title: Autoinhibition and signaling by the switch II motif in the G-protein chaperone of a radical B12 enzyme.
abstract: MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5'-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlFx(-) to form the putative transition state analog, GDP·AlF4(-). The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.
authors.affiliation: From the Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0600 and.
authors.forename: Michael
authors.initials: M
authors.lastname: Lofgren
authors.forename: Markos
authors.initials: M
authors.lastname: Koutmos
authors.forename: Ruma
authors.initials: R
authors.lastname: Banerjee
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006153:Guanosine Diphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008765:Methylmalonyl-CoA Mutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020601:Methylobacterium extorquens / Q000201:enzymology* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D014805:Vitamin B 12 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D006153:Guanosine Diphosphate; D019883:Alkyl and Aryl Transferases; D008765:Methylmalonyl-CoA Mutase; D014805:Vitamin B 12
keywords: Adenosylcobalamin; Crystal Structure; G-proteins; Signaling; Small GTPases