Unique architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Masahiro Naganumaa, Shun-ichi Sekinea,b, Ryuya Fukunagaa,1, and Shigeyuki Yokoyamaa,b,2

aDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; and bRIKEN Systems and Structural Biology Center, Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan

Edited by Paul R. Schimmel, The Scripps Research Institute, La Jolla, CA, and approved April 6, 2009 (received for review February 17, 2009) Alanyl-tRNA synthetase (AlaRS) specifically recognizes the major nants of other aaRS–tRNA pairs. It was also striking that the identity determinant, the G3:U70 , in the acceptor stem of predominant identity determinant of a tRNA exists in the tRNAAla by both the tRNA-recognition and editing domains. In this acceptor–stem duplex, rather than the anticodon and the dis- study, we solved the crystal structures of 2 halves of Archaeoglo- criminator base (9, 10). Actually, AlaRS can aminoacylate small, bus fulgidus AlaRS: AlaRS-⌬C, comprising the aminoacylation, isolated portions of tRNA, such as a ‘‘minihelix’’ and a ‘‘micro- tRNA-recognition, and editing domains, and AlaRS-C, comprising helix,’’ as long as they have the G3:U70 base pair (11). The the dimerization domain. The aminoacylation/tRNA-recognition G3:U70 base pair is considered to be recognized from the minor domains contain an insertion incompatible with the class-specific groove side (12, 13). tRNA-binding mode. The editing domain is fixed tightly via hydro- An E. coli AlaRS fragment comprising the aminoacylation and phobic interactions to the aminoacylation/tRNA-recognition do- tRNA-recognition domains (the N-terminal 461 residues) can mains, on the side opposite from that in threonyl-tRNA synthetase. specifically aminoacylate tRNAAla (1). The crystal structure of A groove formed between the aminoacylation/tRNA-recognition the corresponding fragment (AlaRS-N) from the bacterium domains and the editing domain appears to be an alternative Aquifex aeolicus was reported (14, 15). It revealed that AlaRS tRNA-binding site, which might be used for the aminoacylation does not dimerize through the aminoacylation domain, in con- and/or editing reactions. Actually, the amino acid residues required trast to the other class II aaRSs. The structures of amino acid- for the G3:U70 recognition are mapped in this groove. The dimer- and ATP-bound AlaRS-N revealed how the cognate alanine and ization domain consists of helical and globular subdomains. The the noncognate glycine and serine interact with the aminoacy- helical subdomain mediates dimerization by forming a helix– lation site. AlaRS is one of the aaRSs that use the proofreading loop–helix zipper. The globular subdomain, which is important for mechanism, in that mischarged products, such as Gly-tRNAAla the aminoacylation and editing activities, has a positively-charged and Ser-tRNAAla, are transferred to the editing domain, where face suitable for tRNA binding. the ester bond is hydrolyzed (2). A defect in the AlaRS editing activity causes cell death in the mouse nervous system (16). It was crystal structure ͉ dimerization domain ͉ aminoacyl-tRNA synthetase ͉ recently reported that the E. coli AlaRS editing domain pos- proofreading ͉ wobble base pair sesses a region, distinct from the N-terminal domains, that recognizes the G3:U70 base pair (17). Therefore, AlaRS may Ala minoacyl-tRNA synthetases (aaRSs) catalyze the ligation of transfer the acceptor stem of tRNA from the first binding site Acognate amino acids and tRNAs, and thus establish the in the aminoacylation domain to the second site in the editing genetic code in protein biosynthesis. They are modular domain, in contrast to the other editing aaRSs (classes I and II), composed of an aminoacylation domain and a few additional which have been proposed to shuttle the flexible single-stranded domains for discrete functions, such as tRNA binding, oligomer- CCA terminus of the tRNA between the aminoacylation and ization, and amino acid proofreading (1, 2). The 20 aaRSs are editing catalytic sites (18–22). The C-terminal domain of AlaRS divided into 2 classes, I and II, based on the 2 unrelated types of is not only essential for the oligomerization, but also important BIOCHEMISTRY aminoacylation domains (3, 4). The aminoacylation reaction for the aminoacylation and editing reactions (17, 23). Small occurs at the catalytic site on the aminoacylation domain, and proteins homologous to the AlaRS editing domain, designated the reaction generally consists of 2 steps: the initial activation of AlaX, are found in many organisms (24, 25). They are active in Ala the amino acid with ATP to generate the aminoacyl-adenylate, the trans hydrolysis of misacylated tRNA in vitro (24). The Ala followed by the transfer of the aminoacyl moiety to the 3Ј end crystal structures of AlaX-S (specific to Ser-tRNA ) and Ala Ala of the tRNA. Although the aminoacylation is generally accurate, AlaX-M (specific to Ser-tRNA and Gly-tRNA ) from the several aaRSs cannot completely avoid the misactivation of a archaeon Pyrococcus horikoshii have been reported (26, 27). noncognate amino acid, when it is similar to the cognate one. To The structures of the editing and oligomerization domains, the solve this problem, these aaRSs use a proofreading mechanism, basis of oligomerization, and the domain arrangement in the in which the incorrect products are hydrolyzed at the active site full-length AlaRS have remained elusive. We previously suc- in the editing domain. ceeded in the crystallization of 2 fragments of AlaRS from the Alanyl-tRNA synthetase (AlaRS) is one of the class II aaRSs and consists of 4 domains: the N-terminal class II aminoacylation Author contributions: S.Y. designed research; M.N., S.S., and R.F. performed research; M.N., domain, the tRNA-recognition domain, the editing domain, and S.S., and S.Y. analyzed data; and M.N., S.S., and S.Y. wrote the paper;. the C-terminal oligomerization (dimerization or tetrameriza- The authors declare no conflict of interest. tion) domain (Fig. 1A) (1, 2). AlaRS occupies a special position This article is a PNAS Direct Submission. in the history of aaRS research. Escherichia coli AlaRS was Data deposition: The coordinates and structure factors have been deposited in the Protein among the first aaRSs that were cloned, sequenced, and char- Data Bank, www.pdb.org (PDB ID codes 2ZTG and 2ZVF). acterized genetically and biochemically (1, 5, 6). tRNAAla con- 1Present address: Department of Biochemistry and Molecular Pharmacology, University of serves a unique G3:U70 wobble base pair in the acceptor stem, Massachusetts Medical School, Worcester, MA 01605. and this base pair dictates the tRNA identity toward AlaRS 2To whom correspondence should be addressed. E-mail: [email protected]. (7, 8). This remarkable finding, that a small number of nucleo- u-tokyo.ac.jp. tide residues serve as the predominant determinant for the This article contains supporting information online at www.pnas.org/cgi/content/full/ tRNA identity, accelerated the search for the identity determi- 0901572106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901572106 PNAS ͉ May 26, 2009 ͉ vol. 106 ͉ no. 21 ͉ 8489–8494 Downloaded by guest on October 6, 2021 A Aminoacylation tRNA recognition Editing Oligomerization A B

