the crystal structure: 0.2 and 1.3 A˚ for optimization was necessary after introduction of the backbone and side chains, respectively. The imidaz- mutated residues, since the model was already pre- ole ring is slightly tilted around the ␹1 dihedral in equilibrated by the earlier simulations. the simulations. In the cutoff simulation, a localized The loop 273–281 that anchors the putative Mg2ϩ distortion occurred due to a 180 degree rotation of ligand Asp/Glu281 has two possible conformations in the C5’ around the ␣ torsion angle of the phosphate the E. coli HisRS:HisAMP crystal structure (Arnez, group, which displaces the C5’ by 1.5 A˚ and twists Francklyn and Moras, unpublished data). The major the ribose downwards somewhat. This is compen- conformation differs from that of the equivalent loop sated by rotation around the ␹ glycosyl torsion angle, in SerRS, while the less-populated one is similar to so that the distortion is limited to a few atoms, and that in SerRS. They will be referred to as the major the most important ligand atoms (e.g., the adenine and minor conformations, respectively. In the minor N9, the P, the carboxyl C, and the imidazole N␦) are 366 J.G. ARNEZ ET AL.

Fig. 2. Stereo views of the active site region of the native (black bonds), the residues mutated in mutants 1–4 further on HisRS:HisAMP complex. a: Crystal structure.5 b: Snapshot after (yellow bonds), the three class II conserved arginines (orange 200 ps of molecular dynamics simulation with no electrostatic bonds), and some other important residues that interact with cutoff. c: Snapshot after a further 200 ps with a 16 A˚ cutoff for histidyl-adenylate (green bonds). d: Superposition of a, red; b, electrostatic interactions. Shown are the ligand, histidyl-adenylate green; and c, blue. ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 367

Fig. 3. Stereo views of the active site region of the native on (yellow bonds), the three class II conserved arginines (orange HisRS:His and HisRS:HisOH complexes. a: Crystal structure of bonds), and some other important residues that interact with the HisRS:HisOH:ATP complex.5 b: Snapshot of molecular dynam- histidine or ATP (green bonds). d: Superposition of a, red; b, ics simulation of HisRS:His. c: Idem, HisRS:HisOH. Shown are the green; and c, blue. ligands (black bonds), the residues mutated in mutants 1–4 further 368 J.G. ARNEZ ET AL.

TABLE II. Magnesium Environment in Different Complexes†

Mutant 3 Protein Native Mutant 1 Mutant 2 HisAMP His/ATP Mutant 4 Ligand(s) HisAMP HisAMP HisAMP His HisAMP Loop conf.a major minor major Major Minor Major Minor major major Ligand rmsdb 0.55 0.46 0.92 0.67 0.49 0.85/1.10 0.81/1.15 0.98 0.70 Ligand rmsfc 0.34 0.35 0.33 0.30 0.33 0.17/0.41 0.21/0.43 0.49 0.30 Mg2ϩ rmsdb 0.52 (R259)d 0.87 1.06 1.53 1.38 1.63 0.18 0.43 1.42 Mg2ϩ rmsfc 0.41 (R259)d 0.28 0.44 0.26 0.38 0.28 0.29 0.33 0.36 Mg2ϩ-S284e naf 2.15 (9) — 2.21 (8) — 2.15 (9) 2.20 (10) 2.10 (7) 2.10 (7) Mg2ϩ-E270 na 1.87 (4) 1.92 (5) — — — — — — Mg2ϩ-E281 na — — — 1.91 (5) — 1.91 (4) — — Mg2ϩ-O1P na — 1.91 (4) 1.88 (3) 1.89 (3) 1.87 (3) 1.89 (4)/1.91 (4)h na 1.87 (3) Mg2ϩ-water na 2.02–2.03 (4)i 2.02–2.04 (4) 2.02–2.03 (4) 2.03–2.04 (4) 2.01–2.03 (4) 2.03 (4) 2.02–2.03 (4) 2.01–2.04 (4) No. of watersg na44444 2 54 R113-OxPj 2.70 (12) 3.17 (71) — 2.73 (11) 2.70 (10) 2.75 (14) — na 3.77 (25) k Y264-N␦ 1.91 (11) 2.02 (20) — 1.88 (11) 2.20 (43) 1.92 (12) 1.90 (11) 1.92 (12) 1.91 (11)

†All distances in Å. aConformation of the 273–281 loop (major or minor; see text). brms deviation at the end of the simulation from starting structure. crms fluctuation around mean structure. dResult for R259 side chain charge. e 2ϩ Distance from Mg to the nearest atom of ligating group (S284-O␥, E270-O⑀, E281-O⑀, O1P, water oxygen). Standard deviation in parentheses expressed in significant digits. When the group is too far to coordinate the ion, ‘‘—’’ is indicated. fGroup not present. gNumber of waters coordinating Mg2ϩ. hThe Mg2ϩ bridges the ␣- and ␤-phosphate groups, each of which coordinates the ion with an O1P atom; both distances are reported. iRange of distances corresponding to the different coordinating waters. j Distance between Arg113-H␩ and the nearest phosphate oxygen. k Distance between Tyr264-OH and imidazole N␦.

