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

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, isobutyrylCoA mutase fused (IcmF) has a G -protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180 degrees 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 Gnucleotide 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'-[(beta,gamma)-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.