title: G-domain dimerization orchestrates the tRNA wobble modification reaction in the MnmE/GidA complex.
abstract: 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.
authors: Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
authors: Simon
authors: S
authors: Meyer
authors: Alfred
authors: A
authors: Wittinghofer
authors: Wim
authors: W
authors: Versées
mesh_terms: 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
chemical_list: 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