The structure of alanyl-tRNA synthetase with editing domain

Abstract

Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNA^Ala and hydrolysis of mis-acylated Ser- and Gly-tRNA^Ala at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned >35 Å from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.

Alanyl-tRNA synthetase (AlaRS) belongs to the class II aminoacyl-tRNA synthetases (aaRSs) and specifically attaches Ala to the 3′-end of the cognate tRNA^Ala (1). AlaRS has an additional catalytic site (termed an editing site), which specifically hydrolyzes a mischarged Ser- or Gly-tRNA^Ala, because the accuracy of discrimination of cognate Ala from noncognate Ser/Gly at the aminoacylation site is not sufficient (2). Because even a mild defect in the editing activity has been shown to cause neural degeneration in mice, such quality control in AlaRS is crucial, especially for higher eukaryotes (3). The protein is made up of 4 functional modules (Fig. S1), which are responsible for aminoacylation catalysis, tRNA recognition, editing, and oligomerization (2, 4). Eubacterial AlaRS form tetramers (5) whereas those in eukaryotes are monomers (4). It has been shown that the C-terminal half, the region responsible for editing and oligomerization, is not necessary for aminoacylation, even though the activity is significantly decreased in its absence (6). The anti-codon stem of tRNA^Ala is also not necessary for aminoacylation, and thus, the acceptor stem provides sufficient information for specific recognition by AlaRS, which is particularly dependent on the G3:U70 base pair known as the second genetic code (7).

A tRNA-binding model has been proposed by superposition of the AspRS-tRNA complex onto the structure of the N-terminal half of Aquifex aeolicus AlaRS (N453). The acceptor stem of the tRNA binds to a concave surface made by the activation domain and the C-terminal helical domains, which approach the G3:U70 base pair from the major groove side (8). In most aaRS harboring an editing domain, the editing site approaches the acceptor stem of the bound tRNA from the side opposite that for aminoacylation so that the 3′-end of the tRNA can be translocated rapidly from 1 site to the other site by simply flipping its CCA tail without significantly moving its body (9). For example, in class II ThrRS, ProRS, and PheRS, the activation domain approaches from the major groove side whereas the editing domain is located at the minor groove side. The situation in AlaRS remains unclear because structures containing either the editing domain or the tRNA have not been reported. However, it has been shown recently that the C-terminal half fragment, which lacks the region essential for aminoacylation, still has considerable editing activity. Surprisingly, this fragment can specifically recognize Ser-tRNA^Ala, at least in part, in a manner dependent on the G3:U70 base pair, suggesting that the editing domain has its own tRNA-binding capability mostly independent of the activation site (10).

Misactivation of noncognate amino acids at the activation site in AlaRS has been studied with the structures of N453 in complex with Ala/Ser/Gly (11). The structures show the accommodation of a Ser hydroxyl by an induced-fit involving the Asn-194′ amide group (hereafter, prime refers to N453), which forms a hydrogen bond with the Ser hydroxyl. Discrimination of Ala- from Ser-tRNA^Ala at the editing site has been studied in our group by determining the structure of an autonomous homolog of the editing domain (AlaXS) in a complex with Ser (12). The structure suggested that the conserved Thr-30 in AlaXS (conserved as Gln-633 in Pyrococcus horikoshii AlaRS) is important for discrimination. Indeed, T30V/Q633M mutants of AlaXS/AlaRS show mis-editing of a cognate Ala-tRNA^Ala (12). The catalytic mechanism of deacylation remains controversial. The structure of the editing domain in ThrRS, which has homology with that in AlaRS, in complex with SerA76 (a nonhydrolyzable analog of the 3′-terminus of Ser-tRNA) suggests that the conserved ₇₃HXXXH₇₇ and ₁₈₂CXXXH₁₈₆ motifs do not bind zinc for deacylation, but rather are directly involved in both recognition and catalysis of the substrate (13, 14). In contrast, in the structure of AlaXS-Ser, the binding of zinc to the motif is apparently concomitant with the binding of Ser (12). Although AlaRS has also been suggested to bind zinc at the editing site (2), whether it is required for the deacylation activity is unclear.