AlaRS 1 906 AddA1 InsB/E1 AlaRS-ΔC 1 739

AlaRS-C 736 906 InsA1 B Aminoacylation Editing InsB/E2 InsA1 Linker N motif1 AddA1 β barrel InsA2 Mid2 Mid2 A.fulgidus A.aeolicus

Fig. 2. The aminoacylation and tRNA-recognition domains. (A) The amino- acylation and tRNA-recognition domains of A. fulgidus AlaRS, colored as in InsA2 C Fig. 1B, are shown. (B) The A. aeolicus AlaRS-N structure, shown in the same orientation. The 2 regions missing in A. fulgidus (InsB/E1 and InsB/E2) are colored brown, and Mid2 is shown in gold.

Editing core Mid1 AlaRS independently recognize the G3:U70 base pair of Mid2 Helix-loop tRNAAla. tRNA recognition Results C Structure Determination. A. fulgidus AlaRS is a homodimer of 906 C amino acid residue polypeptides (23). It was genetically divided into 2 parts, AlaRS-⌬C (residues 1–739) and AlaRS-C (residues 736–906) (23), and both structures were solved (Table S1). Globular subdomains AlaRS-⌬C is composed of the class-II specific aminoacylation domain, the tRNA-recognition domain, and the editing domain. The structure of AlaRS-⌬C complexed with an alanyl-adenylate C analog, 5Ј-O-[N-(L-alanyl)sulfamoyl] adenosine (Ala-SA), was determined at 2.2 Å (Fig. 1B). The crystallographic asymmetric unit contained 1 AlaRS-⌬C molecule. The refined model has R and Rfree factors of 21.5% and 26.4%, respectively. AlaRS-C comprises the dimerization domain, and its structure was deter- N mined at 3.2 Å (Fig. 1C). Two AlaRS-C molecules form a Helical subdomains homodimer, and there are 4 dimers in the asymmetric unit. The N refinement converged to R and Rfree factors of 20.5% and 27.6%, respectively (Table S1). Fig. 1. The structures of AlaRS-⌬C and AlaRS-C. (A) Domain organizations of A. fulgidus AlaRS, AlaRS-⌬C, and AlaRS-C. Shown are the aminoacylation The Aminoacylation Domain. The A. fulgidus AlaRS-⌬C structure domain (green), Mid1 (blue) and Mid2 (cyan) in the tRNA-recognition domain, revealed the aminoacylation domain (residues 1–257), composed ␤ the -barrel (yellow) and editing-core (orange) subdomains in the editing of a central antiparallel ␤-sheet (␤1–␤8 and ␤10) and 5 ␣-helices domain, and the helical (midnight blue) and globular (light blue) subdomains ␣ ␣ in the dimerization domain. (B) A ribbon representation of AlaRS-⌬C. The ( 1– 5), which is typical of the class-II aaRSs. It is superposable model is colored as in A, and the N-terminal addition (AddA1) and insertions on that of A. aeolicus AlaRS-N, with an rmsd of 1.9 Å for 179 (InsA1 and InsA2) are highlighted in purple and brown, respectively. Motif 1 C␣ atoms. A. fulgidus AlaRS possesses an archaea-specific in the aminoacylation domain (violet), the helix–loop in the editing domain N-terminal extension (AddA1, residues 1–58), including ␣1, ␣2, (salmon), and the linker connecting the tRNA-recognition and editing do- ␤1, and ␤2 (Fig. 2 and Fig. S1A). ␤1 and ␤2 are integrated in the mains (red) are shown. Ala-SA in the aminoacylation site and the editing-site central ␤-sheet, and thus AddA1 is part of the aminoacylation zinc ion are depicted as cpk models. (C) The AlaRS-C dimer is shown as a ribbon domain. Although the bacterial and eukaryal AlaRSs lack model. One molecule of the dimer is colored as in A, and the other molecule AddA1, they instead possess 2 insertions (depicted as InsB/E1 is colored gray. and InsB/E2 in A. aeolicus AlaRS), which occupy the corre- sponding space. A. fulgidus AlaRS also contains an insertion archaeon Archaeoglobus fulgidus, AlaRS-⌬C, comprising the (InsA1, residues 226–232) including ␤9 (Fig. 2 and Fig. S1A). aminoacylation, tRNA-recognition, and editing domains, and Lys-229, at the tip of InsA1, seems to occupy the position of AlaRS-C, comprising the dimerization domain (23). In the Lys-73 in the E. coli enzyme, which cross-links to tRNAAla (28). present study, we determined their crystal structures at 2.2- and A clear electron density corresponding to Ala-SA is visible in 3.2-Å resolutions, respectively. The AlaRS-⌬C structure re- the active-site cleft (Fig. 1B and Fig. S2). The manner of vealed a unique arrangement of the editing domain, relative to interaction with Ala-SA in the aminoacylation active site is the aminoacylation/tRNA-recognition domains, and the ar- described in SI Text. chaea-specific insertions/deletions. The AlaRS-C structure pro- vided the basis of dimerization, via the formation of a helix– The tRNA-Recognition Domain. The ␣-helix-rich middle domain of loop–helix zipper (HLHZ). The structures suggested the domain AlaRS-⌬C (residues 258–484), which is supposed to interact organization in the full-length AlaRS dimer, and thus could with the tRNA acceptor arm, is composed of 11 ␣-helices and a serve as a platform for future analyses of how the aminoacyla- 2-stranded short parallel ␤ sheet (Fig. 1B and Fig. S1A). This tion/tRNA-recognition domains and the editing domain of domain can be divided into 2 subdomains, designated here as

