title: Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-L-homocysteine.
abstract: The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-L-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-L-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 A resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 A, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central alpha/beta-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.
authors.affiliation: RIKEN Genomic Sciences Center, Yokohama, Japan.
authors.forename: Augen A
authors.initials: AA
authors.lastname: Pioszak
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Murayama
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakagawa
authors.forename: Akio
authors.initials: A
authors.lastname: Ebihara
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003460:Crystallization; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D016963:Thermus thermophilus / Q000201:enzymology*; D014461:Ultracentrifugation; D014529:Uridine / Q000737:chemistry; D012359:tRNA Methyltransferases / Q000737:chemistry*
chemical_list: D012313:RNA; D002244:Carbon; D012435:S-Adenosylhomocysteine; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase; D014529:Uridine