Here, we describe the crystal structures of a C-terminal truncated AlaRS from archaea, which harbors the regions essential for editing as well as for aminoacylation, in its apo form (2.70-Å resolution), in complex with an alanyl-adenylate analog (3.10 Å), and in a high-resolution lysine-methylated form (2.16 Å), showing that the predicted tRNA-binding sites for aminoacylation and editing are far apart in the monomer structure.

Results

Overall Structure.

We have crystallized the N-terminal 752 (out of 915) residues of AlaRS from the archaeon P. horikoshii (N752), which shows both aminoacylation and editing activities (12). We analyzed 3 different crystal forms of N752: the apo form at 2.70-Å resolution (N752-Zn) with R and R_free factors of 20.6 and 26.3%; in complex with the alanyl-adenylate analog AlaSA (5′-O-[N- l-alanyl-sulfamoyl]-adenosine) at 3.10-Å resolution (N752-AlaSA) with R and R_free factors of 20.0 and 27.2%; and a high-resolution lysine-methylated form at 2.16-Å resolution (N752m) with R and R_free factors of 18.9 and 22.7%. The methylated and nonmethylated forms showed essentially the same structure, indicating that the methylation did not perturb the structure significantly. However, in the methylated structure, the Cys-717 side chain, which is thought to bind zinc at the editing site, appears to be oxidized to cysteic acid, probably due to the long incubation for the methylation reaction and thus does not bind zinc. Hereafter, we generally do not distinguish the structures unless otherwise indicated.

The structure consists of 2 regions (Fig. 1A). The N-terminal region (almost corresponding to N453 fragment) is comprised of 3 consecutive domains: the activation domain (AD, residues 1–263), the helical domain N-terminal half (HN, 264–423), and the helical domain C-terminal half (HC, 424–496). HN and HC are separated by Tyr-423 in the middle of the α15 helix, which is kinked as in the N453 structure (8). The C-terminal part is comprised of the β-barrel domain (BD, 504–602) and the editing domain (ED, 603–752). An interdomain loop between these N- and C-terminal regions (497–503) is disordered and could not be modeled, although SDS/PAGE analysis of the N752-Zn crystals clearly showed that the protein molecules in the crystals are not degraded at all. Considering the distance between both ends of the disordered region (≈20 Å for 7 residues), it is also not feasible that the BD-ED region of the adjacent molecules is located at this position. In each crystal form, each molecule does not make significant contact with another molecule, and thus appears to be a monomer. Indeed, gel-filtration analysis showed that N752 is most likely to be monomer in solution (Fig. S2). However, gel-filtration of full-length AlaRS (from the archaeon Sulfolobus tokodaii) showed that it is most likely to be homodimer in solution (Fig. S2), indicating that the extreme C-terminal domain (OD) in archaeal AlaRS is indeed responsible for dimerization. The structures of AD-HN and BD-ED regions are highly similar to corresponding regions in N453 and AlaXM (8, 15), respectively, whereas HC shows a significantly different conformation as compared with N453 (Fig. 2B).

Fig. 1.

Overall structure of N752. (A) Stereo ribbon diagram of N752 structure. AD, HN, HC, BD, and ED are colored blue, red, yellow, orange, and magenta, respectively. The secondary structures are also indicated (see also Fig. S4). The N- and the C-termini are indicated as “N” and “C”, respectively. Motifs 1, 2, and 3 are colored cyan. The archaea- and P. horikoshii-specific insertions are colored green and yellow green, respectively. Zinc atoms in NX and ED are shown as red balls. The disordered loop between HC and BD is shown as dashed lines. (B) Interactions between ED and AD/HN. Color coding is the same as in A. The residues involved in the interactions are shown as stick models, along with hydrogen bonds indicated as dashed lines. Water molecules are shown as red balls.

Fig. 2.

Comparison with N453. (A) Superposition of AD of N752 (blue) and N453 (purple), where NX and the regions in N453 corresponding to the deletions in N752 are colored green and orange, respectively. (B) Comparison of HC of N752 (yellow) and N453 (purple) by superposition of HN. α16′ in N453 is colored as orange.

Interactions Between the Editing Domain and the N-terminal Half.