8490 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901572106 Naganuma et al. Downloaded by guest on October 6, 2021 Mid1 (residues 258–419) and Mid2 (residues 420–484). These subdomain structures in A. fulgidus AlaRS are similar to their A counterparts in A. aeolicus AlaRS (14), as revealed by the rmsds of 2.4 and 2.3 Å, respectively. Mid1 tightly contacts the amino- acylation domain by hydrophobic interactions, whereas Mid2 protrudes from the rest of the protein body. The ␣-helix (␣13) connecting Mid1 and Mid2 is continuous, whereas the corre- sponding helix is distorted in the middle in A. aeolicus AlaRS-N. Thus, Mid2 in A. fulgidus AlaRS-⌬C is oriented outward by Ϸ20°, compared with that of A. aeolicus AlaRS-N (Fig. S3). It is further tilted by Ϸ50°, because the last ␣-helix of Mid2 is connected to the editing domain by the linker. In the beginning ThrRS editing domain AlaRS-∆C editing domain of the subdomain, the Mid1, archaeal AlaRSs possess an inser- Ϸ tion of 50 amino acid residues, which is missing in the bacterial Ile670 AlaRSs. In the A. fulgidus AlaRS-⌬C structure, this insertion B (InsA2, residues 277–330) assumes a helix–loop–helix structure Tyr681 (␣8–␣9) and is bent back to form part of the active-site cleft. The Val112 Phe107 presence of this insertion seems to be incompatible with the Arg679 tRNA interaction manner proposed previously for the bacterial Val685 AlaRS (14), as discussed below. Phe678 Tyr84 Trp619 The Editing Domain. The editing domain of A. fulgidus AlaRS (residues 501–737) consists of 2 subdomains, the N-terminal Trp90 Arg89 ␤-barrel subdomain (residues 501–588) and the C-terminal ␣/␤ subdomain (the editing core, residues 589–737), composed of 2 central ␣-helices (␣17 and ␣18, residues 590–614 and 642–657, C respectively) sandwiched by 3- and 4-stranded antiparallel Thr635 ␤-sheets (Fig. 1B and Fig. S1B). The editing domain is super- Asp727 posable on P. horikoshii AlaX-M [ (PDB) ID Asn616 Val356 His617 code 2E1B], with an rmsd of 1.5 Å for 202 C␣ atoms. The ␣/␤ Arg638 P. horikoshii subdomain also superposes well on AlaX-S (PDB Phe637 ID code 1WXO) lacking the ␤-barrel subdomain, with an rmsd Val403 of 1.4 Å for 134 C␣ atoms. A cavity is formed at the subdomain interface. At the Arg367 Asp402 Ile411 Thr407 bottom, His-600, His-604, His-707, and Cys-703 coordinate a Leu414 Glu410 metal ion, which is supposed to be the editing active center (Fig. S4A). The tetrahedral coordination and the intense anomalous peak observed at the metal site suggested that the Fig. 3. The editing domain. (A) The position of the AlaRS editing domain. The structure of AlaRS-⌬C, depicted as a tube model, is superposed on that of metal is a zinc, as in the case of the AlaX proteins. Thr-603, ThrRS by the aminoacylation domain. The AlaRS-⌬C model is colored as in Fig. Gln-620, Gln-682, and Gln-701 constitute the cavity wall. 1B, and ThrRS is colored gray. (B) The interface of the editing and aminoacy- His-600, Thr-603, His-604, Gln-620, His-707, and Cys-703 are lation domains. The helix–loop in the editing domain and motif 1 in the conserved among the AlaRSs. Glu/Gln occupies the position aminoacylation domain are colored salmon and brown, respectively. The corresponding to Gln-701. Gln-620, Gln-682, and Gln-701 residues involved in the interactions are shown as white stick models. (C) correspond to Thr-30, Asp-92, and Asp-114, respectively, in P. The interface of the editing and tRNA-recognition domains. The editing core BIOCHEMISTRY horikoshii AlaX-S, which