Abstract

G-proteins function as molecular switches to power cofactor translocation and confer fidelity in metal trafficking. MMAA, a G-protein motor, together with MMAB, an adenosyltransferase, orchestrate cofactor delivery and repair of B12-dependent human methylmalonyl-CoA mutase (MMUT). The mechanism by which the motor assembles and moves a >1300 Da cargo, or fails in disease, are poorly understood. Herein, we report the crystal structure of the human MMUT-MMAA nanomotor assembly, which reveals a dramatic 180{degrees} rotation of the B12 domain, exposing it to solvent. The nanomotor complex, stabilized by MMAA wedging between two MMUT domains, leads to ordering of the switch I and III loops, revealing the molecular basis of mutase-dependent GTPase activation. The structure explains the biochemical penalties incurred by methylmalonic aciduria-causing mutations that reside at the newly identified MMAA-MMUT interfaces.