The C-terminal region interacts with the N-terminal half solely through ED, which interacts with AD and HN from the ventral side of the N-terminal half with a total interface of 2,060 Ų (Fig. 1B and Fig. S3A). The region between β24 and β25 (α22, η7, and the following loop) in ED interacts with the C-terminal part of motif 1 and the following α3-η1 in AD. Therefore, motif 1, which is generally involved in dimerization of the activation domain in class II aaRS, is unlikely to mediate dimerization in AlaRS. The loop regions in the 4-stranded sheet (β22, β23, β27, and β28) in ED form another interface with the N-terminal half of α15 (α15N) and the α11-loop-α12 in HN, which encompasses a HEAT-like motif (8). In addition, Arg-692 in the η7, Trp-632 in the 4-stranded sheet, and Arg-89 in the C terminus of motif 1 form iminoaromatic stacking at the center of these 2 interfaces, and make overall contact to form a continuous interface (Fig. 1B). The domain interface involves an extensive hydrophobic core and a dense hydrogen bond network (some are mediated by water molecules) through residues especially conserved among archaea (Fig. 1B and Fig. S3A), indicating that this interface is fairly stable. Similar conservation is also discernible in a corresponding region in the eubacterial N453 structure (Fig. S3B), suggesting that similar interdomain contact is also likely for eubacterial AlaRS. It has been shown that mutations in Escherichia coli AlaRS in the region corresponding to α3 show defect in the oligomerization state (5). Because these mutations are likely to affect the interaction between ED and AD, the proper orientation of these domains would be important for AlaRS oligomer formation.

Comparison with the Eubacterial AlaRS.

Although the overall structures of AD and HN are highly similar to those of eubacterial N453, N752 has 2 large insertions (the N-terminal extension (NX: residues 1–54), and the α8-α10 insertion between α7 and α11) and 2 large deletions (35 residues between β6 and β7, and 19 residues between β9 and β10 as compared with E. coli AlaRS) within the N-terminal half, all of which are characteristic of archaeal AlaRS (Fig. 1A and Fig. S4). NX is comprised of the α1 helix, 2 anti-parallel strands (β1 and β2) that are part of the central anti-parallel β sheet in AD, and the following 25-residue-long loop (Fig. 2A). Further, NX contains a conserved Cys₄ zinc binding motif (₂₀CXXCG₂₄ between β1 and β2 and ₃₇CGDXPC₄₂ in the long loop), which physically connects the β1-β2 turn and the long loop and thus would be important for the structural stability of the NX region. The binding of a zinc ion was confirmed by X-ray absorption spectroscopy and anomalous difference Fourier map analysis (Fig. S5A). The long loop makes conservative hydrophobic interactions, including Tyr-45, Phe-47, Ile-48, Pro-51, and Ile-53, with a cavity formed by α8 and a loop between β9 and β10, both of which are also characteristic parts of archaeal AlaRS (one is the insertion, and the other is the deletion). Interestingly, the region in N453 corresponding to the deletion between β6 and β7 in N752 consists of α4′ and β7′, which appear to mimic α1 and β2 in NX, although β7′ is in the opposite orientation (Fig. 2A). The α8-α10 insertion is comprised of the 3-helix bundle lying at the concave surface made by AD, HN, and HC, and interacts with the N terminus of α18 in HC, the interdomain loop between AD and HN, the loop between β4 and β5 in motif 2, and the long loop in NX. This insertion shows a relatively low level of sequence conservation (no strictly conserved residues within this region), although the lengths are almost same among species. Further, temperature factors of the α8–α9 region, which interact mainly with motif 2 and α18, are relatively high (≈42 Ų, whereas the total average value is 33 Ų). Together, these 2 archaea-specific insertions wrap around 1 side of the N-terminal half and form extensive bridges across HC to AD (Fig. 1A).

HC in N752 shows a significantly different conformation from that of N453 (Fig. 2B), although it still consists of an α15C-α17 3-helix bundle and an additional α18 helix as in N453 (α12′-α14′ and α15′). Compared with N453, i) the axis of α15C is rotated by ≈30° away from the concave; ii) α15C is rotated by ≈30° around its helix axis; iii) the position of α16–α17 relative to α15C is rotated by ≈20° toward the concave surface around the axis of α15C; and iv) α18 shows a different orientation. Consequently, α17 and the following loop face the concave surface, which is in contrast to N453, in which α12′ and α14′ (α15 and α17 in N752) face the concave surface instead. In fact, HC shows relatively high temperature factors as observed in N453, suggesting that HC has intrinsic flexibility, which was already suggested based on the susceptibility to proteolysis at the kink in α15.

