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.