Structural plasticity of an aminoacyl-tRNA synthetase active site

Abstract

Recently, tRNA aminoacyl-tRNA synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl aminoacyl-tRNA synthetases that charge the unnatural amino acids p-bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substrate-bound WT synthetase, as well as a mutant synthetase that charges p-acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the α₈-helix, which spans the WT active site. The high degree of structural plasticity that is observed in these aminoacyl-tRNA synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids.

With the rare exceptions of selenocysteine (1) and pyrrolysine (2, 3), the common 20 amino acids are conserved across all known organisms. However, there does not appear to be an inherent limit to the size or chemical nature of the genetic code, since it has been shown that additional amino acids can be genetically encoded in both prokaryotic and eukaryotic organisms in response to nonsense or frameshift codons (4, 5). This requires a unique codon-suppressor tRNA pair and the corresponding aminoacyl-tRNA synthetase, which do not crossreact with the amino acids, tRNAs, or synthetases of the host organism (4, 5). The specificity of the aminoacyl tRNA synthetase is then altered by generating large libraries of active-site mutants and passing them through positive and negative selections to identify synthetases that selectively acylate the cognate tRNA with the unnatural amino acid but not any of the common amino acids. This approach has been used to add >30 unnatural amino acids to the genetic codes of bacteria, yeast, and mammalian cells with high fidelity and efficiencies.

In Escherichia coli, a tyrosyl aminoacyl-tRNA synthetase (TyrRS) tRNA_CUA^Tyr pair from the archea Methanococcus jannaschii (Mj) was used as an orthogonal tRNA synthetase pair (6). Mutant synthetases have been evolved that selectively aminoacylate their cognate suppressor tRNAs with glycosylated (7), photoreactive (8), and metal-binding amino acids, as well as amino acids with unique functional groups (9–11) . To establish the molecular basis for the surprising adaptability of this synthetase, we solved (12) the structure of a mutant Mj TyrRS that is selective for p-acetylphenylalanine (p-AcPhe). The x-ray crystal structure revealed significant structural changes within the enzyme active site that result from the mutations Y32L, D158G, I159C, and L162R. The Y32L and D158G mutations remove two hydrogen bonds (H-bonds) with the Tyr hydroxyl group to disfavor recognition of the natural substrate. The Y32L mutation also forms van der Waals interactions with the methyl group of the p-AcPhe amino acid, and D158G enlarges the binding pocket to sterically accommodate the acetyl group, as well as allow formation of a H-bond between the keto oxygen and Q109. The D158G mutation in the p-AcPhe synthetase also prematurely terminates the α₈-helix. As a result, several residues in the p-AcPhe TyrRS (including H160, Y161, and L162R) move up to 4 Å away from their position in the structure of the WT TyrRS–Tyr complex to optimize binding interactions with bound p-AcPhe.

Because the mutations in the Mj p-AcPhe TyrRS active site result in significant alterations in both side-chain and backbone conformations, we sought to determine the extent to which this structural plasticity can be observed in other mutants of the Mj TyrRS. Thus, the structures of Mj TyrRSs specific for 3-(2-naphthyl)Ala (NpAla) (13) and p-bromophenylalanine (p-BrPhe) (11) were solved and compared with structures of the WT and p-AcPhe-specific Mj TyrRS. This analysis suggests that altered amino acid recognition by the Mj TyrRS mutants may generally result from a large degree of structural plasticity in the backbone and side-chain conformations in this active site.

Results and Discussion

Expression and Crystallization of Mutant Mj TyrRSs.

