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PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism

S. Muench,S. Rawson,V. Eyraud,A. Delmas,P. D. da Silva,C. Phillips,J. Trinick,M. Harrison,F. Gressent,M. Huss

Published 2014 in Journal of Biological Chemistry

ABSTRACT

Background: Pea albumin 1b (PA1b) is a potent and selective inhibitor of insect vacuolar ATPase, but its mechanism is poorly understood. Results: PA1b binds the extracellular surface of the c ring and contacts subunit e. Conclusion: PA1b inhibits V-ATPase by blocking transition of its rotor past subunit e. Significance: This reveals insights into the mechanics and mode of inhibition of the V-ATPase. The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.

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