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

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.
Document type :
Journal articles
Complete list of metadatas

https://hal.archives-ouvertes.fr/hal-01179517
Contributor : Isabelle Frapart <>
Submitted on : Wednesday, July 22, 2015 - 4:36:04 PM
Last modification on : Tuesday, June 4, 2019 - 4:30:08 PM

Links full text

Identifiers

Citation

Stephen P. Muench, Shaun Rawson, Vanessa Eyraud, Agnès F. Delmas, Pedro da Silva, et al.. PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals its Insecticidal Mechanism. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2014, 289 (23), pp.16399-16408. ⟨10.1074/jbc.M113.541250⟩. ⟨hal-01179517⟩

Share

Metrics

Record views

180