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Structural Basis for Methyl-Donor–Dependent and Sequence-Specific

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Structural Basis for Methyl-Donor–Dependent and Sequence-Specific Structural basis for methyl-donor–dependent and PNAS PLUS sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD Takuhiro Itoa,b,c, Isao Masudad, Ken-ichi Yoshidaa,b, Sakurako Goto-Itoa,b, Shun-ichi Sekinea,b,c, Se Won Suhe, Ya-Ming Houd, and Shigeyuki Yokoyamaa,b,f,1 aRIKEN Systems and Structural Biology Center, Tsurumi-ku, Yokohama 230-0045, Japan; bGraduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; cDivision of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Tsurumi-ku, Yokohama 230-0045, Japan; dDepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107; eDepartment of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea; and fRIKEN Structural Biology Laboratory, Tsurumi-ku, Yokohama 230-0045, Japan Edited by Joseph D. Puglisi, Stanford University School of Medicine, Stanford, CA, and approved June 19, 2015 (received for review December 10, 2014) The deep trefoil knot architecture is unique to the SpoU and tRNA the N1 position of Ψ1191 in the 18S rRNA (14, 15), but the im- methyltransferase D (TrmD) (SPOUT) family of methyltransferases portance of Nep1 actually resides in its chaperone function, (MTases) in all three domains of life. In bacteria, TrmD catalyzes the rather than its methylation function. The TrmD and Nep1 di- N1-methylguanosine (m1G) modification at position 37 in transfer mers share quite similar SPOUT domain structures (Fig. S1B), 36 37 RNAs (tRNAs) with the GG sequence, using S-adenosyl-L-methio- but they exhibit largely different intersubunit orientations with 1 nine (AdoMet) as the methyl donor. The m G37-modified tRNA func- distinct elements added to the SPOUT domain (colored blue in tions properly to prevent +1 frameshift errors on the ribosome. Here Fig. S1B). Therefore, it is intriguing to investigate how AdoMet we report the crystal structure of the TrmD homodimer in complex binding may affect substrate RNA binding by TrmD. with a substrate tRNA and an AdoMet analog. Our structural analysis Here, we report the crystal structures of TrmD in complex revealed the mechanism by which TrmD binds the substrate tRNA in with an AdoMet analog, sinefungin (Fig. 1A), in the presence of an AdoMet-dependent manner. The trefoil-knot center, which is struc- wild-type and variant tRNA substrates. These structures revealed BIOCHEMISTRY turally conserved among SPOUT MTases, accommodates the adenosine that the TrmD-specific structural features are the major de- moiety of AdoMet by loosening/retightening of the knot. The TrmD- terminants of tRNA binding and recognition. Furthermore, we specific regions surrounding the trefoil knot recognize the methionine performed structure-guided kinetic analyses of TrmD mutants moiety of AdoMet, and thereby establish the entire TrmD structure for and tRNA variants. Based on these results, we have elucidated global interactions with tRNA and sequential and specific accommoda- tions of G37 and G36, resulting in the synthesis of m1G37-tRNA. the mechanism by which the SPOUT MTase TrmD captures AdoMet in the deep trefoil knot fold and ensures the subsequent 1 36 37 RNA modification | SPOUT methyltransferase | TrmD | X-ray crystallography m G37 methylation of GG -containing tRNAs. Results and Discussion t is highly exceptional for proteins to have a knot in their Structure Determination and Overall Structures. We purified the TrmD Ifolding patterns. Actually, all of the proteins found to possess a proteins from two species, Thermotoga maritima and Haemophilus deep trefoil knot (1–5) belong to a single family of RNA methyl- influenzae, to crystallize in the complex with the T. maritima transferases (MTases), named SPOUT after SpoU (currently called TrmH) and tRNA methyltransferase D (TrmD) (6). SPOUT MTases methylate the base or ribose moiety of ribosomal RNA Significance (rRNA) or transfer RNA (tRNA) in all three domains of life. The deep trefoil knot of the SPOUT MTases provides the binding site In bacterial tRNAs with the 36GG37 sequence, where positions for the methyl donor, S-adenosyl-L-methionine (AdoMet). The 36 and 37 are, respectively, the third letter of the antico- SPOUT MTase transfers the methyl moiety of AdoMet onto its don and 3′ adjacent to the anticodon, the modification of substrate RNA, and produces the methylated RNA and S-adeno- N1-methylguanosine (m1G) at position 37 prevents +1 frame- 1 syl-L-homocysteine (AdoHcy). SPOUT MTases usually function as shifts on the ribosome. The m G37 modification is introduced homodimers, although as an exception, yeast Trm10 functions as by the enzyme TrmD, which harbors a deep trefoil knot within a monomer (7). In the