Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy

Simon Meyer,Johann P Klare,André Krüger,Heinz-Jürgen Steinhoff,Sabine Böhme,Alfred Wittinghofer,Gregory A Petsko

doi:10.1371/journal.pbio.1000212

Abstract

Author SummaryMnmE is an evolutionary conserved G protein that is involved in modification of the wobble U position of certain tRNAs to suppress translational wobbling. Despite high homology between its G domain and the small G protein Ras, MnmE displays entirely different regulatory properties to that of many molecular switch-type G proteins of the Ras superfamily, as its GTPase is activated by nucleotide-dependent homodimerization across the nucleotide-binding site. Here we explore the unusual G domain cycle of the MnmE protein by combining X-ray crystallography with pulse electron paramagnetic resonance (EPR) spectroscopy, which enables distance determinations between spin markers introduced at specific sites within the G domain. We determined the structures of the full-length MnmE dimer in the diphosphate and triphosphate states, which represent distinct steps of the G domain cycle, and demonstrate that the G domain cycle of MnmE comprises large conformational changes and domain movements of up to 18 Å, in which the G domains of the dimeric protein traverse from a GDP-bound open state through an open/closed equilibrium in the triphosphate state to a closed conformation in the transition state, so as to assemble the catalytic machinery.

Highlights

Cells devote substantial biosynthetic effort and resources to posttranscriptional modification of tRNAs [1]
We explore the unusual G domain cycle of the MnmE protein by combining X-ray crystallography with pulse electron paramagnetic resonance (EPR) spectroscopy, which enables distance determinations between spin markers introduced at specific sites within the G domain
We determined the structures of the full-length MnmE dimer in the diphosphate and triphosphate states, which represent distinct steps of the G domain cycle, and demonstrate that the G domain cycle of MnmE comprises large conformational changes and domain movements of up to 18 A, in which the G domains of the dimeric protein traverse from a GDP-bound open state through an open/closed equilibrium in the triphosphate state to a closed conformation in the transition state, so as to assemble the catalytic machinery

Summary

Introduction

Cells devote substantial biosynthetic effort and resources to posttranscriptional modification of tRNAs [1]. MnmE is an evolutionary conserved G protein found in bacteria, fungi, and humans, which together with the protein GidA catalyzes the formation of a carboxymethylaminomethyl-group (cmnm) at the 5 position of the wobble uridine (U34) of tRNAs reading 2-fold degenerated codons ending with A or G, i.e., tRNAArg(UCU), tRNAGln(UUG), tRNAGlu(UUC), tRNALeu(UAA), and tRNALys(UUU) [4,5,6] This modification (cmnm5U34) together with a thiolation at the 2 position favours the interaction with A and G, but suppresses basepairing with C and U [3,7,8,9,10]. Eucaryotic homologues of MnmE and GidA (termed MSS1 and Mto, respectively, in yeast) are targeted to mitochondria [12,13], and the human homologues (termed hGTPBP3 and Mto, respectively) have been implicated in the development of severe mitochondrial myopathies such as MERRF (myoclenic epilepsy ragged red fibres), MELAS (mitochondrial encephalomyopathy lactic acidosis stroke), and nonsyndromic deafness [14,15,16,17,18]

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