are involved in interactions with subdomain and Mid1 are colored orange and blue, respectively. serine (Fig. S4B) (27). In AlaX-S, Thr-33 is also involved in the serine interaction, but AlaRS lacks the corresponding residue. The conserved Thr-603 in AlaRS could structurally compen- It is remarkable that the position of the editing domain sate for the absence of this residue. The glycine-rich loop of the relative to the aminoacylation domain differs from those in ␤-barrel subdomain resides at the entrance of the cavity and other class II aaRSs with reported structures. For example, might interact with 3Ј end of the tRNA. compared with E. coli threonyl-tRNA synthetase (ThrRS) (20), the editing domain resides on the opposite side of the ⌬ Position of the Editing Domain. The editing domain of A. fulgidus aminoacylation domain in AlaRS- C (Fig. 3A). The amino- Ϸ AlaRS is connected to the last ␣-helix of the tRNA-recognition acylation active site is 37 Å away from the editing active site Ϸ domain by a 38-Å-long loop, consisting of 16 amino acid in A. fulgidus AlaRS, which is comparable with the 39 Å residues (residues 485–500). The editing domain contacts the distance in ThrRS (20, 29). We previously obtained a 3.7-Å aminoacylation domain to form a hydrophobic core (Fig. 3B). dataset from an AlaRS-⌬C crystal belonging to a different Ile-670, Tyr-681, Phe-678, and Val-685, from a helix–loop space group (23). The structure was solved by molecular structure (residues 669–689) in the editing domain, interact replacement, using the present AlaRS-⌬C structure. The with Tyr-84 in motif 1 (residues 61–88), Trp-90, Phe-107, and position of the editing domain relative to the aminoacylation Val-112 of the aminoacylation domain. Arg-89, Trp-619, and domain and their interface are the same. Arg-679 are stacked. The editing domain also contacts Mid1 In most cases, the dimerization of class II aaRSs is mediated of the tRNA-recognition domain (Fig. 3C). His-617, Thr-635, through motif 1 (30, 31). Nevertheless, A. aeolicus AlaRS-N and Phe-637 in the editing domain and Val-356, Val-403, reportedly does not form a dimer, because the tRNA- Thr-407, Ile-411, and Leu-414 in Mid1 form a hydrophobic recognition domain hinders dimerization through motif 1 (14). core. Asn-616, Arg-638, and Asp-727 in the editing domain Consistent with this finding, A. fulgidus AlaRS-⌬C motif 1 does interact with Asp-402, Glu-410, and Arg-367, respectively, in not mediate dimerization, but interacts with the helix–loop Mid1. structure (residues 668–688) in the editing domain to form an

Naganuma et al. PNAS ͉ May 26, 2009 ͉ vol. 106 ͉ no. 21 ͉ 8491 Downloaded by guest on October 6, 2021 A Ala-SA N N

α α

InsA2

α α 1 2 Arg731

Val460 Mid2 Arg371

Fig. 5. Models of tRNA binding. A tRNA model was created by superposition of the aminoacylation domains of AlaRS-⌬C and the E. coli ThrRS⅐tRNAThr complex, and the acceptor-arm portion of the tRNA (residues 1–7 and 66–76) is shown as a dark-yellow transparent surface model (mode 1). The second tRNA model, bound to an alternative tRNA-binding site, is also depicted as a C C blue surface model (mode 2). The A76 residues in the first and second models are highlighted in yellow and blue, respectively. Amino acid residues impor- B tant for the aminoacylation or editing activity (17, 28, 38, 39, 45, 46) are shown as stick models. Ala-SA in the aminoacylation site and the editing-site zinc ion are depicted as cpk models. A stereo version of this figure is presented as Fig. S6.

