Journal Article

Visualization of a radical B₁₂ enzyme with its G-protein chaperone

Marco Jost, Ruma Banerjee, Catherine L. Drennan, Valentin Cracan, Paul A. Hubbard

2015

Overview

Significance Metalloproteins are ubiquitous, accounting for about 30–50% of all proteins. Their functions are wide-ranging, but metalloproteins are frequently used to carry out challenging molecular transformations. Metalloprotein reactivity comes at a price, however, often requiring specialized molecular machinery for holoenzyme assembly. G-protein metallochaperones are an important part of this assembly apparatus, but an understanding of their molecular mechanisms has been hindered by a lack of structural data. Here, we describe crystal structures of a G-protein metallochaperone together with a target enzyme, in this case an adenosylcobalamin-dependent radical enzyme, thereby providing a visualization of the molecular architecture of the G-protein:target enzyme complex. G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

• Publisher: National Academy of Sciences,National Acad Sciences,National Academy of Sciences, Washington, DC (United States)
• Subject: Apoproteins - chemistry / Apoproteins - metabolism / BASIC BIOLOGICAL SCIENCES / Biological Sciences / Catalytic Domain / Coenzymes - metabolism / Conserved Sequence / crystal structure / crystallography / Crystallography, X-Ray / Cupriavidus - enzymology / G protein / G-proteins / GTP Phosphohydrolases - chemistry / GTP-Binding Proteins - chemistry / GTP-Binding Proteins - metabolism / Guanosine Diphosphate - chemistry / Guanosine Diphosphate - metabolism / metallochaperone / metallocofactor delivery / metalloproteins / Methylmalonyl-CoA Mutase - chemistry / Methylmalonyl-CoA Mutase - metabolism / Mitochondrial Membrane Transport Proteins - chemistry / Models, Molecular / Molecular Chaperones - chemistry / Molecular Chaperones - metabolism / Protein Binding / Protein Structure, Tertiary / Protein Subunits - chemistry / Protein Subunits - metabolism / vitamin B12