The Activation Site.

The activation site is located at the center of the antiparallel β sheet in AD, and consists of motif 2 (β4 and β5), motif 3 (β13), β7, and β10. AlaSA binds to the pocket in a manner similar to Ala and the AMP moiety of ATP in the N453-Ala/ATP structures, although both moieties in AlaSA come slightly closer to make a covalent bond with each other (Fig. 3A). The residues interacting with AlaSA are spatially well conserved compared with those interacting with Ala and ATP in N453, except Trp-192 (Trp-161′ in N453) that interacts with a carboxyamino ester of AlaSA. In N453-Ala, Trp-161′ is located farther from Ala and interacts indirectly with the amino group by a water-mediated hydrogen bond. In fact, the Trp192s in the absence of AlaSA (N752-Zn and N752m) are less well ordered and show conformational variability among each molecule, suggesting a propensity to fluctuate.

Fig. 3.

The catalytic sites. (A) The activation site of N752-AlaSA. AlaSA and interacting residues are shown as gray and yellow stick models, respectively. Thr-213 and an interacting water molecule in N752m are superposed as green stick and red ball models (marked as “W”), respectively. Hydrogen bonds in N752-AlaSA and N752m are indicated as black and red dashed lines, respectively. The omit map of AlaSA (at 3.1 Å resolution, contoured at 3 σ) is also shown. (B) Comparison of the editing sites of N752-Zn (yellow) and ThrRS-SerA76 complex (light blue). Only the zinc-binding motifs and SerA76 are shown for ThrRS. The water molecule coordinating to zinc in N752-Zn, and the nucleophile in ThrRS-SerA76 are marked as “W” and “N”, respectively. The GGQ loop is colored green. Interactions in N752-Zn and ThrRS-SerA76 are shown as black and blue dashed lines, respectively.

Interestingly, Asn-194′ in N453, which is strictly conserved among bacteria and eukaryote, is replaced by Thr-213 in most archaeal AlaRS (49/51 species investigated, otherwise it is conserved as Asn). In the N453-Ser complex structure, the Asn-194′ side chain exhibits an induced-fit upon Ser binding to form a hydrogen bond with the Ser γ-hydroxyl group, providing the structural basis of Ser misactivation in eubacterial AlaRS. In N752-AlaSA, the β- and γ-carbons of Thr-213 face the amino acid binding pocket and thus offer an aliphatic environment suitable for an Ala side chain. However, in 1 molecule in N752m, the side chain of Thr-213 is flipped ≈120°, and thus its hydroxyl group faces the binding pocket (Fig. 3A). Moreover, this Thr-213 hydroxyl group forms a hydrogen bond with a water molecule positioned near the γ-hydroxyl of the bound Ser in N453-Ser. Therefore, this situation is likely to mimic Ser accommodation in N752, and thus suggests a different mechanism of Ser misactivation in archaeal AlaRS that involves the intrinsic flexibility of the Thr-213 side chain.

The Editing Site.

The editing site harbors the ₆₁₃HXXXH₆₁₇ and ₇₁₇CXXXH₇₂₁ zinc binding motif, which is conserved through AlaRS, AlaX and even ThrRS, whereas the opposite side of the editing site consists of conservative hydrophilic side chains, such as Thr-616, Gln-633, Ser-636, Gln-695, and Gln-715 (Fig. 3B). Again, anomalous difference Fourier map analysis clearly demonstrates the presence of a zinc ion (Fig. S5B), which is tetra-coordinated by His-613, His-617, Cys-717, and 1 water molecule in a tetrahedral geometry. Interestingly, His-721 in the motif does not bind zinc, but forms a hydrogen bond with Cys-717 Sγ instead. A similar coordination was observed in the AlaXM structure (15), although the fourth ligand, a water molecule, was absent in AlaXM probably due to data quality. However, the situation is different in AlaXS, which binds zinc by all 4 residues in the motif in a tetrahedral manner (12). In ThrRS, it has been well established that the motif should not bind zinc but rather is involved directly in the catalysis of deacylation (13, 14), even though a possible zinc-binding capability in the motif has been suggested by atomic absorption spectroscopy and mass spectrometry (13, 16). In N752, the side chain of Gln-555 in the strictly conserved ₅₅₃GGQ₅₅₅ loop in BD, which is absent in ThrRS and AlaXS, approaches the editing site and forms a hydrogen bond with His-613 Nδ in the motif, causing the imidazolium ring to adopt a specific conformation. Again, a similar interaction between the GGQ loop and the zinc binding motif is observed in AlaXM (15), suggesting that this interaction would be important for the specific zinc coordination in AlaRS/AlaXM.

