G-domain dimerization orchestrates the tRNA wobble modification reaction in the MnmE/GidA complex.
MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation between MnmE and GidA involves large conformational changes that induce G-domain dimerization of MmnE and that GidA co-stimulates GTP hydrolysis on MnmE. Starting from a structural model of the complex, we identify interface mutations disrupting complex formation or communication. Although GidA does not directly contact the G-domains, conformational changes in MnmE, induced by G-domain dimerization in the triphosphate state, regulate the affinity for GidA. We developed a tRNA modification assay and demonstrate for the first time in vitro that the MnmE/GidA complex catalyzes incorporation of glycine into tRNA. An intact MnmE/GidA complex rather than their sequential action is crucial for in vitro modification. Since only GTP, but not GDP or non-hydrolyzable GTP analogs, drives the MnmE/GidA-catalyzed modification reaction, we conclude that GTP hydrolysis is essential for activity. We finally show that an active GTPase, an intact MnmE/GidA communication, and dimerization of G-domains are necessary for in vivo functioning since mutations disrupting either result in a respiratory deficient phenotype in yeast.
Meyer
Simon
S
Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Wittinghofer
Alfred
A
Versées
Wim
W
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D012343:RNA, Transfer / Q000378:metabolism*; D015003:Yeasts
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; C108512:glucose-inhibited division protein A, bacteria; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D020558:GTP Phosphohydrolases; C492932:MnmE protein, E coli