Crystal structures of the conserved tRNA-modifying enzyme GidA: implications for its interaction with MnmE and substrate.

TitleCrystal structures of the conserved tRNA-modifying enzyme GidA: implications for its interaction with MnmE and substrate.
Publication TypeJournal Article
Year of Publication2008
AuthorsMeyer, S., A. Scrima, W. Versées, and A. Wittinghofer
JournalJ Mol Biol
Volume380
Issue3
Pagination532-47
Date Published2008 Jul 11
ISSN1089-8638
KeywordsAmino Acid Motifs, Amino Acid Sequence, Bacterial Proteins, Binding Sites, Chlorobium, Crystallography, X-Ray, Dimerization, Escherichia coli, Escherichia coli Proteins, Flavin-Adenine Dinucleotide, GTP Phosphohydrolases, Histidine, Models, Molecular, Molecular Sequence Data, NADP, Protein Binding, Protein Conformation, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Fusion Proteins, RNA, Transfer, Sequence Homology, Amino Acid, Substrate Specificity
Abstract

GidA is a flavin-adenine-dinucleotide (FAD)-binding protein that is conserved among bacteria and eucarya. Together with MnmE, it is involved in the addition of a carboxymethylaminomethyl group to the uridine base in the wobble position (nucleotide 34) of tRNAs that read split codon boxes. Here, we report the crystal structures of the GidA proteins from both Escherichia coli and Chlorobium tepidum. The structures show that the protein can be divided into three domains: a first FAD-binding domain showing the classical Rossmann fold, a second alpha/beta domain inserted between two strands of the Rossmann fold, and an alpha-helical C-terminal domain. The domain inserted into the Rossmann fold displays structural similarity to the nicotinamide-adenine-dinucleotide-(phosphate)-binding domains of phenol hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, and, correspondingly, we show that GidA binds NADH with high specificity as an initial donor of electrons. GidA behaves as a homodimer in solution. As revealed by the crystal structures, homodimerization is mediated via both the FAD-binding domain and the NADH-binding domain. Finally, a large patch of highly conserved, positively charged residues on the surface of GidA leading to the FAD-binding site suggests a tRNA-binding surface. We propose a model for the interaction between GidA and MnmE, which is supported by site-directed mutagenesis. Our data suggest that this interaction is modulated and potentially regulated by the switch function of the G domain of MnmE.

DOI10.1016/j.jmb.2008.04.072
Alternate JournalJ. Mol. Biol.
PubMed ID18565343