all correctly positioned, with shifts of just 0.3–0.4 A˚ interacts with Arg113 through a bridging water (Fig. 2d). The experimental 3’-endo sugar pucker is molecule; hydrogen bonds to Gln127 and Arg259 are preserved in all cases. Moreover, all the main adenyl- present initially, but break after about 120 ps as the ate interactions observed experimentally are pre- carboxylate becomes solvated. In the HisOH complex served in both simulations. The HisAMP imidazole (Fig. 3c), the ligand is shifted almost 1 A˚ further back group hydrogen bonds to Glu131 in the back of the into the binding pocket. In a simulation where His binding pocket and to Tyr264 in the front. Tyr264 is starts out in the HisOH position, the opposite shift is anchored in place by a hydrogen bond to Arg259, observed (not shown). This shift appears to be driven which also hydrogen bonds to the adenylate ␣-phos- by optimization of the interactions of the HisOH phate and to Glu270 below. The ammonium group of ammonium, given that the constraint to maintain the His moiety of HisAMP hydrogen bonds to Thr85, strong interactions between the His carboxylate Glu83, and a stable water molecule. This water fills a group and water or arginines is removed. Arg259 cavity in the back of the binding pocket, bridging the does not hydrogen bond to histidinol at all, and its ligand ammonium, the Gln127 amide group, and the hydrogen bond to Tyr264 is only present 20% of the Tyr107 hydroxyl; it is seen in the same position in time. The rest of the time it is solvated. The histidi- the crystal structure. The adenylate phosphate also nol hydroxyl interacts with water. Compared with interacts with the motif 2 Arg113 and (more weakly) the crystal structure of HisRStt, both the His and with Gln127, and the adenine moiety stacks on the HisOH are shifted by 1 A˚ and rotated slightly; all conserved Phe125. This active site network is very three imidazole rings are nearly superimposable. stable, as indicated by the small atomic fluctuations about their mean positions (0.3–0.5 A˚ for all atoms; Mutants 1–4 Complexed With HisAMP Table II) and the stability of the hydrogen bonds mentioned (90–100% occupancy over 2 ϫ 200 ps). As described in Materials and Methods, the 273– In the simulation of the His complex, the interac- 281 loop occupies two distinct conformations in tions of the amino acid are very similar to those native HisRS, which place the carboxylate side chain outlined above and to the crystal structure of the of residue 281 in two different positions with respect HisRS:HisOH:ATP complex (Fig. 3). A typical snap- to the ␣-phosphate and the putative Mg2ϩ site. Since shot from the simulation is shown in Figure 3b. The this carboxylate coordinates the principal Mg2ϩ in imidazole is tilted downwards slightly around ␹1, on SerRS, it is important to investigate both loop confor- average, in the opposite direction from the adenylate mations explicitly. We first describe simulations of simulations. However, the hydrogen bonds to Glu131 all four mutants in complex with HisAMP with the and Tyr264 are preserved, and the ammonium makes 273–281 loop in its major conformation (the predomi- the same interactions as before. The main differ- nant conformation in E. coli HisRS, as seen in the ences are around the carboxylate group. This group crystal structure). Simulations with the loop in its ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 369 minor conformation are described further on. All coordination sites are occupied by water molecules, four mutants behaved stably throughout the simula- one of which interacts with Asp281. The coordination tions, as illustrated by the small rms deviations of sphere is distorted and not as good as that in the the structures from their starting positions (Table other mutants. The ␣-phosphate has shifted 1 A˚ II). These values ranged from 1.1 to 1.2 A˚ for all downwards, losing its direct interaction with the atoms, from 0.7 to 0.9 A˚ for the backbone, and from class II Arg113, which becomes mediated by two 0.7 to 1.0 A˚ for the HisAMP. The rms fluctuations water molecules. Ser284 donates its hydrogen bond around the mean structure for the heavy atoms alternately to Glu270 and to the ␣-phosphate of the inside the unrestrained simulation region are 0.32– adenylate. Lys168, which is only present in mutants 0.39 A˚ for the HisAMP complexes. Slightly larger 2 and 4 (Table I), appears to orient Gln268 such that fluctuations are observed below with the His and the latter interacts with Glu270. This places Glu270 ˚ HisϩATP complexes, 0.36–0.40 A. These are similar in a different orientation with respect to the metal to the value observed in recent simulations of an ion, compared with the mutant 1 simulation; this ˚ 33 Asp-AspRS complex, 0.42 A. The final resulting may play a role in enabling the Mg2ϩ ion to coordi- coordinates from each simulation are shown in Fig- nate the ␣-phosphate directly. Tyr264 donates its ure 4. hydrogen bond to Ser284 instead of to the N␦ of the 2ϩ Data pertaining to the Mg coordination and His moiety; the latter interacts with a water mol- substrate deviations are summarized in Table II. In ecule instead. the mutant complexes, the adenylate has only slightly Mutant 3 (R259G, E270D, T281E, A284S, V268Q, larger deviations from its starting position than in Fig. 4c) has shifted from the starting structure by an the native simulations, and similar rms fluctuations. rms deviation of 1.3 A˚ for heavy atoms and 0.7 A˚ for the The interactions of the ligand imidazole were largely adenylate. In the starting structure, the Mg2ϩ ion is 2ϩ preserved in all cases. However, the Mg coordina- coordinated to the ␣-phosphate, Ser284, three wa- tion sphere underwent some rearrangements in each ters, and Glu281. Asp270 is too short to coordinate case. the ion directly and interacts through a water mol- Mutant 1 (R259G, T281D, A284S, V268Q, Fig. 4a) ecule instead. Although the 273–281 loop is in its has shifted from the starting structure by an rms major conformation, Glu281 reaches far enough to deviation of 1.2 A˚ for all heavy atoms and 0.9 A˚ for coordinate the ion when it is built in a fully extended the adenylate. In the starting structure, the Mg2ϩ conformation. Within the first 20 ps of simulation, ion is coordinated to Ser284, Glu270, and the ␣-phos- however, Glu281 bends back and a water molecule phate of the adenylate. The remaining Mg2ϩ coordi- becomes inserted between it and the Mg2ϩ ion. The nation sites are occupied by water molecules. The ion remains coordinated to Ser284 and the ␣-phos- 273–281 loop is in its major conformation, and phate of the adenylate. The remaining Mg2ϩ coordi- Asp281 is too far away to coordinate the Mg2ϩ nation sites are occupied by water molecules, one of directly (unlike the equivalent Glu in SerRS). Within the first 20 ps, the Mg2ϩ ion shifts 1.5 A˚ left- and which interacts with Glu281 part of the time. After downwards, losing its coordination to the ␣-phos- the initial rearrangement, the coordination sphere is phate, but remaining coordinated to Ser284 and very stable throughout the simulation, with the Glu270 and to four waters. After the initial shift, the lowest rms fluctuation and no significant drift, al- structure is mostly stable, as shown by the lack of though the rms deviation from the starting structure drift of the ion (not shown) and the small fluctua- is the highest of the four mutants (Table II). The rms tions of the Mg2ϩ-ligand distances (Table II). One of fluctuation of the histidyl-adenylate and its rms the Mg2ϩ-coordinated waters interacts with Asp281 deviation from the starting structure are also the and two of them form bridges to the ␣-phosphate. lowest. Ser284 interacts with Asp270, which presum- Ser284 hydrogen bonds part of the time to the ably helps to fix the Ser284 orientation and stabi- ␣-phosphate and part of the time to a water molecule lizes its interaction with the Mg2ϩ. Another Mg2ϩ- that interacts with Tyr264. Tyr264 does not interact coordinated water molecule donates a hydrogen bond directly with any of the Mg2ϩ-coordinated water to the phenolic OH of Tyr264, which in turn hydro- molecules. Arg113 interacts directly with the ␣-phos- gen bonds to the N␦ of the histidine moiety of the phate most of the time, although the two are occasion- adenylate. The class II Arg113 interacts directly ally bridged by water molecules. with the ␣-phosphate as in the native structure. Mutant 2 (R259G, T281D, A284S, V268Q, I168K, Mutant 4 (R259G, E270D, T281E, A284S, V268Q, Fig. 4b) has shifted from the starting structure by I168K, Fig. 4d) behaves similarly to mutant 3. After an rms deviation of 1.2 A˚ for heavy atoms and 1.0 A˚ for 200 ps, it has shifted from the starting structure by the adenylate. The Mg2ϩ ion has the same initial an rms deviation of 1.1 A˚ for heavy atoms and 0.7 A˚ for cooordination as mutant 1. Within the first 20 ps, the the adenylate. The initial Mg2ϩ coordination is the coordination sphere rearranges. The Mg2ϩ remains same as for mutant 3. After 20 ps of simulation, the coordinated to Glu270 and the ␣-phosphate of the Mg2ϩ is coordinated to Ser284 and the ␣-phosphate adenylate, but not to Ser284. The remaining Mg2ϩ of the adenylate, but not to Asp270 or Glu281, which 370 J.G. ARNEZ ET AL.