␣21 (Pro-762, Leu-765, Pro-766, and Val-769) in 1 protomer also interacts with the C-terminal portion of ␣20 (Ala-754, Ile-757, and Leu-758). The linker mediates the formation of the hydro- phobic core at the ␣20–␣21 junction, where the coiled-coil is twisted (Fig. 4A). Similar HLHZ structures are also present in several transcriptional regulators, such as the Myc protoonco- product and its relatives (32). The C-terminal globular subdomain is composed of a 6-stranded antiparallel ␤-sheet and 3 ␣-helices (Fig. 4B and Fig. S1B). This subdomain tightly packs against ␣21 of the HLHZ to Fig. 4. The C-terminal dimerization domain. (A) The dimer interactions via form a hydrophobic core. Trp-794, Leu-798, and Met-799, in the helical subdomains. An HLHZ formed in the dimer is shown in a stereoview. ␣21, and Val-811, Val-815, Leu-829, Leu-838, and Phe-851, in One molecule is colored midnight blue, and the side chains are shown as red the globular subdomain, are involved in the hydrophobic inter- stick models. The other molecule is colored gray. (B) The globular subdomain actions. One surface of the globular subdomain is positively is shown in a ribbon representation. Basic amino acid residues forming the charged, by the contributions of Lys-855, Arg-859, Arg-863, basic patch are shown as stick models. The conserved Gly-rich segment (870KGSGGGR876) is highlighted in red. Arg-867, Lys-870, Arg-876, and Lys-877. It is interesting that the conserved glycine-rich segment (870KGSGGGR876) forms a ␤-strand that is part of the ␤-sheet (Fig. 4B). A structural interdomain interface. It is interesting to note that AlaX-M, similarity search using the DALI server (33) revealed that the which lacks the helix–loop structure, exists as a monomer in globular subdomain is similar to that of the ssDNA 5Ј-3Ј solution (26). In contrast, the helix–loop mediates the ho- exonuclease RecJ (34) and exopolyphosphatase (35), with high modimerization of AlaX-S (27). Z scores of 11.5 and 9.1, respectively.

The C-Terminal Dimerization Domain. A. fulgidus AlaRS forms a Discussion dimer through an interaction between the C-terminal dimeriza- tRNA Interactions. In the crystal structures of tRNA-bound tion domains of the 2 molecules (23). AlaRS-⌬C, lacking the class-II aaRSs, including ThrRS, seryl-tRNA synthetase, and dimerization domain, exists as a monomer in solution (23). We aspartyl-tRNA synthetase, the amino acid acceptor arm of the first determined the structure of the isolated dimerization tRNA binds to a common site on the class II aminoacylation domain of A. fulgidus AlaRS, AlaRS-C (Fig. 1C). The structure domain (20, 36, 37). The binding site corresponds to the groove revealed that the dimerization domain consists of a long helical formed between Mid1 and Mid2 in the tRNA-recognition subdomain and a globular subdomain. The helical subdomain domain of A. fulgidus AlaRS. However, if the tRNA acceptor contains 2 ␣-helices of 32 and 53 Å and a linker in between. This arm binds to the groove of A. fulgidus AlaRS via the common subdomain exclusively mediates the dimer interaction to form a mode (mode 1), then the nucleotides at positions 1–5 and 68–73 characteristic HLHZ (Fig. 4A and Fig. S5). Val-744, Met-747, have a serious steric clash with the archaea-specific insertion of Leu-750, and Leu-751 in ␣20, and Leu-765, Val-769, Phe-772, a helix–loop–helix (InsA2) within the groove (Fig. 5). To avoid Phe-773, Trp-776, Gln-779, Ile-783, Leu-786, Val-789, Ile-790, the putative clash, a drastic conformational change should occur. Leu-793, and Ile-797 in ␣21, in 1 protomer of the dimer, Because InsA2 interacts with ␣7 and ␣10 to form a hydrophobic respectively, form leucine-zipper-like interactions with their core, the reorientation of InsA2 seems to be unlikely. When the counterparts in the other monomer. The N-terminal portion of tRNA relocates to the other tRNA-binding site for editing, it

