Metastable asymmetrical structure of a shaftless V₁ motor.

Abstract

V₁-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A₃B₃ complex and a central DF shaft. The nucleotide-free A₃B₃ complex of <i>Enterococcus hirae</i>, composed of three identical A₁B₁ 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 A₃B₃. The nucleotide-free mutant A₃B₃ 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 A₃B₃ forms a metastable (stressed) asymmetric structure composed of unstable A₁B₁ 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.