Fig. 4. Stereo views of the active site region of mutant T281E, A284S, V268Q, I168K. Shown are the ligand, histidyl- HisRS:HisAMP complexes after 200 ps of molecular dynamics adenylate (black bonds), the mutated residues (yellow bonds), the simulations. a: Mutant 1: R259G, T281D, A284S, V268Q. b: Mutant three class II conserved arginines (orange bonds), and some other 2: R259G, T281D, A284S, V268Q, I168K. c: Mutant 3: R259G, important residues that interact with histidyl-adenylate (green E270D, T281E, A284S, V268Q. d: Mutant 4: R259G, E270D, bonds). The metal ions are drawn as light blue spheres. ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 371

Fig. 5. Stereo views of (a) the active site of mutant 3 HisAMP complex from the simulation with the minor 273–281 loop conformation and (b) comparison of the simulations of the mutant 3 HisAMP complex with the major (blue) and minor (red) loop conformation.

Fig. 6. Alternative 273–281 loop structures. Stereo views of the major (blue) and minor (red) loop conformations in mutant 3:HisAMP and HisRS from T. thermophilus (green). has bent away as before. The remaining Mg2ϩ coordi- OH of Tyr264, which in turn donates its hydrogen nation sites are occupied by water molecules, one of bond to the N␦ of the histidine moiety of the adenyl- which occasionally interacts with Glu281 and an- ate. Lys168 does not seem to play any role in the other hydrogen bonds with Asp270. Ser284 also active site; instead, it interacts with the backbone interacts with Asp270. The coordination sphere is carbonyl oxygen of Tyr264 and otherwise faces the very stable through the dynamics simulation. Much solvent. Arg113 contacts the ␣-phosphate directly as in mutant 3, another Mg2ϩ-coordinated water but weakly (average O2P-N␩ distance of 3.8 A˚ , molecule donates a hydrogen bond to the phenolic compared to 2.7 A˚ in mutant 3). 372 J.G. ARNEZ ET AL.

