Atsuko Nakanishi

Molecular Basis of ADP Inhibition of Vacuolar (V)-type ATPase/Synthase

Background: ADP inhibition of rotary ATPases is a common mechanism to avoid wasteful ATP hydrolysis. Results: Domain swap approaches in V1 showed that domain interaction plays a key role in sensitivity of ADP inhibition. Conclusion: Increasing the affinity of V1 for phosphate correlates with reducing sensitivity to ADP inhibition. Significance: The molecular basis of ADP inhibition of V0V1 is clarified. Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis.

F-subunit reinforces torque generation in V-ATPase.

Vacuolar-type H+-pumping ATPases (V-ATPases) perform remarkably diverse functions in eukaryotic organisms. They are present in the membranes of many organelles and regulate the pH of several intracellular compartments. A family of V-ATPases is also present in the plasma membranes of some bacteria. Such V-ATPases function as ATP-synthases. Each V-ATPase is composed of a water-soluble domain (V1) and a membrane-embedded domain (Vo). The ATP-driven rotary unit, V

General anesthetics cause mitochondrial dysfunction and reduction of intracellular ATP levels

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Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus

1, is composed of A, B, D, and F subunits. The rotary shaft (the DF subcomplex) rotates in the central cavity of the A3B3-ring (the catalytic hexamer ring). The D-subunit, which has a coiled-coil domain, penetrates into the ring, while the F-subunit is a globular-shaped domain protruding from the ring. The minimal ATP-driven rotary unit of V

Single particle analysis of V-type ATP synthase from Thermus thermophilus by cryo-EM

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Rotation of xenogeneic subunits in rotary motor proteins

1 is comprised of the A3B3D subunits, and we therefore investigated how the absence of the globular-shaped F-subunit affects the rotary torque generation of V