The specificity of the Mj TyrRS was altered (14) by generating two synthetase libraries, each of which is formed by randomizing five residues within the synthetase active site (Library 1: Tyr-32, Glu-107, Asp-158, Ile-159, and Leu-162; Library 2: Tyr-32, Asp-158, Ile-159, Leu-162, and Ala-167) based on the crystal structure of the homologous Bacillus stearothermophilus TyrRS–tyrosyl adenylate complex. These libraries were separately subjected to subsequent rounds of positive and negative selection to identify aminoacyl tRNA synthetases that, together with a cognate Mj tRNA_CUA^Tyr, selectively incorporate into proteins in E. coli the unnatural amino acids p-BrPhe (Library 1) or NpAla (Library 2), in response to the amber nonsense codon. The positive selection was based on the suppression of an amber codon at a permissive site in the chloramphenicol acetyl transferase gene (CmR) in the presence of the unnatural amino acid; the negative selection involved suppression of amber codons at permissive sites in the barnase gene in the absence of the unnatural amino acid. A final round of shuffling with subsequent positive and negative selections was carried out also for the NpAla TyrRS (by using four active-site mutants as templates), but no additional mutations beyond the randomized five amino acids were found. The mutant Mj TyrRS that resulted from selections with NpAla had the following mutations: Y32L, D158P, I159A, L162Q, and A167V; the mutant Mj TyrRS that resulted from selections with p-BrPhe had the following mutations: Y32L, E107S, D158P, I159L, and L162E. Subsequent experiments showed that the latter synthetase also recognizes the unnatural amino acid p-iodoPhe, which is not surprising given the similarity in van der Waals radii between bromine and iodine (difference ≈ 0.2 Å). Both mutant synthetases incorporate their respective amino acids with good efficiency and high fidelity (<0.2% incorporation of 1 of the 20 common amino acids). The p-BrPhe and NpAla Mj aminoacyl-tRNA synthetases were expressed with a C-terminal His-6 fusion tag in E. coli and purified by affinity chromatography over Ni-NTA resin. Subsequent purification by cation ion-exchange chromatography provided protein for crystallization. Crystals were grown either in the presence of 2 mM p-BrPhe or NpAla and belong to space group P4₃2₁2, containing one molecule per asymmetric unit.

Structure Determination of Mutant Mj TyrRSs.

The structure of the p-BrPhe TyrRS–p-BrPhe complex was solved to 1.9 Å, with R_cryst = 0.21 and R_free = 0.25. The structure of the NpAla TyrRS–NpAla complex was solved to 1.9 Å, with R_cryst = 0.22 and R_free = 0.28 (Table 1). The final models for both structures had no residues in disallowed regions of the Ramachandran plot as well as a strong difference electron density for the bound amino acid. Like the WT Mj TyrRS (15, 16), the NpAla and p-BrPhe Mj synthetases are divided into five regions (the Rossmann-fold catalytic domain; the short N-terminal region; the connective polypeptide 1 domain, which forms the dimer interface; the C-terminal domain; and the KMSKS loop, which links the Rossmann-fold domain to the C-terminal domain). Both mutant synthetases superimpose well with the WT TyrRS–Tyr complex in all regions except the active site (rms deviations were as follows: WT/pBrPhe, 0.83 Å, over 299 aligned C^α atoms; and WT/NpAla, 0.77 Å, over 294 aligned C^α atoms), where the mutations in the p-BrPhe TyrRS and NpAla TyrRS significantly reconfigure the Tyr-binding pocket to selectively bind p-BrPhe or NpAla, respectively (Fig. 1).

Table 1.

Data and refinement statistics for the p-BrPhe Mj TyrRS–p-BrPhe and NpAla Mj TyrRS–NpAla complexes

Parameter	p-BrPhe	NpAla
Space group	P43212	P43212
Unit-cell parameters, Å	a = b = 102.98, c = 70.71	a = b = 103.0, c = 71.4
Wavelength, Å	1.0	1.0
Resolution range, Å	70.0–1.9	70.0–1.9
Rsymm (highest-resolution shell)	0.072 (0.74)	0.053 (0.67)
Unique reflections (observed)	30,607 (166,252)	31,188 (91,993)
Completeness (highest-resolution shell), %	99.4 (98.3)	97.1 (93.2)
Highest-resolution shell, Å	1.97–1.9	2.03–1.9
Mean I/σ(I)	39.9 (2.6)	32.8 (2.0)
Refinement parameters
Reflections, total	28,644	28,143
Reflections, test	1,538	1,507
Rcryst* (Rfree†)	0.21 (0.25)	0.22 (0.28)
No. of protein atoms	2,464	2,461
No. of heterogen atoms	13	24
No. of water atoms	142	115
rmsd bonds, Å	0.013	0.015
rmsd angles, °	1.47	1.57
Average isotropic B value, Å2	41.5	50.7
Protein	40.9	55.0
Amino acid	33.2	44.0
Waters	45.9	57.9

