PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals its Insecticidal Mechanism
2014
Muench, Stephen P. | Rawson, Shaun | Eyraud, Vanessa | Delmas, Agnès F. | da Silva, Pedro | Phillips, Clair | Trinick, John | Harrison, Michael A. | Gressent, Frédéric | Huss, Markus | School of Biomedical Sciences ; University of Leeds | Biologie Fonctionnelle, Insectes et Interactions (BF2I) ; Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées de Lyon (INSA Lyon) ; Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA) | Centre de biophysique moléculaire (CBM) ; Université d'Orléans (UO)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie - CNRS Chimie (INC-CNRS)-Centre National de la Recherche Scientifique (CNRS) | School of molecular and cellular biology ; University of Leeds | Abteilung Tierphysiologie, Fachbereich Biologie/Chemie ; Universität Osnabrück - Osnabrück University | Medical Research Council [G1000567]; British Biotechnology Research Council (BBSRC) Grant [BB/D016142/1]; Deutsche Forschungsgemeinschaft [SFB 431, SFB 944]; Wellcome Trust [099752/Z/12/Z]
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Show more [+] Less [-]English. 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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