Discussion

Binding of tRNA to the Activation Site.

According to the previous tRNA docking model with N453 (8), the tRNA acceptor stem binds on its major groove side to the concave surface made by AD, HN, and HC, whereas the anticodon stem does not make significant contact with the protein. In the model, motif 2 and α14′ in HC (α17 in N752), which have been suggested to be involved in tRNA binding, approach the major groove side of the third and the fourth base pairs of the modeled tRNA. In archaeal AlaRS, motif 2 is also highly conserved and shares residues important for amino acid transfer [such as Arg-69, Asp-76, and Phe-90 in E. coli AlaRS (17), corresponding to Arg-128, Asp-131, and Phe-145 in N752, respectively (Fig. S4)]. Indeed, the activation site and motif 2 in N752 comprise a continuous conserved surface resembling that in N453 (Fig. 4A and Fig. S3C), suggesting that motif 2 in archaeal AlaRS would also be important for tRNA recognition. However, the α8–α10 insertion in N752 covers motif 2, and thus severely interferes with the acceptor stem of the modeled tRNA (Fig. 4A). Because this insertion has very low sequence conservation among archaea, it is difficult to envisage that the insertion alternatively comprises an archaea-specific tRNA interface (compare the surface around the insertion and motif 2 in Fig. 4 A and B). Thus, it is more likely that the insertion exhibits a conformational change upon tRNA binding (the most plausible direction of the conformational change is shown in Fig. 4A). In fact, the region covering motif 2 (α8–α9) shows relatively high temperature factors, implying its flexibility.

Fig. 4.

The tRNA complexes. (A) tRNA^Asp (orange) was modeled by superposition of AspRS-tRNA complex onto the AD-HN region. Surface representation of N752 without the α8-α10 insertion is shown and was colored by the sequence conservation among archaeal AlaRS using the program ConSurf 3.0 (23): from low to high, cyan, white, and purple. The α8-α10 insertion is shown as a ribbon diagram (green). The proposed direction of conformational change of the α8-α9 region is indicated by a black dashed arrow. (B) tRNA^Thr (blue) is modeled by superposition of ThrRS-tRNA complex onto ED. Surface representation of the whole N752 (including the α8-α10 insertion) is shown as in A. Possible orientation of the CCA tail is shown as blue dashed lines. Proposed orientation of OD is shown as gray dashed circle connecting to the C terminus of N752 by dashed lines. (C) Gel-shift assay. N752 (50 μM) was mixed with tRNA^Ala (100 μM) and then analyzed. The same gel was stained with TB (Left) and then CB after destaining TB (Right). The major and the minor (blurry) complex bands are indicated by orange and blue arrows, respectively. The image was arranged from the single larger gel image.

In N752, the surface of HC seems to be compatible with the modeled tRNA (Fig. 4A). The α17 approaches the minor groove of the T stem, and the following loop and the N terminus of α18 lie along the backbone of the acceptor stem from the major groove side. In the model with N453, HC shows a different structure and thus α14′ (α17 in N752) interacts with tRNA by inserting its hydrophilic side chains into the major groove within a contact distance to the third and the fourth base pairs (8). Although either model is consistent with the significance of HC in tRNA recognition, it is envisaged that HC undergoes conformational change upon tRNA binding when considering its possible flexibility. This possibility will be clarified directly by the structure of the tRNA complex in future studies. Regarding HC, it should be mentioned here that the structure of HC in N453 might involve partial artifacts probably due to the C-terminal truncation at the middle of BD (Fig. S1). In N453, the C-terminal 20 residues comprise α16′ packed into the cleft between HC and HN (Fig. 2B) whereas residues between α15′ and α16′ are disordered. In N752, the corresponding region (residues 509–523) comprises an integral part of BD N terminus (β14–α19), which is ≈50 Å far from where α16′ is in N453 (compare Figs. 1A and 2B). Such a dramatic conformational change seems unfeasible, and thus the truncation in N453 might cause unnatural conformations of α16′ region and hence the interacting HC.