*R_cryst = ΣΣ|I_i − 〈I_i〉|/Σ|I_i|, where I_i is the scaled intensity of the ith measurement, and 〈I_i〉 is the mean intensity for that reflection.

^†As for R_cryst, but for 5.0% of the total reflections chosen at random and omitted from refinement.

Fig. 1.

Comparison of p-BrPhe, NpAla, and WT Mj TyrRSs bound to their respective substrates. (A) Structure of the p-BrPhe Mj TyrRS bound to p-BrPhe. Residues Y32L, E107S, D158P, I159L, L162E, H160, and Y161 are indicated. A sigmap-weighted F_o − F_c electron-density map (40) (contoured at 3σ) for p-BrPhe is also shown. The electron-density map was derived before the inclusion of p-BrPhe. (B) Structure of the NpAla Mj TyrRS bound to NpAla. Residues Y32L, D158P, I159A, L162Q, A167V, H160, and Y161 are indicated. A sigmap-weighted F_o − F_c electron-density map (40) (contoured at 3σ) for NpAla is also shown. The electron-density map was derived before the inclusion of NpAla. (C) Structure of the WT Mj TyrRS (16) bound to Tyr. Residues Y32, E107, D158, I159, L162, H160, and Y161 are indicated. Images were generated by bobscript and raster3d (45–47).

Comparison of WT and p-BrPhe Mj TyrRSs.

In the WT structure (16), recognition of Tyr involves a combination of H-bonding interactions and van der Waals contacts. The amino group of Tyr forms H-bonds with Y151 OH, Q155 Oε, and Q173 Oε; the Tyr carboxylate group forms a H-bond with Q173 Nε; and the Tyr hydroxyl group forms H-bonds with Y32 OH and D158 Oδ. There are van der Waals contacts between the Tyr side chain and G34, L65, Q155, and H70. The mutations to the p-BrPhe Mj TyrRS alter the active-site structure to specifically recognize p-BrPhe vs. Tyr while maintaining contacts to the conserved regions of the two amino acid substrates. For example, the amino and carboxylate groups of p-BrPhe form the same H-bonds as in the WT complex, and the aromatic ring of p-BrPhe again forms van der Waals contacts with G34, L65, Q155, and H70. However, the D158P and Y32L mutations in the p-BrPhe TyrRS enlarge the binding site to accommodate p-BrPhe and remove the two H-bonds to the hydroxyl oxygen of Tyr that are present in the WT structure (Fig. 2) (16). The D158P mutation also terminates the α₈-helix, resulting in formation of a short 3₁₀-helix consisting of residues 157–161. The H-bonds that are disrupted within the α₈-helix (Met154O–Asp158NH, Gln155O–Ile159NH, Val156O–His160NH, Asn157O–Tyr161NH, and Ile159O–Gly163NH) are partially compensated by H-bond capping interactions (17–20) from H160 and Y161 (His160Nδ–Met154O, Tyr161OH–Gln155O), as well as an additional side-chain H-bond (Tyr114OH–Met154O). The short 3₁₀-helix (after α₈-helix) is formed by two H-bonds (Asn157O–His160NH and Pro158O–Tyr161NH) and is capped at the N and C termini by Asn157Oδ–Leu159NH and Tyr161O–Val164NH, respectively. Consistent with this observation, it is known that there is a 3.5:1 preference for Asn as the N-capping residue and a 2.6:1 preference for Pro as the subsequent residue of an α-helix (17); studies of 3₁₀-helices have shown similar results (21). Other examples of proteins containing a 3₁₀-helix sequence that is contiguous with an α-helix sequence (21) are hemoglobin (22) and Arthromyces ramosus peroxidase (23).

