Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase

• Published in: The Journal of biological chemistry 2023-09, Vol.299 (9), p.105109-105109, Article 105109
• Main Authors: Vaccaro, Francesca A., Faber, Daphne A., Andree, Gisele A., Born, David A., Kang, Gyunghoon, Fonseca, Dallas R., Jost, Marco, Drennan, Catherine L.
• Format: Article
• Language: English
• Subjects: adenosylcobalamin (AdoCbl); Catalytic Domain; chaperone; Cobamides - metabolism; Coenzymes - metabolism; cofactor delivery; cryo-electron microscopy; Cupriavidus - chemistry; Cupriavidus - enzymology; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; GTPase; Guanosine Triphosphate - metabolism; Isomerases - chemistry; Isomerases - metabolism; maturation; metalloenzyme; Methylmalonyl-CoA Mutase - chemistry; Methylmalonyl-CoA Mutase - metabolism; Models, Molecular; Molecular Chaperones - metabolism; Protein Structure, Quaternary; Protein Subunits - chemistry; Protein Subunits - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2023.105109

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(β,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein–assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.