Mutants 1 and 3 Complexed With directly with the ion. Asp281 interacts through a HisAMP With the 73–281 Loop in its water molecule, and the ␣-phosphate is separated Minor Conformation from the class II Arg113 by a water molecule, as in mutant 2. The question arises why Glu281 in the simula- The rearrangements described above do not ap- tions of mutants 3 and 4 does not maintain a pear to be strongly dependent on the loop reconstruc- conformation similar to that of its homologue Glu345 tion, since the structures sampled are similar to in SerRS, which coordinates the principal metal ion. mutant 1 and 2 structures with the major loop The ‘‘major’’ conformation of its anchoring loop 273– conformation; rather, they probably reflect an alter- 281 is different from that in SerRS, and in HisRS nate stable arrangement sampled as a result of from T. thermophilus, as discussed. The loop started slightly different initial conditions. It appears that out in this conformation and did not undergo any the main factor influencing the Mg2ϩ coordination in significant rearrangements during the simulations. mutants 1 and 2, and differentiating it from mutants With this backbone conformation, Glu281 can only 3 and 4, is the nature of the 270–281 carboxylate 2ϩ coordinate Mg in a completely extended conforma- pair: (Glu-Asp). If so, the structures sampled in this tion. The ability of the loop to adjust during the simulation and the previous mutant 1 and 2 simula- simulations may be limited because the backbone of tions can be viewed as alternate possible arrange- residues 275–276 is in the outer part of the simula- ments of the active site for this carboxylate se- tion model and was constrained during the calcula- quence. The magnitude of the rearrangements tions. Despite the constraints, a significant portion of between the three simulations then gives a measure ˚ the loop was in the inner 15 A free dynamics sphere, of the uncertainty of the simulation model, with and so it was free in principle to adjust in order to respect to the exact geometry of the active site. optimize the Glu281 position. However, the simula- In the mutant 3 simulation, the effect of the tion length may be insufficient for this to occur. changed loop conformation is also small. A typical Therefore, to address the effect of loop conformation snapshot of the structure is shown in Figure 5a, and 2ϩ on the Mg coordination, additional simulations of the structure is compared with the previous mutant mutants 1 and 3 were performed. The 273–281 loop 3 simulation in Figure 5b. A detailed comparison of was rebuilt into the weak experimental electronic the loop regions is shown in Figure 6. density that represents the minor loop conformation The main differences from the previous mutant 3 in the native structure. This alternate conformation simulation are the replacement of a water in the is close to that of the homologous loop in SerRS. In Mg2ϩ coordination sphere by Glu281, a downward this geometry, Glu281 of mutant 3 initially coordi- shift of the ␣-phosphate and the Mg2ϩ by1A˚, and a nates the principal Mg2ϩ, while Asp281 of mutant 1 rotation of the coordination sphere so that Ser284 interacts through a water molecule. Simulations now interacts through a water molecule. The rms were then initiated from this starting conformation. deviation from the starting structure is reduced from The two loop conformations are compared in Figure 1.3 A˚ to 0.9 A˚ (nonhydrogen atoms; Table II), and the 5. The mutant 1 and 3 simulations are expected to be average potential energy is reduced by 42 kcal/mol, approximately representative of mutants 2 and 4, as or 1.4 standard deviations, compared with the major well. Indeed, mutant 4 behaves very similarly to loop conformation (-5,373 vs. –5,331 kcal/mol). These mutant 3 in the simulations above and has the same results suggest that the present structure may be side chain (Glu) at position 281. Mutant 2 has the the more stable. The Mg2ϩ fluctuations are increased same side chain (Asp) as mutant 1, which is too short from 0.28 A˚ to 0.38 A˚ , but the fluctuations of the to directly coordinate the Mg2ϩ, even with the loop in ion-ligand distances are of the same magnitude as its minor conformation (see below). previously. The range of active site fluctuations is Within the first 20 ps of the mutant 1 simulation, illustrated by a series of snapshots in Figure 7a. The the loop conformation relaxes into a structure very waters coordinating the ligand ammonium and the similar to the major conformation occupied in the Mg2ϩ are seen to be very stable, typical of all the previous simulation. The resulting active site struc- simulations. ture is intermediate between those of mutants 1 and 2 in the previous simulations. The Asp281 side chain Mutant 3 in Complex With His and HisϩATP is2A˚ from its position in the previous mutant 1 Mutant 3 was selected for further study, since its simulation, and only 1 A˚ from its position in the Mg2ϩ ion coordination most closely matches that of mutant 2 simulation. The overall rms deviations the equivalent ion in SerRS.6 In addition, Tyr264 from the previous mutant 1 and 2 simulations are accepts a hydrogen bond from the Mg2ϩ-hydrate, 0.6 and 0.8 A˚ , respectively, for non-hydrogen atoms which in this case plays the role of Arg259 in the within 6 A˚ of the ligand. The Mg2ϩ coordination is wild-type HisRS.5 This in turn enables Tyr264 to very similar to the previous mutant 2 simulation and donate its phenolic proton to the N␦ of the histidine differs from the previous mutant 1 simulation, with moiety of the adenylate and thus participate in the Ser284, Glu270 and the ␣-phosphate interacting specific recognition of the imidazole group. By con- ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 373

Fig. 7. Stereo views of ten snapshots at 10 ps intervals of the conserved arginines, and some other important residues that active site region of mutant 3 (minor loop conformation) com- interact with histidine and ATP, the metal ions, and selected waters plexed (a) with histidyl-adenylate and (b) with histidine and ATP. coordinating the principal Mg2ϩ and the ligand ammonium. Waters Shown are the ligand(s), the mutated residues, the three class II are shown as small spheres.