Fig. 2.

Active sites of the p-BrPhe and NpAla Mj TyrRSs superimposed on the active site of the WT Mj TyrRS (16). H-bonds between Q173 of the p-BrPhe and NpAla synthetases and both the amine and carboxylate of p-BrPhe and NpAla, respectively. (A) Structure of the p-BrPhe TyrRS bound to p-BrPhe superimposed on the WT TyrRS (yellow) bound to Tyr (green). Residues Y32, D158, H160, and Y161 of the WT synthetase and Y32L, D158P, H160, and Y161 of the p-BrPhe synthetase are indicated. (B) Structure of the NpAla TyrRS (teal) bound to NpAla (red) superimposed on the WT TyrRS (yellow) bound to Tyr (green). Residues Y32, D158, H160, and Y161 of the WT synthetase and Y32L, D158P, H160, and Y161 of the NpAla synthetase are indicated. For both synthetase active sites, the D158P mutation alters local backbone conformation, inserting residues H160 and Y161 into the active site. Images were generated by bobscript and raster3d (45–47).

As a result of the D158P mutation and the resulting 3₁₀-helix, there are significant translational and rotational movements of several active residues (Fig. 3). In the WT TyrRS, D158, I159, and L162 are proximal to Tyr in the active site. In the structure of the p-BrPhe TyrRS, D158P, I159L, and L162E move away from the active site and are solvent-exposed (D158_WT C^α/D158P_p-BrPhe C^α distance, 5.4 Å; I159_WT C^α/I159L_p-BrPhe Cα distance, 6.0 Å; and L162_WT Cα/L162E_p-BrPhe C^α distance, 3.7 Å). Conversely, residues H160 and Y161 of the WT TyrRS do not make any contacts with bound Tyr, but in the p-BrPhe TyrRS these residues line the active site (H160_WT C^α/H160_p-BrPhe C^α distance, 4.7 Å; and Y161_WT C^α/Y161_p-BrPhe C^α distance, 4.6 Å) and form van der Waals contacts with the bromine of p-BrPhe. The oxygen of a Tyr would be too distant (>4 Å) to form either a H-bond or van der Waals interactions with H160 or Y161 in the active site of the p-BrPhe TyrRS. These residues are restricted from reorienting to bind Tyr as a result of H-bond capping interactions with the C terminus of the α₈-helix (H160Nδ-M154O and Y161OH-Q155O). Also, the bromine atom of p-BrPhe interacts with the π-system of the imidazole side chain of H160. C-halogen–π interactions have been described in the crystal structures of proteins and small molecules (24–26).

Fig. 3.

Backbone structural plasticity at the C terminus of the α₈-helix of the Mj TyrRS. (A) Structure of the p-BrPhe TyrRS bound to p-BrPhe (cyan) superimposed on the WT TyrRS (green) (16). The deviation in C^α position for residues 158–163 is indicated by dashed lines. The distances between the C^α positions of the WT and p-BrPhe TyrRSs are as follows: 1, D158_WT–D158P_p-BrPhe, 5.4 Å; 2, I159_WT–I159L_p-BrPhe, 6.0 Å; 3, H160_WT–H160_p-BrPhe, 4.7 Å; 4, Y161_WT–Y161_p-BrPhe, 4.6 Å; 5, L162_WT–E162_p-BrPhe, 3.7 Å; and 6, G163_WT–G163_p-BrPhe, 3.1 Å. (B) Structure of the NpAla TyrRS bound to NpAla (pink) superimposed on the WT TyrRS (green). The deviation in C^α position for residues 158–163 is indicated by dashed lines. The distances between the C^α positions of the WT and NpAla TyrRSs are as follows: 1, D158_WT–P158_NpAla, 5.1 Å; 2, I159_WT–A159_NpAla, 6.2 Å; 3, H160_WT–H160_NpAla, 4.8 Å; 4, Y161_WT–Y161_NpAla, 4.5 Å; 5, L162_WT–Q162_NpAla, 3.3 Å; and 6, G163_WT–G163_NpAla, 2.8 Å. (C) Structure of the WT TyrRS bound to Tyr (16). The protein backbone is colored from blue to red, according to increasing backbone structural diversity at each residue [defined as the rms distance between the C^α atoms of the WT Mj TyrRS and the p-AcPhe (12), p-BrPhe, and NpAla TyrRSs]. Images were generated by pymol (48).