8492 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901572106 Naganuma et al. Downloaded by guest on October 6, 2021 should dissociate to move over the protuberant Mid2 subdomain and then rebind. However, an alternative mode (mode 2) is that the tRNA acceptor stem binds to a groove formed between the Mid2 subdomain and the editing domain (‘‘alternative groove’’) of A. fulgidus AlaRS (Fig. 5). In the present structure, the linker connecting Mid2 and the editing domain is located in the alternative groove, but the linker appears to be quite flexible and to change its conformation upon tRNA binding. The alternative groove has entrances to both the aminoacylation and editing active sites, which would facilitate tRNA relocation between them. Consequently, the alternative mode 2 is more likely than the common mode 1 for A. fulgidus AlaRS. For the bacterial AlaRS from A. aeolicus, the tRNA was docked in mode 1 (14). Because A.aeolicus AlaRS lacks the archaea-specific insertion (InsA2), tRNA binding is not hindered. In addition, the acceptor stem could be proximal to Asp-398, corresponding to Ala-409 in E. coli AlaRS, which is thought to be indirectly involved in the G3⅐U70 interaction (38). The aminoacylation and tRNA- recognition domains of A.aeolicus AlaRS were successfully sepa- Fig. 6. A model of the full-length AlaRS dimer. Two copies of AlaRS-⌬C, rated from the editing domain for crystallography (14), whereas the which are correlated by the crystallographic 2-fold axis, and an AlaRS-C dimer, corresponding aminoacylation/tRNA-recognition fragment and are shown. The N termini of AlaRS-C were placed near the C termini of AlaRS-⌬C. The model was colored as in Fig. 1. the editing domain fragment of E. coli AlaRS were both prepared for functional studies (17, 38). In contrast, in the case of the archaeal AlaRS from A. fulgidus, it was difficult to prepare the corresponding tant from the 2-fold axis. In the AlaRS-⌬C crystal structure, the fragments, probably because of the hydrophobic interaction be- editing domain interacts back-to-back with that of the symmetry- tween the aminoacylation/tRNA-recognition domains and the ed- related molecule correlated by the crystallographic 2-fold axis. iting domain (Fig. 3). Therefore, the bacterial AlaRSs might have Met-650, Ile-656, Leu-657, and Met-716 mediate the interaction, the editing domain in a different location from that in the archaeal and the buried surface area is Ϸ400 Å2. In the crystal lattice, the AlaRS, relative to the aminoacylation/tRNA-recognition domains, distance between the editing-domain C-termini is Ϸ19 Å, which thus allowing the tRNA to shift easily between the 2 active sites. In allows their connection to the dimerization-domain N termini E. coli AlaRS, the aminoacylation/tRNA-recognition domains and ⅐ without a large conformation change. Overall, the full-length the editing domain are both able to recognize the G3 U70 base pair AlaRS dimer is likely to assume a butterfly-like structure (Fig. in the tRNA acceptor stem (17), involving Arg-314 on the tRNA- 6). Although this model still requires validation, it could serve as recognition domain and Arg-693 on the editing domain of E. coli a platform for future analyses. AlaRS (17, 39). Intriguingly, in the present A. fulgidus AlaRS structure, Arg-371 (Mid1) and Arg-731, which correspond to the Materials and Methods ⅐ G3 U70 recognition residues Arg-314 and Arg-693, respectively, are Protein Preparation. See SI Text. close to the putative tRNA binding site in the alternative groove (Fig. 5, mode 2). The tRNA-recognition domain including Arg-371 Crystallization and Data Collection. See SI Text. resides on the minor groove side of the acceptor stem, and this binding mode (mode 2) is more preferable for the minor groove Structure Determination and Refinement. The structure of AlaRS-⌬C was solved recognition of the G3⅐U70 base pair than mode 1 (12, 13). There- by the single-wavelength anomalous dispersion method. The Se-site and fore, we cannot exclude the possibility that the bacterial AlaRSs initial phase determinations and solvent flattening were performed with the BIOCHEMISTRY have a similar domain arrangement to that of A. fulgidus AlaRS, and AutoSHARP program (41). All 15 of the Se sites were identified. Density bind tRNA via mode 2. However, the editing domain could interact modification and initial model building using the RESOLVE program placed with the G3⅐U70 base pair in the major groove (Fig. 5). Arg-371 and 51% of the amino acid residues, and the remaining residues were built Arg-731 are too far from each other to simultaneously interact with manually with the COOT program (42, 43). Structure refinement was carried the G3⅐U70 base pair. Our model in mode 2 is compatible with the out with the CNS program (44). A randomly-chosen 5% of the data were set fact that E. coli AlaRS aminoacylates the 3Ј-OH of A76 (40). aside for cross-validation. The refinement included several rounds of simulat- ed-annealing, positional, and individual B factor refinements. The refinement The C-terminal dimerization domain of A. fulgidus AlaRS is converged to final R and Rfree factors of 21.5% and 26.4%, respectively (Table also crucial for the tRNA interaction, because the deletion of the S1). In the Ramachandran plot, 87.4%, 11.9%, and 0.6% of the residues fell in domain dramatically reduces the aminoacylation activity (23). the most favored, additional allowed, and generously allowed regions, re- The A. fulgidus AlaRS-C structure reveals that the globular spectively. No residues were in the disallowed region. subdomain of the dimerization domain possesses a positively- The structure of AlaRS-C was solved by the SAD method with the AutoSHARP charged face, including the conserved Gly-rich segment (Fig. program (41). Of the 56 Se sites, 48 were identified. Model building was per- 4B). The globular subdomain, therefore, is a candidate for the formed manually by using the COOT program (42). Refinement was done with tRNA-binding site, to support aminoacylation reactions. For E. the CNS program, and the R and Rfree factors for the final model are 20.5% and coli AlaRS, the region of residues 808–875 is a nonspecific 27.6%, respectively (Table S1). In the Ramachandran plot, 88.5%, 11.2% and tRNA-binding site (17), which includes the Gly-rich segment. 0.2% of the residues fell in the most favored, additional allowed, and generously allowed regions, respectively. No residues were in the disallowed region. The Full-Length AlaRS Structure. The present structures of ⌬ ACKNOWLEDGMENTS. We thank the staffs of the Photon Factory (Tsukuba, AlaRS- C and AlaRS-C provide clues to infer the full-length Japan) and SPring-8 BL41XU (Hyogo, Japan) beam lines for assistance with our AlaRS structure. The distance between the N termini in the data collection. This work was supported in part by a Ministry of Education, AlaRS-C dimer is only 14 Å, which could restrict the positions Culture, Sports, Science, and Technology Global Centers of Excellence Program of the other domains. Because the editing domain C terminus is (Integrative Life Science Based on the Study of Biosignaling Mechanisms), a Ministry of Education, Culture, Sports, Science, and Technology Grant-in-Aid for connected to the dimerization domain N terminus, the 2 editing Scientific Research, and the Ministry of Education, Culture, Sports, Science and domains in the dimer should be close to each other, whereas the Technology Targeted Proteins Research Program. R.F. was supported by Research aminoacylation and tRNA-recognition domains would be dis- Fellowships from the Japan Society for the Promotion of Science.