trast, in mutant 2 and occasionally in mutant 1, ion-ligand distances are very stable as a function of Tyr264 does not fill this role as effectively. Two time (Fig. 9a). The main differences with SerRS are molecular dynamics simulations of mutant 3 com- the missing Glu281 coordination and a 1.5 A˚ shift of plexed with HisϩATP were therefore performed, the ion towards the ␣-phosphate in the simulation; with the 273–281 loop initially in its major and in SerRS, the ion bridges the ␣- and ␤-phosphates. minor conformations, respectively. These simula- Nevertheless, comparing the present reactant simu- tions lasted 600 ps each, and for the last 400 ps all lation with the corresponding product (i.e., HisAMP) constraints on the 273–281 loop (i.e., on residues simulation above, the active site appears preorga- 275–276 in the outer buffer region) were removed so nized to carry out the adenylation reaction. For that the loop was free to adjust its conformation. example, the principal Mg2ϩ ion and the reactive P␣ Even so, no significant loop rearrangements were are shifted by just 1 and 2 A˚ , respectively, compared seen in either HisϩATP simulation. The principal with the corresponding HisAMP simulation. A 1 A˚ Mg2ϩ occupies different positions in the two simula- Mg2ϩ shift was recently observed experimentally tions, as described below. A single simulation of between the equivalent states in GlyRS (Arnez, mutant 3 complexed with His was also performed. Dock-Bregeon, and Moras, in preparation). The His In the HisϩATP complex with the major loop carboxylate interacts directly with the principal conformation, Glu281 was initially built in an ex- Mg2ϩ hydrate, while the (ATP)P␣-OT(His) distances tended conformation, coordinating the principal fluctuate around 4 A˚ and frequently reach values of Mg2ϩ. It shifted away within 20 ps of dynamics as less than 3.5 A˚ (Fig. 9b). The Mg2ϩ hydrate continues before, to be replaced by a water molecule. The to orient properly Tyr264, which maintains its spe- coordination sphere of the principal Mg2ϩ includes cific interaction with the imidazole group. Arg113 the ATP ␣-phosphate, Ser284, and four waters, only interacts directly with the ␣-phosphate for the first approximately matching that observed in the experi- 500 ps, and then shifts away slightly so that the mental SerRS:ATP structure. A typical snapshot interaction becomes mediated by a water molecule. from the simulation is shown in Figure 8a. The One of the remaining Mg2ϩ ions bridges the ␤- and 374 J.G. ARNEZ ET AL.

Fig. 8. Stereo views of the active site region of mutant 3 bonds), the mutated residues (yellow bonds), the three class II complexed (a) with histidine and ATP, with the 273–281 loop in its conserved arginines (orange bonds), and some other important major conformation; (b) with the same, but in the minor loop residues that interact with histidine and ATP (green bonds). The conformation; (c) with His alone. Shown are the ligand(s) (black metal ions are drawn as light blue spheres.

␥-phosphates, with the other coordination sites filled water molecule that is coordinated to the principal by water molecules. These waters hydrogen-bond to Mg2ϩ. All three Mg2ϩ ion sites are highly stable Glu66 and Glu115. The former is invariant among throughout the 600 ps simulation (Fig. 9a). prokaryotic HisRS, and the latter is highly con- In the HisϩATP complex with the minor loop served in class II aaRSs (being part of motif 2).4 The conformation, the Mg2ϩ coordination sphere matches third ion coordinates the ␥-phosphate; this differs that of SerRS more closely: the principal Mg2ϩ from the coordination geometry observed in SerRS bridges the ␣- and ␤-phosphates as in SerRS, while (bridging the ␤- and ␥-phosphates)6 or HisRS (mostly the other coordination sites are occupied by Ser284, on the ␤-phosphate).5 Glu281 is coordinated to the two waters, and Glu281, which consistently coordi- latter Mg2ϩ and also occasionally interacts with a nates both the principal and the third Mg2ϩ ions. A ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 375