Comparison of WT and NpAla Mj TyrRSs.

The NpAla TyrRS forms both H-bonds and van der Waals contacts with NpAla. Like the structures of the WT and p-BrPhe synthetases bound to their respective amino acids, the amino group of NpAla forms H-bonds with Y151 OH, Q155 Oε, and Q173 Oε (distance, 2.6–2.8 Å), and the carboxylate group forms a H-bond with Q173 Nε (distance, 3.0 Å). The first aromatic ring of the naphthyl side chain (closest to C^α) also forms van der Waals contacts with G34, L65, Q155, and H70 of the NpAla TyrRS, analogous to the WT and p-BrPhe TyrRS. The Y32L mutation removes a potential H-bond to Tyr and expands the active site, allowing favorable van der Waals contacts with the second aromatic ring of NpAla. This case is different from the p-BrPhe TyrRS, in which the Y32L mutation opens the binding pocket to provide room for the bromine atom, but it does not form van der Waals contacts (Y32L_p-BrPhe C^δ/p-BrPhe Br distance, 4.4 Å).

The effects of the D158P mutation on the structure of the NpAla TyrRS are similar to the effects that were detected in p-BrPhe TyrRS. This mutation prevents formation of a H-bond to Tyr, and it truncates the α₈-helix to form a subsequent 3₁₀-helix with residues 157–161 (Figs. 1 and 2). The H-bonds that are broken within the α₈-helix of the NpAla TyrRS are the same as the H-bonds that are broken by the D158P in the p-BrPhe TyrRS. Residues D158P, I159A, and L162Q move out of the active site in the WT structure to become solvent-exposed in the NpAla TyrRS structure (D158_wt C^α/D158P_NpAla C^α distance, 5.1 Å; I159_WT Cα/I159A_NpAla Cα distance, 6.2 Å; and L162_WT C^α/L162Q_NpAla distance, 3.3 Å) (Fig. 3). Residues H160 and Y161, which are solvent exposed in the WT TyrRS, move to form part of the active-site binding pocket of the NpAla TyrRS (H160_WT C^α/H160_NpAla C^α distance, 4.8 Å; and Y161_WT C^α/Y161_NpAla C^α distance, 4.5 Å) with van der Waals contacts to NpAla. In a similar fashion to the p-BrPhe TyrRS structure, the Y161_NpAla hydroxyl group forms a H-bond with Q155_NpAla O (distance, 2.6 Å). However, unlike the p-BrPhe TyrRS structure, the H160_NpAla Nδ forms only a long H-bond with M154_NpAla O (H160_NpAla Nδ-M154_NpAla O distance, 3.4 Å; and H160_pBrPhe Nδ-M154_pBrPhe O distance, 3.0 Å). This difference is likely to be due to the larger size of the NpAla side chain compared with p-BrPhe, as well as the A167V_NpAla mutation. Although A167V_NpAla does not interact with NpAla, this mutation forces Y161_NpAla to rotate ≈45° compared with the position of Y161_pBrPhe, allowing the NpAla side chain to occupy the active site. This rotation causes Q155_NpAla to move to maintain the Y161–Q155 H-bond. Therefore, the adjacent M154_NpAla moves and is unable to form an optimal H-bond with H160_NpAla.