Naganuma et al. PNAS ͉ May 26, 2009 ͉ vol. 106 ͉ no. 21 ͉ 8493 Downloaded by guest on October 6, 2021 1. Jasin M, Regan L, Schimmel P (1983) Modular arrangement of functional domains 24. Ahel I, Korencic D, Ibba M, So¨ll D (2003) Trans-editing of mischarged tRNAs. Proc Natl along the sequence of an aminoacyl tRNA synthetase. Nature 306:441–447. Acad Sci USA 100:15422–15427. 2. Beebe K, Ribas De Pouplana L, Schimmel P (2003) Elucidation of tRNA-dependent 25. Schimmel P, Ribas De Pouplana L (2000) Footprints of aminoacyl-tRNA synthetases are editing by a class II tRNA synthetase and significance for cell viability. EMBO J 22:668– everywhere. Trends Biochem Sci 25:207–209. 675. 26. Fukunaga R, Yokoyama S (2007) Structure of the AlaX-M trans-editing enzyme from 3. Cusack S (1995) Eleven down and nine to go. Nat Struct Biol 2:824–831. Pyrococcus horikoshii. Acta Crystallogr D 63:390–400. 4. Eriani G, Delarue M, Poch O, Gangloff J, Moras D (1990) Partition of tRNA synthetases 27. Sokabe M, Okada A, Yao M, Nakashima T, Tanaka I (2005) Molecular basis of alanine into two classes based on mutually exclusive sets of sequence motifs. Nature 347:203– discrimination in editing site. Proc Natl Acad Sci USA 102:11669–11674. 206. 28. Hill K, Schimmel P (1989) Evidence that the 3Ј end of a tRNA binds to a site in the 5. Jasin M, Regan L, Schimmel P (1984) Dispensable pieces of an aminoacyl tRNA syn- adenylate synthesis domain of an aminoacyl-tRNA synthetase. Biochemistry 28:2577– thetase which activate the catalytic site. Cell 36:1089–1095. 2586. 6. Putney SD, et al. (1981) Primary structure of a large aminoacyl-tRNA synthetase. Science 29. Dock-Bregeon AC, et al. (2004) Achieving error-free translation; the mechanism of 213:1497–1501. proofreading of threonyl-tRNA synthetase at atomic resolution. Mol Cell 16:375–386. 7. Hou YM, Schimmel P (1988) A simple structural feature is a major determinant of the 30. Logan DT, Mazauric MH, Kern D, Moras D (1995) Crystal structure of glycyl-tRNA identity of a transfer RNA. Nature 333:140–145. synthetase from Thermus thermophilus. EMBO J 14:4156–4167. 8. McClain WH, Foss K (1988) Changing the identity of a tRNA by introducing a G-U 31. Mosyak L, Reshetnikova L, Goldgur Y, Delarue M, Safro MG (1995) Structure of wobble pair near the 3Ј acceptor end. Science 240:793–796. phenylalanyl-tRNA synthetase from Thermus thermophilus. Nat Struct Biol 2:537–547. 9. Vasil’eva IA, Moor NA (2007) Interaction of aminoacyl-tRNA synthetases with tRNA: 32. Nair SK, Burley SK (2003) X-ray structures of Myc-Max and Mad-Max recognizing DNA. General principles and distinguishing characteristics of the high-molecular-weight Molecular bases of regulation by protooncogenic transcription factors. Cell 112:193– substrate recognition. Biochemistry (Moscow) 72:247–263. 205. 10. Beuning PJ, Musier-Forsyth K (1999) Transfer RNA recognition by aminoacyl-tRNA 33. Holm L, Sander C (1998) Touring protein fold space with Dali/FSSP. Nucleic Acids Res synthetases. Biopolymers 52:1–28. 26:316–319. 11. Francklyn C, Schimmel P (1989) Aminoacylation of RNA minihelices with alanine. 34. Yamagata A, Kakuta Y, Masui R, Fukuyama K (2002) The crystal structure of exonu- Nature 337:478–481. clease RecJ bound to Mn2ϩ ion suggests how its characteristic motifs are involved in 12. Musier-Forsyth K, Schimmel P (1992) Functional contacts of a transfer RNA synthetase exonuclease activity. Proc Natl Acad Sci USA 99:5908–5912. with 2Ј-hydroxyl groups in the RNA minor groove. Nature 357:513–515. 