Binding of tRNA to the Editing Site.

Recently, it has been shown that the C-terminal half fragment of E. coli AlaRS is still highly active in deacylation of exogenous Ser-tRNA^Ala and is able to specifically recognize the cognate tRNA^Ala moiety at least in part in a manner dependent on the G3:U70 base pair (10). This study strongly suggested that the C-terminal half has a distinct tRNA binding capability mostly independent from the N-terminal half, which bind tRNA for aminoacylation. The interaction of tRNA with ED has originally been proposed with the structure of AlaXS by its superposition onto the editing domain of the ThrRS-tRNA complex, showing binding of the strand-loop-strand region (corresponding to β27–β28 in N752) around the G3:U70 base pair from the minor groove side (12). An R693K mutation (corresponding to R745K in N752) in this region in the E. coli C-terminal fragment confers relaxed specificity for Ser-tRNA^Thr, suggesting that the previous model with AlaXS is also likely for AlaRS (10). In N752, the region around this strand-loop-strand shows a positively charged concave surface formed by β27, the loop between β23 and α21 in ED, and α15 in HN, which is generally suitable for binding negatively charged nucleic acids. These regions and the editing site also constitute a highly conserved surface (Fig. 4B). A tRNA molecule could reasonably be modeled to this concave surface without severe steric clash by superposition of ThrRS-tRNA (Fig. 4B). In this model, the strand-loop-strand region approaches the first to the fourth base pairs from the minor groove side, as proposed (10, 12). Furthermore, Lys-421, Arg-428, and Arg-429 in the middle region of α15 are adjacent to the backbone phosphates of nucleotides 63–65 in the T arm. A conservative hydrophilic patch, consisting of the N terminus of α12 and the C terminus of α16 in HC (Fig. S3A), is also located near the 5′-terminus of the modeled tRNA. A similar hydrophilic patch (consisting of a10′ and a13′) is also discernible in N453 (Fig. S3B), although it shows a slightly different conformation due to the flexibility of HC. Although these 2 regions in HC are candidates for the tRNA interface, they may have a minor contribution when considering the dispensability of the N-terminal half for editing (10). Recently, elimination of the C-terminal OD was shown to markedly reduce the editing activities of the C-terminal half fragment possibly due to reduced affinity to tRNA (10). It is still unclear whether OD has a tRNA binding activity or the oligomerization per se promotes tRNA binding. In our model, the anticodon stem is oriented toward the solvent and could be within a distance suitable for interaction with OD (Fig. 4B). This implies that the former possibility is conceivable, although it does not exclude the latter possibility. The possible interaction between the anticodon stem and OD is consistent with the previous observation that AlaRS requires a whole L-shaped tRNA for efficient deacylation, unlike aminoacylation (18).

