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

J Biol Chem. 2023 Sep;299(9):105109. doi: 10.1016/j.jbc.2023.105109. Epub 2023 Jul 28.


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.

Keywords: GTPase; adenosylcobalamin (AdoCbl); chaperone; cofactor delivery; cryo-electron microscopy; maturation; metalloenzyme.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Cobamides* / metabolism
  • Coenzymes / metabolism
  • Cupriavidus / chemistry
  • Cupriavidus / enzymology
  • GTP-Binding Proteins / chemistry
  • GTP-Binding Proteins / metabolism
  • Guanosine Triphosphate / metabolism
  • Isomerases / chemistry
  • Isomerases / metabolism
  • Methylmalonyl-CoA Mutase* / chemistry
  • Methylmalonyl-CoA Mutase* / metabolism
  • Models, Molecular*
  • Molecular Chaperones* / metabolism
  • Protein Structure, Quaternary
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism


  • Cobamides
  • GTP-Binding Proteins
  • Guanosine Triphosphate
  • Isomerases
  • Methylmalonyl-CoA Mutase
  • Molecular Chaperones
  • Protein Subunits
  • Coenzymes