Metastable asymmetrical structure of a shaftless V 1 motor

Abstract

V 1 -ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A 3 B 3 complex and a central DF shaft. The nucleotide-free A 3 B 3 complex of Enterococcus hirae, composed of three identical A 1 B 1 heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A 3 B 3 . The nucleotide-free mutant A 3 B 3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A 3 B 3 forms a metastable (stressed) asymmetric structure composed of unstable A 1 B 1 conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.