In class II aaRS, such as ThrRS, the editing site is generally adjacent to the activation site so that the CCA tail of the bound tRNA can be flipped from 1 site to the other site without moving its body. However, at least as the N752 fragment, which lacks OD and thus is a monomer, the editing site in AlaRS is located on the opposite side as compared with that of ThrRS with a distance of ≈35 Å (Fig. 4A), and the proposed tRNA binding sites for the 2 reactions are too far for such tRNA translocation (Fig. 4 A and B). Interestingly, in the gel-shift assay, the N752 fragment displayed 2 types of complex band, which can be stained with both Toluidine blue (TB, for nucleic acids) and Coomassie blue (CB, for proteins), when mixed with the excess amount of tRNA (Fig. 4C, major and minor blurry bands). The different mobility of these 2 complexes implies the structural difference among them, which is consistent with the above 2 tRNA binding models. Although the minor band is weak and blurry, it is conceivable that binding of tRNA to the editing site would be weak due to lacking of mischarged amino acid moiety and OD. Considering the strong interaction between ED and the N-terminal half, a dramatic movement of ED also seems to be infeasible. One possibility is that, in the dimer (or tetramer in the case of E. coli AlaRS), the editing site in 1 subunit is closely located to the activation site in the other subunit. To assess this idea, a dimer structure was modeled by the superposition of each AD and ED in 2 N752 molecules onto those in the ThrRS-tRNA, by the structural homology of each domain (Fig. S6A). However, the resulting dimer showed a completely asymmetric structure, which is unusual for a homodimer, even though it did not show severe clash among subunits. Indeed, the C-termini of subunits, which are followed by OD in the full-length protein, are far apart, inconsistent with the OD-mediated dimerization in archaeal AlaRS (Fig. S2). Further, the tetramer model showed serious overlap among subunits (Fig. S6B), suggesting that these types of oligomer are unlikely. Indeed, eukaryotic AlaRS is a monomer in solution (4), implying that oligomerization per se is not necessary for AlaRS function. In the canonical CCA flipping, the body of the tRNA is thought to remain bound to the activation domain even after flipping. In this situation, editing would be significantly dependent on the activation domain, which is not the case in AlaRS (10). Therefore, it is suggested that, at least with regard to CCA flipping, either internal or intersubunit tRNA translocations are unlikely to occur in AlaRS, and thus a misacylated tRNA would have to dissociate from the activation site to rebind the editing site. This idea implies that editing in AlaRS is not efficient as other aaRS, partly rationalizing the conservation of autonomous trans-editing AlaX proteins in many organisms, which would compensate for the editing activity (19).

Editing Reaction.

In ThrRS, it has been well established that the ₇₃HXXXH₇₇ and ₁₈₂CXXXH₁₈₆ motifs in the editing site do not bind zinc but are rather involved directly in the catalysis of deacylation (13, 14). In fact, a presence of zinc inhibited the binding of Ser to the editing site (13). His-73 in the motif (His-613 in N752, which interacts with the GGQ loop) has been shown to serve as a general base for a nucleophilic water molecule whereas Cys-182 (Cys-717 in N752) recognizes the 2′-OH of the ribose (13). Although a similar mechanism is conceivable for AlaRS when considering the functional and sequence homologies, little information has been reported to date regarding the AlaRS editing mechanism. Our structure, along with anomalous difference Fourier analysis, clearly demonstrated the binding of zinc to the editing site in AlaRS (Fig. 3B and Fig. S5B), in which the zinc ion is coordinated by the 3 residues in the zinc-binding motif (His-613, His-617, and Cys-717) and 1 water molecule. This specific coordination seems to be facilitated by the interaction between the coordinating His-613 and Gln-555 in the GGQ loop, which is conserved among AlaRS and AlaXM but not in ThrRS. The presence of the coordinating water molecule suggests that this zinc has a functional rather than a structural role, in which zinc coordination is generally completed by 4 protein residues. However, when we compared the deacylation activities against Ser-tRNA^Ala in the presence and absence of zinc, the results were ambiguous. Because the protein was purified in buffer containing zinc, it likely already contained zinc. Indeed, the addition of zinc (10 μM) did not affect the deacylation rate significantly as compared with that in its absence (90 ± 5.8%). However, elimination of zinc by 10 mM EDTA resulted in only a slight reduction of the deacylation rate to 73 ± 15%. Similar ambiguity of zinc requirement has also been observed for AlaXS (12), although it binds zinc by all 4 residues in the zinc-binding motif. However, in clearly contrast to ThrRS, zinc binding did not interfere with deacylation at all in AlaRS, implying that the mechanism may be different.