throughout the simulation. The rms deviation be- tween the structures sampled in the two simulations is 0.6 A˚ for non-hydrogen atoms within 6 A˚ of the ligands. The (ATP)P␣-OT(His) distances are about 0.5 A˚ longer than in the previous simulation (Fig. 9c), and the His carboxylate is slightly farther from the Mg2ϩ hydrate. The shift between the present HisϩATP reactant complex and the corresponding HisAMP product complex is comparable to above. For example, the principal Mg2ϩ shifts by 1.9 A˚ and the P␣ by 1.5 A˚ . The average potential energy of the present structure is significantly lower than that in the previous simulation with the major loop confor- mation (-7,423 vs. –7,250 kcal/mol), the difference being five times the standard deviation (ca. 35 kcal/mol). The protein structure also remains closer to its starting structure than in the previous simula- tion, with an average rms deviation of 1.0 A˚ for nonhydrogen atoms vs. 1.2 A˚ above (Table II). This is similar to the HisAMP case above. These results suggest that the present structure is more stable than that sampled with the major loop conformation, and more representative of the mutant 3 HisϩATP complex. The other two Mg2ϩ ions occupy essentially the same positions in the simulations with the major and minor loop conformations, and have very similar coordination spheres. A single His simulation was performed, with the 273–281 loop in its major conformation. Despite the absence of ATP, the principal Mg2ϩ remains in essen- tially the same position as in the HisϩATP simula- tion above with the major loop conformation (Fig. 8a,c). The interaction with Ser284 is maintained, the ␣-phosphate is replaced by a water molecule, and the orientation of the five coordinating waters is very close to that observed with HisϩATP. Thus, the His carboxylate is pre-positioned to interact with the ATP ␣-phosphate. Although the minor loop conforma- tion was not simulated for the His complex, we expect that rebuilding the loop in its minor conforma- tion will not modify the Mg2ϩ coordination strongly. In the mutant 3 HisAMP simulations above, the Fig. 9. Selected interatomic distances as a function of time. main effect of rebuilding the loop was to replace a Distances between (a) the carboxylate oxygens OT1 and OT2 of His and the ␣-phosphorus of ATP; and (b) and (c) between the water ligand by Glu281, without perturbing the principal Mg2ϩ ion and its ligands, as indicated: one or two of the other ligands. ␣-phosphate oxygens, the O␥ of Ser284, a carboxylate oxygen O⑀ of Glu281, and 2–4 water oxygens, with the 273–281 loop in its (b) DISCUSSION major and (c) minor conformations. In the course of the four native simulations, all complexes studied have dynamically stable struc- typical snapshot from the simulation is shown in tures and interactions with the ligands. The active Figure 8b, and a series of snapshots illustrating the site structure was observed to be robust with respect range of fluctuations are shown in Figure 7b. The to the exact treatment of electrostatic interactions, simulation and SerRS:ATP structures are compared by comparing simulations with and without an elec- in Figure 10. trostatic cutoff. The structures sampled were in good The principal Mg2ϩ in the simulation is just 0.7 A˚ agreement with the available experimental struc- away from its position in SerRS. The main difference tures. The amino acid position is essentially the with the SerRS active site is a weaker interaction same in the His and HisAMP simulations, indicating here between the ␣-phosphate and the class II that this position is selected prior to ATP binding. Arg113, which is mediated by a water molecule The catalytic carbon of His is within 0.75 A˚ of its 376 J.G. ARNEZ ET AL.

Fig. 10. Stereo view of the superposition of the active sites of HisRS mutant 3 (loop 273–281 in the minor conformation, black) and SerRS6 (gray), both complexed with ATP.