35. Ugochukwu E, Lovering AL, Mather OC, Young TW, White SA (2007) The crystal 13. Musier-Forsyth K, et al. (1991) Specificity for aminoacylation of an RNA helix: An structure of the cytosolic exopolyphosphatase from Saccharomyces cerevisiae reveals unpaired, exocyclic amino group in the minor groove. Science 253:784–786. the basis for substrate specificity. J Mol Biol 371:1007–1021. 14. Swairjo MA, et al. (2004) Alanyl-tRNA synthetase crystal structure and design for 36. Cavarelli J, et al. (1994) The active site of yeast aspartyl-tRNA synthetase: Structural and acceptor-stem recognition. Mol Cell 13:829–841. functional aspects of the aminoacylation reaction. EMBO J 13:327–337. 15. Swairjo MA, Schimmel PR (2005) Breaking sieve for steric exclusion of a noncognate 37. Biou V, Yaremchuk A, Tukalo M, Cusack S (1994) The 2.9-Å crystal structure of T. amino acid from active site of a tRNA synthetase. Proc Natl Acad Sci USA 102:988–993. thermophilus seryl-tRNA synthetase complexed with tRNASer. Science 263:1404–1410. 16. Lee JW, et al. (2006) Editing-defective tRNA synthetase causes protein misfolding and 38. Ho C, Jasin M, Schimmel P (1985) Amino acid replacements that compensate for a large neurodegeneration. Nature 443:50–55. polypeptide deletion in an enzyme. Science 229:389–393. 17. Beebe K, Mock M, Merriman E, Schimmel P (2008) Distinct domains of tRNA synthetase 39. Ribas de Pouplana L, Buechter D, Sardesai NY, Schimmel P (1998) Functional analysis of recognize the same base pair. Nature 451:90–93. peptide motif for RNA microhelix binding suggests new family of RNA-binding do- 18. Fukai S, et al. (2000) Structural basis for double-sieve discrimination of L-valine from mains. EMBO J 17:5449–5457. L-isoleucine and L-threonine by the complex of tRNAVal and valyl-tRNA synthetase. Cell 40. Hecht SM, Chinualt AC (1976) Position of aminoacylation of individual Escherichia coli 103:793–803. and yeast tRNAs. Proc Natl Acad Sci USA 73:405–409. 19. Fukunaga R, Yokoyama S (2005) Aminoacylation complex structures of leucyl-tRNA 41. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta synthetase and tRNALeu reveal two modes of discriminator-base recognition. Nat Crystallogr D 60:2126–2132. Struct Mol Biol 12:915–922. 42. Vonrhein C, Blanc E, Roversi P, Bricogne G (2006) Automated structure solution with 20. Sankaranarayanan R, et al. (1999) The structure of threonyl-tRNA synthetase-tRNAThr autoSHARP. Methods Mol Biol 364:215–230. complex enlightens its repressor activity and reveals an essential zinc ion in the active 43. Terwilliger TC (2000) Maximum-likelihood density modification. Acta Crystallogr D site. Cell 97:371–381. 56:965–972. 21. Silvian LF, Wang J, Steitz TA (1999) Insights into editing from an Ile-tRNA synthetase 44. Brunger AT, et al. (1998) Crystallography and NMR system: A new software suite for structure with tRNAIle and mupirocin. Science 285:1074–1077. macromolecular structure determination. Acta Crystallogr D 54:905–921. 22. Tukalo M, Yaremchuk A, Fukunaga R, Yokoyama S, Cusack S (2005) The crystal structure 45. Davis MW, Buechter DD, Schimmel P (1994) Functional dissection of a predicted of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing con- class-defining motif in a class II tRNA synthetase of unknown structure. Biochemistry formation. Nat Struct Mol Biol 12:923–930. 33:9904–9911. 23. Fukunaga R, Yokoyama S (2007) Crystallization and preliminary X-ray crystallographic 46. Shi JP, Musier-Forsyth K, Schimmel P (1994) Region of a conserved sequence motif in a study of alanyl-tRNA synthetase from the archaeon Archaeoglobus fulgidus. Acta class II tRNA synthetase needed for transfer of an activated amino acid to an RNA Crystallogr F 63:224–228. substrate. Biochemistry 33:5312–5318.

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