Open AccessStructures of a putative RNA 5‐methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S ‐adenosyl‐ l ‐homocysteine
Author(s) -
Pioszak Augen A.,
Ebihara Akio,
Murayama Kazutaka,
Nakagawa Noriko,
Kuramitsu Seiki,
Shirouzu Mikako,
Yokoyama Shigeyuki
Publication year - 2005
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309105029842
Subject(s) - thermus thermophilus , methyltransferase , rna , chemistry , biochemistry , thermus , stereochemistry , enzyme , methylation , dna , gene , thermophile , escherichia coli
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 Å resolution. The SeMet‐ApoII and Native‐AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å, 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 α/β‐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.