†TABLE III. Mg2؉ Ion-Ligand Distances experimental position in three of four native simula- tions, and 1.2 A˚ away in the HisOH simulation. The AspRS:AspAMP HisRS mutant same preorganization was observed for the Asp crystal 3:HisAMP substrate in AspRS in X-ray structures at 100 K34 Ser NA (Gly) (3.74) and molecular dynamics simulations at room tem- Glu 2.46 1.90 perature.33 No significant alternate structures around OxP 2.33 1.84 the His moiety were sampled during the native or H2O NA 2.01–2.05 mutant trajectories. A similar stability (and similar SerRS:ATP HisRS mutant rms atomic fluctuations) was observed in simula- crystal 3:His:ATP tions of AspRS complexed with Asp, but not with Site 1 Asn, where many alternate structures were found Ser 2.07 2.12 over a similar time scale.33 Some rearrangements Glu 2.29 1.91 occur here during equilibration. For example, HisOH, O␣P 2.18 1.90 initially placed in the His position, shifts 1 A˚ further O␤P 2.09 2.01 H O 2.21 2.01–2.03 back into the active site pocket, and His, if initially 2 Site 2 placed in the HisOH position, shifts outwards by the Glu 2.45 1.96 same amount. These data suggest that the simula- O␤P 2.94 1.97 tion duration was adequate to study the structure of O␥P 2.39 1.88 ˚ the imidazole binding site. A similar 1 A shift of H2O 2.09 2.01–2.12 HisOH compared with the histidine moiety of Site 3 HisAMP was observed experimentally.5 O␤P 2.27 1.84 The mutant simulations illustrate some of the O␥P 2.34 1.88 possibilities and limitations of computer experi- H2O 2.09 2.03–2.06 ments for metalloprotein design. Despite the simplic- †The HisRS mutant 3 data are for that with the minor ity of the molecular mechanics description, both the loop conformation. octahedral geometry of the Mg2ϩ coordination sphere and the Mg2ϩ-ligand distances (reported in Table III) are in good agreement with those observed in the Mg2ϩ-water clusters has been shown to be small.37 The relevant SerRS, AspRS, and HisRS crystal struc- CHARMM22 force field has also been used successfully tures. Similar agreement has been found in nucleic to study zinc binding by several proteins,32 yielding a acid simulations,35 confirming that the Mg2ϩ-phos- zinc coordination sphere in good agreement with experi- phate geometry is well reproduced by the force field. ment, despite the neglect of explicit electronic polarizabil- The CHARMM22 force field is also known to repro- ity. These results suggest that the Mg2ϩ parameters are duce the solvation structure around Mg2ϩ in bulk adequate for the present study. water (A. MacKerell, personal communication). Wa- In the nine mutant simulations, the coordination ter-ion distances are slightly shorter here (2.01— sphere of the engineered Mg2ϩ sampled several 2.04 A˚ ) than in bulk water (2.1 A˚ )36; this could be due alternate arrangements, which correlate with the to slightly stronger electrostatic interactions in the nature of the carboxylates (Asp or Glu) at positions HisRS active site compared with high-dielectric bulk 270 and 281, and with the conformation (major or water. Electronic polarization induced by the diva- minor) of the 273–281 loop, which anchors residue lent Mg2ϩ ion in its environment is not explicitly 281. Indeed, the presence of Glu281 in the coordina- included in the force field; however, the effect of elec- tion sphere depends on the loop conformation. When tronic polarizability on the structure and energetics of the 273–281 loop is built in its major HisRS conforma- ENGINEERING AN Mg2ϩ SITE BY COMPUTER EXPERIMENTS 377 tion, neither an Asp nor a Glu at position 281 are this conformation is the most stable for mutant 3. A close enough to coordinate the engineered Mg2ϩ ion high degree of preorganization of the catalytic groups directly in a stable fashion, although they coordinate is observed when one compares the mutant 3 reac- it indirectly through a bridging water part of the tant and product complexes (HisRS:His:ATP and time. When the 273–281 loop is built in its minor, HisRS:HisAMP), as well as the His complex (simu- alternate conformation similar to the conformation lated only with the major loop conformation). For of the analogous loop in SerRS, Glu281 coordinates example, the His carboxylate approaches the ATP-P␣ the ion directly in a stable fashion. The simulations within 3.5–3.7 A˚ several times during the mutant 3 were unable to select spontaneously between these reactant state simulations, and the principal Mg2ϩ two loop conformations, as a consequence of the ion position differs by 1.9 A˚ between the reactant and finite simulation length. product states. A 1 A˚ shift was observed experimen- Three different positions of the principal Mg2ϩ are tally recently in the corresponding GlyRS complexes seen in the simulations. In mutants 1 and 2 (which have (Arnez, Dock-Bregeon and Moras, unpublished data). the carboxylate pair Glu270-Asp281), the ion is in a The results described here show that the Asp-Glu downwards position: it initially coordinates the ␣-phos- arrangement, in this order (as in Asp270-Glu281), is phate directly, but during equilibration it shifts down- preferable to the Glu-Asp alternative for coordinat- wards to strengthen its interaction with Glu270. In ing the metals in the active site. This could explain mutant 1 with the major loop conformation, it becomes why this sequential arrangement has been observed separated from the ␣-phosphate by a water molecule. In in most class II aaRSs examined, including AspRS,9,10 mutant 2, and mutant 1 with the minor loop conforma- SerRS,6 and GlyRS.38 Two class II aaRSs have tion, the ␣-phosphate shifts along with the ion and loses different residues at the first position: LysRS,39 its direct interaction with the class II Arg113, which which harbors a Glu, and PheRS,40 which harbors a becomes water-mediated. In mutants 3 and 4 (which Gln. This indicates that the second carboxylate is have the carboxylate pair Glu270-Asp281), the ion is more strictly conserved, with the notable exception usually in an upwards position. It coordinates the of HisRS,4,5 where the position is occupied by Thr. ␣-phosphate directly and Asp270 through a bridging The first carboxylate in wild-type HisRS is Glu270. water molecule. The other ligands are Ser284, 3 to 4 We expect that all four mutant constructs consid- waters, and in one case Glu281. Finally, in the mutant 3 ered here would be stable at the protein structure HisϩATP simulation with the minor loop conformation, level. It is more difficult to venture whether they the principal Mg2ϩ shifts into a third position, between would be enzymatically active if tested in vitro for the ␣- and ␤-phosphates, very similar to the experimen- aminoacylation and pyrophosphate exchange activ- tal SerRS:ATP complex. In contrast, with the major loop ity. In that regard, mutant 3 is the most promising. conformation the Mg2ϩ remains close to the ␣-phos- Its conformation is largely preserved in all three phate and does not coordinate the ␤-phosphate. complexes tested, which amount to a total of 2000 ps The positions of the other two Mg2ϩ ions in the ATP of molecular dynamics. It presents two alternate complexes are not significantly different in the mu- structures in complex with HisϩATP, depending on tant 3 structures with either the major or the minor the 273–281 loop conformation; the lowest energy loop conformation. One of them coordinates the structure is very similar to the SerRS active site ␤- and ␥-phosphates and a water molecule that structure. This structure seems to have the at- hydrogen bonds to a conserved motif 2 Glu. This is tributes for carrying out the activation of histidine, very similar to the Mn2ϩ coordination observed in although it may not perform as well as wild-type the crystal structures of the SerRS:ATP and HisRS: HisRS. To test these predictions, the mutants will be ATP complexes. The other metal ion’s coordination is engineered and studied biochemically. more idiosyncratic. In the SerRS:ATP complex it bridges the ␤- and ␥-phosphates; in the crystal ACKNOWLEDGMENTS structure of the HisRS:HisOH:ATP complex it coordi- We thank Prof. C. Francklyn for discussions and nates the ␤-phosphate, and in the simulations it careful reading of the manuscript. Some of the coordinates the ␥-phosphate. In SerRS and the mu- simulations were performed at the Centre National tant 3 HisRS simulation it also coordinates the Universitaire Sud de Calcul (Ministe`re de l’Education nearly invariant carboxylate. Nationale). The CHARMM program was kindly pro- Among the alternate structures sampled with the vided by Prof. M. Karplus. different mutants, ligands, and loop conformations it is difficult to predict unambiguously the most stable REFERENCES and catalytically favorable arrangement. Mutant 3 1. 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TABLE V.Aminoacyl-Adenylate Force Field Parameters: CHARMM22-Compatible Parameters for the Region of the Adenylate Linking the Amino Acid and the AMP

