High resolution structures of rat brain V-ATPase with concanamycin.

Abstract

Vacuolar-type ATPase (V-ATPase) is a rotary proton pump that acidifies intracellular organelles and is an important therapeutic target. Concanamycin A is a potent macrolide inhibitor of V-ATPase, but the structural basis of its strong inhibitory activity has remained unclear. Here, we determined high-resolution cryo-EM structures of rat brain V-ATPase in apo, concanamycin-bound, and BafA1-bound states. Concanamycin occupies multiple binding pockets within the c-ring and additionally binds at the a-c interface, where its extended tail interacts with both stator and rotor components of the proton pump. These interactions provide a structural explanation for its enhanced potency. In addition, we identified macrolide occupancy at the c9-c″ interface in both concanamycin- and BafA1-bound V-ATPase structures, leading to a revised model of inhibitor binding in mammalian V-ATPase. Furthermore, an in situ structure of concanamycin-bound V-ATPase in synaptic vesicles revealed conserved binding modes but altered inhibitor occupancy, demonstrating that membrane context can influence drug binding.

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