Dimerisation-dependent GTPase reaction of MnmE: how potassium acts as GTPase-activating element.
MnmE, a Guanine nucleotide-binding protein conserved between bacteria and man, is involved in the modification of tRNAs. Here we provide biochemical and X-ray structural evidence for a new GTP-hydrolysis mechanism, where the G-domains of MnmE dimerise in a potassium-dependent manner and induce GTP hydrolysis. The structure in the presence of GDP-AlFx and potassium shows how juxtaposition of the subunits induces a conformational change around the nucleotide which reorients the catalytic machinery. A critical glutamate is positioned such as to stabilise or activate the attacking water. Potassium provides a positive charge into the catalytic site in a position analogous to the arginine finger in the Ras-RasGAP system. Mutational studies show that potassium-dependent dimerisation and GTP hydrolysis can be uncoupled and that interaction between the G-domains is a prerequisite for subsequent phosphoryl transfer. We propose a model for the juxtaposition of G-domains in the full-length protein and how it induces conformational changes in the putative tRNA-modification centre.
Scrima
Andrea
A
Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
Wittinghofer
Alfred
A
D017607:Aluminum Compounds / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005459:Fluorides / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D020836:Protein Structure, Quaternary
D017607:Aluminum Compounds; D029968:Escherichia coli Proteins; D009711:Nucleotides; D020558:GTP Phosphohydrolases; C492932:MnmE protein, E coli; D005459:Fluorides; D011188:Potassium; C032311:aluminum fluoride