Abstract
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.
Highlights
Pea albumin 1b (PA1b) is a potent and selective inhibitor of insect vacuolar ATPase, but its mechanism is poorly understood
We show that PA1b binds at the base of the c ring, the first direct visualization of inhibitor binding to V-ATPase
Measurement of M. sexta V-ATPase activity in the presence of the PA1b-biotin derivative showed no significant difference in activity from native PA1b (Fig. 2A)
Summary
Pea albumin 1b (PA1b) is a potent and selective inhibitor of insect vacuolar ATPase, but its mechanism is poorly understood. 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 VATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. The vacuolar Hϩ-ATPase (V-ATPase) is a complex molecular machine responsible for the transmembrane movement of protons against a gradient, fuelled by ATP It plays a central role in the physiology of virtually all eukaryotic cells, performing such critical functions as acidification of endosomal compartments and energization of secondary active transport [1, 2]. A number of potent inhibitors have been shown to bind tightly to the c ring (14 –16), presumably preventing proton translocation by obstructing procession of the rotor
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