homodimeric SPOUT MTases, the helix the S-adenosyl-L-methionine (AdoMet)-binding site. We de- located at the carboxyl terminus of the deep trefoil knot is in- termined the crystal structure of the TrmD homodimer in com- volved in the dimerization (1–5). plex with a substrate tRNA and an AdoMet analog. The structure TrmD, the product of the trmD gene, is one of the broadly revealed how TrmD, upon AdoMet binding in the trefoil knot, conserved SPOUT MTases in bacteria. It is responsible for the obtains the ability to bind the substrate tRNA, and interacts with methylation at the N1 position of G37 in tRNA, in cases where G37 and G36 sequentially to transfer the methyl moiety of 1 the third anticodon letter, 5′ adjacent to G37, is also guanosine, AdoMet to the N position of G37. G36 (Fig. 1A). The modified nucleotide N1-methylguanosine at 1 Author contributions: T.I., S.W.S., Y.-M.H., and S.Y. designed research; T.I., I.M., K.-i.Y., position 37 (m G37) is present in all three domains of life. Re- S.G.-I., S.-i.S., Y.-M.H., and S.Y. performed research; T.I., I.M., K.-i.Y., S.G.-I., S.-i.S., Y.-M.H., markably, mutations in trmD frequently result in growth defects, and S.Y. analyzed data; and T.I., S.W.S., Y.-M.H., and S.Y. wrote the paper. associated with increased +1 frameshift errors (8–12). Each TrmD The authors declare no conflict of interest. monomer consists of two globular domains, the N-terminal do- This article is a PNAS Direct Submission. “ main (NTD), which harbors the SPOUT fold, called the SPOUT Freely available online through the PNAS open access option. ” domain, and the TrmD-specific C-terminal domain (CTD) (Fig. Data deposition: The atomic coordinates and structure factors have been deposited in the S1A) (3, 5). Protein Data Bank, www.pdb.org (PDB ID codes 4YVG, 4YVH, 4YVI, 4YVJ, and 4YVK). So far, only one SPOUT MTase in complex with a substrate RNA 1To whom correspondence should be addressed. Email: [email protected]. • has been crystallized: the Saccharomyces cerevisiae Nep1 RNA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex (Fig. S1B) (13). Nep1 is the SPOUT MTase specific to 1073/pnas.1422981112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1422981112 PNAS | Published online July 16, 2015 | E4197–E4205 Downloaded by guest on September 29, 2021 ABacceptor NH2 NH2 sinefungin branch subunit A subunit B CH N N 3 N NH2 N - + - tRNA OOC S OOC N N N N + O + O NH3 NH3 OH OH OH OH AdoMet sinefungin anticodon O O branch H N H3C N N TrmD N G37 G36 H H TrmD H H N N N N N N 90° H sugar H sugar G37 NH2 m1G37 N subunit A N - OOC S N N + O NH3 OH OH AdoHcy subunit B C Glu80 Glu80 Phe155 Phe155 Glu80 Phe155 Met1 Ser246 Fig. 1. Crystal structure of the TrmD•sinefungin•tRNA complex. (A) Schematic representation of the synthesis of m1G37-tRNA catalyzed by TrmD. The chemical structures of AdoMet, AdoHcy, and sinefungin are also shown. (B) Ribbon representation of the crystal structure of the ternary complex of TrmD, sinefungin, and tRNA. The TrmD subunits A and B are colored green and salmon, respectively. The acceptor and anticodon branches of tRNA are colored brown and yellow, respectively. G36 and G37 in the tRNA are represented by stick models. The sinefungin molecules in the AdoMet-binding sites of the TrmD subunits A and B are represented by CPK models, colored light green and light salmon, respectively. (C) The backbone wire model of the deep trefoil knot structure of subunit A in the tRNA•sinefungin-bound form of TrmD. The backbone model of the residues from Glu80 to Phe155 is presented in a stereoview, from the same angle as the lower panel of B. The color of the residues from Glu80 to Ala153 gradually changes from yellow to blue, to clarify the relationship between the positions in the primary structure and the knotted part in the tertiary structure, as in the bar indicated below. The bound sinefungin is rep- resented by a stick model, and its simulated-annealing omit map, contoured at 3σ, is illustrated on the model. Gln Gln tRNACUG transcript and sinefungin. T. maritima tRNACUG pos- The crystal structures of the AdoMet-bound and sinefungin- sesses the 36GG37 sequence, which is necessary for recognition bound forms of TrmD are almost the same. The asymmetric unit by TrmD. We successfully crystallized the ternary complex of contains one TrmD subunit bound to one AdoMet or sinefungin Gln molecule, and a homodimer is formed between the symmetry- H. influenzae TrmD, the T. maritima tRNACUG transcript, and sinefungin. Actually, the structure of H. influenzae TrmD was related subunits (Figs. S1A, S3B,andS4A). H. influenzae TrmD – – previously well characterized (3), and became the basis for the consists of the NTD (residues 1 160) and the CTD (residues 169 – further structural characterization.
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