Comparison with Other Mj TyrRS Structures.

A comparison of the amino acid-bound structures of the p-BrPhe, NpAla, p-AcPhe, and WT Mj TyrRSs reveals that the selected mutations introduce significant structural changes into all of the active sites (Fig. 3c). These structural changes alter the active site to recognize regions of the unnatural amino acid that differ from Tyr but do not disrupt interactions with structural elements that are conserved between Tyr and the unnatural amino acids. For example, the H-bonds between the p-AcPhe, p-BrPhe, and NpAla TyrRSs and both the amino and carboxylate groups of their respective substrates are the same as those between the WT TyrRS and Tyr. Furthermore, van der Waals interactions are present between the four substrates G34, L65, Q155, and H70 of the WT and mutant synthetases. However, mutations in the p-AcPhe, p-BrPhe, and NpAla synthetases alter the active-site structure to discriminate between the distinct functional groups (ketone, halogen, and fused aromatic ring) of their respective substrates by altering both H-bonding and packing interactions. For example, mutations to the residues Y32 and D158 are found in all three mutant synthetases. These mutations remove H-bonds to the Tyr hydroxyl that are present in the WT complex and, instead, result in van der Waals contacts with the ketone methyl group, bromine atom, and aromatic ring of p-AcPhe, p-BrPhe, and NpAla, respectively. The D158G mutation in the p-AcPhe TyrRS and the D158P mutation in the p-BrPhe TyrRS and NpAla TyrRS also remove the second H-bond to Tyr. Also, these mutations change the backbone structure and side-chain conformations in the active site. The backbone conformational changes that are due to the D158P mutations in both the p-BrPhe TyrRS and NpAla TyrRS are more prominent than the changes that are due to the D158G mutation in the p-AcPhe TyrRS. Although the p-AcPhe D158G mutation allows the formation of a H-bond between Q109 Nε and the p-AcPhe carbonyl oxygen by removing the intervening side chain of D158, residues H160 and Y161 of the p-AcPhe TyrRS do not move to form part of the new active site (as they do in the p-BrPhe TyrRS and NpAla TyrRS). The C^α of H160_p-AcPhe moves 2.7 Å, and the C^α of Y161_p-AcPhe moves 3.6 Å relative to the WT enzyme; for comparison, H160_pBrPhe C^α and H160_NpAla C^α move 4.7 and 4.8 Å, respectively, and Y161_p-BrPhe C^α and Y161_NpAla C^α move 4.6 and 4.5 Å, respectively. This difference between the p-AcPhe TyrRS and the p-BrPhe and NpAla TyrRS may be a consequence of different mechanisms of recognition. Wheras the WT and p-AcPhe Mj TyrRSs recognize their substrates by H-bonding as well as van der Waals interactions, the hydrophobic nature of p-BrPhe and NpAla may require larger changes in both side-chain and backbone structure in the active site to form complementary van der Waals surfaces as well as a C-halogen–π interaction between H160 and the bromine atom (in the case of the p-BrPhe TyrRS).

Another mutation that changes amino acid specificity without making any contacts with the amino acid is A167V of the NpAla TyrRS. The larger side chain of this mutation causes the Y161 side chain to rotate, which affords additional space for NpAla. Without this mutation, residues H160 and Y161 of the NpAla TyrRS would be likely to occupy the same positions as in the p-BrPhe TyrRS. Superimposition of these two structures reveals a steric clash between NpAla and Y161_p-BrPhe but not Y161_NpAla. Therefore, even though A167V_NpAla does not interact with NpAla, it is important for binding because it expands the active site by rotating Y161_NpAla. I159L and L162E of the p-BrPhe TyrRS, L162R of p-AcPhe TyrRS, and I159A and L162Q of the NpAla TyrRS are all distal to the active site, and these mutations were likely to have been chosen to maximize physical properties of the protein.

Role of Structural Plasticity in Biomolecular Recognition.