Although the zinc requirement is ambiguous, it would be interesting to discuss the possible roles of zinc in AlaRS editing. One possibility might be that zinc mediates hydrolysis, in which an activated water molecule coordinating to zinc serves as a nucleophile. However, we could not identify any candidate as a proton acceptor around the coordinated water molecule, which is generally required for activation of the water molecule in zinc-mediated hydrolases. Another possibility is recognition of the substrate directly by the zinc ion. In the ThrRS activation site, a zinc ion, which is tetra-coordinated by 3 protein residues and 1 water molecule in the absence of a substrate, is penta-coordinated by a side chain hydroxyl and an amino groups of a bound Thr (or Ser) moiety in addition to the 3 residues already coordinating to zinc (20). This specific recognition of γ-hydroxyl and α-amino groups by zinc logically exclude γ-methyl of isosteric Val from the activation site. The situation in the AlaRS editing site is reminiscent of this situation; zinc is coordinated by 3 protein residues and 1 water molecule in the absence of the substrate, and the editing site is thought to recognize Ser but not Ala. Although the side chain of Ala is shorter than that of Val, the β-methyl of Ala would still be within the distance conflicting with zinc when its amino group is coordinated. In this context, Gly would not be rejected because of the absence of the side chain. Interestingly, mutation of Gln-633 to a hydrophobic residue (Q633M) resulted in relaxed specificity to the cognate Ala-tRNA^Ala (12). Because the Gln-633 side chain is located near the coordinating water molecule with a distance of 4.5 Å (Fig. 3B), this residue, and possibly the surrounding hydrophilic residues, may provide an unfavorable environment for the Ala β-methyl. These 2 possibilities of the role of zinc will be confirmed by the structural analysis of SerA76 complex in future studies.

Materials and Methods

Full Details of the Methods Used Are Provided in SI Text.

The gene encoding N752 was expressed in E. coli, and purified by heat treatment followed by cation-exchange and size-exclusion chromatography. The C717A mutant was prepared in a manner similar to the wild type. The C-terminal His-tagged full-length AlaRS from S. tokodaii was expressed in E. coli, and purified by heat treatment followed by Ni-NTA affinity, size-exclusion, and heparin-affinity chromatography. Gel-filtration analysis was performed with a Superdex200 10/300 GL column (GE Healthcare).

For N752m crystals, the purified N752 was subjected to a reductive-methylation procedure targeting its lysine residues (21). Both methylated and nonmethylated N752 proteins were crystallized by the hanging drop vapor diffusion method at 20 °C. The N752-Zn crystals were grown in buffer containing 0.1 M Mes-Na, pH 5.6, 6% PEG6000, 108 mM succinic acid, and 6% glucose using 9 mg/mL protein. The N752-AlaSA crystals were grown under the same condition with buffer containing 1 mM AlaSA. The N752m crystals were grown with 0.1 M tri-sodium citrate dihydrate, pH 5.6, 0.2 M ammonium acetate, and 30% PEG4000, using 5 mg/mL protein.

The X-ray diffraction data were collected at beamlines BL-5A of Photon Factory and BL41XU of SPring-8 at 100 K. The N752-Zn crystals belong to the space group C2 with 1 molecule in an asymmetric unit. The N752-AlaSA crystals belong to the space group P1 with 2 molecules in an asymmetric unit. The N752m crystals belong to the space group P2₁ with 2 molecules in an asymmetric unit. The structure was solved by molecular replacement with the sequential search of N453 and AlaXS as models, yielding 2 sets of unique solutions for the N752m dataset. The structure was refined with program LAFIRE (22) and subsequent cycles of manual fitting and restrained refinement with TLS parameters. The N752-Zn and N752-AlaSA crystals showed high Wilson B-factors (85 Ų and 80 Ų, respectively), which would be a major reason for the relatively high R_free values in these structures. The statistics are summarized in Table S1.

In gel-shift assay, N752 (50 μM) was mixed with tRNA^Ala (100 μM) in 50 mM Tris-borate, pH 7.0, and 2 mM MgOAc₂, and incubated at 4 °C for 1 h. Then, 10% glycerol was added, and aliquots of 20-μL samples were analyzed by 5% polyacrylamide gel electrophoresis in the same buffer at 4 °C. The gel was stained with TB, completely destained, and then stained again with CB.

Deacylation assay was performed essentially as described in ref. 12. ³H Labeled Ser-tRNA^Ala (2.5 μM) was incubated with 200 nM N752 at 55 °C. To eliminate zinc, the purified protein was diluted extensively with buffer containing 10 mM EDTA, and then concentrated. Under the conditions with addition of zinc, the reaction mixture was brought to 10 μM ZnOAc₂. The values were calculated from the averages of 2 independent experiments.