Atom Atom typea Chargeb N NH3 Ϫ0.30 HT1 HC 0.33 HT2 HC 0.33 HT3 HC 0.33 CA CT1 0.21 HA HB 0.10 C C 0.90 OO Ϫ0.60 OX OX Ϫ0.60 P P 1.20 O1P ON3 Ϫ0.75 O2P ON3 Ϫ0.75 O5Ј ON2 Ϫ0.50 C5Ј CN8 Ϫ0.08 H5Ј HN7 0.09 H5Љ HN7 0.09 Force Target Atom types constantc valued Bond C OX 570 1.25 Bond P OX 270 1.60 Angle CT1 C OX 40 110.8 Angle O C OX 100 126.7 Angle C OX P 20 127.5 Angle ON3 P OX 100 105.0 Angle ON2 P OX 80 100.4 Fig. 11. a: Methylacetyl-phosphate. A water ligand is repre- Force Periodi- sented in three possible positions (see text). Ab initio bond lengths, e e e angles, and atomic partial charges are indicated, as well as Atom types constant city Offset water-oxygen distances for positions 2 and 3. b: Partial charges Dihe CT2 CT1 C OX 0.067 3 180. for atoms in the region bridging the amino acid and AMP moieties Dihe NH3 CT1 C OX 0.067 3 180. of the aminoacyl-adenylate. Dihe X OX P X 0.250 3 0. Dihe X OX C X 0.000 3 0. Dihe CN8 ON2 P OX 0.250 3 0. TABLE IV. Water-Methylacetylphosphate Dihe CN8 ON2 P OX 0.750 2 0. Interaction Energies (kcal/mol) Dihe HB CT1 C OX 0.000 1 0. CHARMM22‡ Atom f g h h Large Small MP2/ type ⑀ ␴ ⑀14 ␴14 Position† OX OX 6-311ϩG*§ HF/6-311 ϩ G* § Nonbonded OX 0.1521 3.1538 0.1521 3.1538 1 Ϫ18.86 Ϫ18.83 Ϫ16.02 Ϫ12.67 aCHARMM22 atom type. 2 Ϫ20.13 Ϫ20.11 Ϫ16.29 Ϫ13.30 bFraction of electronic charge. 3 Ϫ17.02 Ϫ17.24 Ϫ15.70 Ϫ12.62 ckcal/mol/Å2 (bonds) or kcal/mol/rad2 (angles). dÅ (bonds) or degrees (angles). †Water position in Figure 11. ekcal/mol. The dihedral energy is given by k(1 ϩ cos(n␾ϩ␦)), ‡Large (⑀ϭ0.1521 kcal/mol, ␴ϭ3.1539 Å) and small (⑀ϭ0.1521 where k, n, and ␦ are the force constant, periodicity, and offset, kcal/mol, ␴ϭ2.7974 Å) CHARMM22 oxygen van der Waals respectively. parameters for OX. fkcal/mol. §Basis set superposition error neglected. gÅ. hParameters for interactions between atoms separated by three covalent bonds. only for histidyl-adenylate, but for all 20 aminoacyl adenylates. Consistent with the methods used to develop the CHARMM22 force field,22,41 we consider molecule; finally, the resulting parameters are trans- a small molecule analogous to the central part of the ferred to the aminoacyl-adenylate and tested in adenylate, methylacetyl-phosphate (MAP; Fig. 11). molecular dynamics simulations (described above) of We first determine initial charges and geometry His-adenylate complexed to HisRS. from ab initio calculations at the 6–31G* level, and The geometry of MAP was optimized at the HF/6– then we consider its interactions with a single water 31G* level using the program Gaussian92,42 and 380 J.G. ARNEZ ET AL. atomic partial charges were obtained by fitting the side of the phosphate (position 3). Minimizing from ab initio electrostatic potential on a grid around the position 1 with HF/3–21G*, the water shifts to the molecule. Results are shown in Figure 11. right, away from OX, closer to the phosphate (not The same procedure, when applied to dimethyl- shown). The HF/6–311ϩG*, MP2/6–311ϩG*, and phosphate, yields charges in good agreement with molecular mechanics energies of positions 1–3 are the CHARMM22 charges (results not shown). shown in Table IV. Basis set superposition error was Stereochemical force constants and van der Waals neglected. Although the molecular mechanics ener- parameters were taken from the CHARMM22 force gies only reproduce the ab initio energies approxi- field by considering the analogous amino acid back- mately, they rank the three conformations in the bone and AMP groups. For the bridging oxygen OX correct order, and furthermore, they are very in- between the amino acid and AMP, two van der Waals sensitive to the choice of OX van der Waals param- assignments are possible: either the ’’large’’ or ’’small’’ eters. Clearly, the energy and position of a water CHARMM22 oxygen van der Waals parameters could close to OX are dominated by the nearby, negatively be used, by analogy to O5’ or O, respectively. Initially charged phosphate group, and the exact van der the ’’large’’ oxygen parameters were chosen. Thus Waals parameters of OX are not critical. Thus, the parameterized, the fragment was immersed in a present parameters (Table V), with the ’’large’’ OX, sphere of modified TIP3P water molecules (see Mate- were transferred without further adjustment to rials and Methods), the energy minimized, and all the histidyl-adenylate and tested in molecular dy- waters except the one closest to OX discarded. This namics simulations of the HisRS:HisAMP complex. water bridges O and O2P (position 1 in Fig. 11). After Good agreement was observed with the crystal struc- molecular mechanics minimization with the ’’large’’ ture for the region around the adenylate, as de- OX, it shifts slightly closer to O (position 2 in Fig. 11). scribed in Results. The parameters for the bridging Minimizing from position 1 with a ’’small’’ OX (analo- region should be suitable for all 20 aminoacyl- gous to O, O1P, O2P), the water shifts to the other adenylates.