Previous structural studies of aminoacyl-tRNA synthetases that recognize unnatural amino acids have shown little change in the backbone structures compared with the WT structure. For example, both the substrate-bound and Tyr-bound structures of a mutant E. coli tyrosyl synthetase that recognizes 3-iodo-l-Tyr have very similar backbone traces as the WT synthetase bound to Tyr (27). The backbone structure of tryptophanyl tRNA synthetase from Deinococcus radiodurans bound to 5-hydroxy-Trp (5HT) is similar to the Trp-bound structure, although the orientation of 5HT in the active site is rotated by almost 180° compared with Trp (28). Indeed, most structurally characterized mutant enzymes with altered substrate specificity [including mutants of isocitrate dehydrogenase, isopropylmalate dehydrogenase (29, 30), and Asp aminotransferase (AspAT) (31)] do not involve significant main-chain conformational changes. For example, the specificity of the WT AspAT was changed from Asp to Val by directed evolution, but a comparison of the backbone structures of the WT and mutant AspAT shows that only one residue (S363G) deviates by >2 Å, which is likely to be due at least in part to this mutation occurring next to an adjacent Gly residue (31).

However, it is clear that mutations and modifications can significantly alter protein backbone conformation and, as a consequence, protein function. For example, Thr phosphorylation activates yeast glycogen phosphorylase by refolding the N-terminal loop into an α-helix (32), and several studies have identified mutations that cause changes in the topological fold of the RNA-binding protein Rop (33, 34). Another example in which both backbone and side-chain conformational changes have a significant role in binding specificity is the affinity maturation of germ-line antibodies (35, 36). In this case, somatic point mutations can act to lock significant conformational changes induced by binding of ligands to the germ-line antibody.

There are two factors (12) that likely lead to the observed structural plasticity of the p-AcPhe, p-BrPhe, and NpAla Mj TyrRSs. First, because the changes are on a solvent-exposed helix, the number of requisite compensatory mutations is reduced, because bulk water can more readily reorganize to solvate various new conformations than adjacent protein residues. Second, the focused library restricts mutations to active-site residues in local proximity, which increases the probability of favorable cooperative interactions. In contrast, mutations that arise from DNA shuffling are recombined from independent selection experiments, and the resulting set of mutations may not be cooperative. Random point mutations also have a low probability of being cooperative because of the limited size of sequence space that can be explored. In contrast, successive rounds of point mutations such as those that occur during affinity maturation are more likely to act cooperatively. In one notable example, iterative rounds of mutations in the Arc repressor homodimer (guided by modeling, NMR, and thermal denaturing studies) showed that interchanging the sequence of a hydrophobic core residue with an adjacent surface polar residue converted an N-terminal β-sheet into a 3₁₀-helix (37).

As in the case of germ-line antibodies, it may be possible that substrate binding induces some of the conformational changes observed in the p-BrPhe and NpAla Mj synthetase structures. However, preliminary low-resolution studies suggest that this structural change is present in the apo forms as well. Also, the WT Mj TyrRS does not show significant backbone changes between the free and Tyr-bound form of the enzyme. High-resolution studies with the apo form of the p-BrPhe and NpAla Mj TyrRSs, as well as other synthetases specific for glycosylated and photoreactive amino acids, may provide additional insight.

The structures of the p-BrPhe, NpAla, and p-AcPhe Mj TyrRSs show that mutation of a few residues within the active site can induce very significant conformational changes in the side chains and the backbone of the enzyme. These changes can alter interactions with amino acid directly, as well as modulate the interactions between the amino acid and other residues. Because these changes are currently difficult to predict, structural characterization of other mutant Mj aminoacyl-tRNA synthetases may reveal unexpected structural motifs that suggest methods to improve and/or expand library design. For example, we have generated a library with a D158P mutation and randomized active-site residues (Y32, H160, and Y161) to take advantage of the scaffold that is present in the p-BrPhe TyrRS and NpAla Mj TyrRS.

Materials and Methods

Expression, Purification, and Crystallization of Mutant Mj TyrRSs.

DNA fragments encoding the p-BrPhe and NpAla TyrRSs were amplified by PCR and cloned into the NdeI/XhoI sites of the expression vector pET22b. This vector was transformed in BL21(DE3) cells (Novagen) and grown to an OD of 0.8–1.0. After induction overnight with 500 mM isopropyl-β-d-thiogalactopyranoside, cells were pelleted by centrifugation and resuspended in lysis buffer [50 mM Hepes, pH 7.9 (Sigma)/500 mM NaCl (Fisher)/10 mM 2-mercaptoethanol (Sigma)/10% glycerol (Sigma)/0.1% Triton X-100 (Sigma)/5 mM imidazole (Sigma)] and frozen overnight at −80°C. After thawing on ice, cells were sonicated, and insoluble cell debris was pelleted by centrifugation. The lysate was incubated with Ni-NTA agarose (Qiagen, Valencia, CA) for 1 h at 4°C, filtered, and washed with wash buffer (50 mM Hepes, pH 7.9/500 mM NaCl/10 mM β-mercaptoethanol/5 mM imidazole). The synthetase was eluted in wash buffer with 250 mM imidazole, dialyzed in buffer A (25 mM Tris, pH 8.5/25 mM NaCl/10 mM 2-mercaptoethanol/1 mM EDTA); purified by anion exchange (MonoQ; Amersham Biosciences) by using buffer A with gradient elution from 25 mM to 1 M NaCl; dialyzed in 20 mM Tris, pH 8.5/50 mM NaCl/10 mM 2-mercaptoethanol; and concentrated to 20–30 mg/ml. Crystals of the mutant TyrRS–amino acid complex were grown at 22°C or 4°C by using the sitting-drop vapor-diffusion technique against a mother liquor composed of 20–16% polyethylene glycol (PEG) 300, 5–3% PEG 8000, 100 mM Tris (pH 8.8–8.2), and 10% glycerol by using a 1:1 mixture of concentrated protein (15 mg/ml) with 2 mM amino acid (p-BrPhe or NpAla) and mother liquor.

Data Collection, Refinement, and Structure Determination.

For the NpAla TyrRS and p-BrPhe Mj TyrRS, data were collected at beamline 5.0.3 of the Advanced Light Source at a temperature of 100 K and a wavelength of 1.0 Å to a maximum Bragg spacing of 1.9 Å. All data were reduced and scaled by using the hkl2000 package (38). The NpAla Mj TyrRS structure was solved by using molrep (39). We used all data with a resolution of 50–4.0 Å, and the WT TyrRS [Protein Data Bank (PDB) ID code 1J1U] (16) was used as a probe model. The p-BrPhe Mj TyrRS structure was solved by rigid body refinement with the NpAla Mj TyrRS. Differences between the WT structure and both the NpAla and p-BrPhe structures were clearly evident in sigmaa weighted electron-density maps (40) at this point. Model-building and refinement were then carried out with o (41) and refmac (42). Automated water-building was carried out with arp/warp (43), and manual inspection of the waters was then performed. All other crystallographic manipulations were carried out with the ccp4 program suite (44). The final model of the p-BrPhe Mj TyrRS had an R_cryst and R_free of 0.21 and 0.25, respectively, whereas the final model of the NpAla Mj TyrRS had an R_cryst and R_free of 0.22 and 0.28, respectively. Both models have no residues in disallowed regions of the Ramachandran plot. Coordinates for the NpAla and p-BrPhe synthetases have been deposited in the PDB (PDB ID codes 1ZH0 and 2AG6, respectively).

Abbreviations

p-BrPhe

p-bromophenylalanine

NpAla

3-(2-naphthyl)alanine

p-AcPhe

p-acetylphenylalanine

TyrRS

tyrosyl aminoacyl-tRNA synthetase

Mj

Methanococcus jannaschii.