title: 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.
authors.affiliation: From the School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT Leeds, West Yorkshire, United Kingdom, s.p.muench@leeds.ac.uk.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Muench
authors.affiliation: From the School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT Leeds, West Yorkshire, United Kingdom.
authors.forename: Shaun
authors.initials: S
authors.lastname: Rawson
authors.affiliation: Institut National de la Recherche Agronomique, Institut National des Sciences Appliquées-Lyon, Université de Lyon, IFR 41, UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, Batiment Louis-Pasteur 20, avenue Albert Einstein, F-69621 Villeurbanne, France.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Eyraud
authors.affiliation: the Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique Unité Propre de Recherche 4301, Rue Charles Sadron, 45071 Orléans cedex 2, France.
authors.forename: Agnès F
authors.initials: AF
authors.lastname: Delmas
authors.affiliation: Institut National de la Recherche Agronomique, Institut National des Sciences Appliquées-Lyon, Université de Lyon, IFR 41, UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, Batiment Louis-Pasteur 20, avenue Albert Einstein, F-69621 Villeurbanne, France.
authors.forename: Pedro
authors.initials: P
authors.lastname: Da Silva
authors.affiliation: From the School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT Leeds, West Yorkshire, United Kingdom.
authors.forename: Clair
authors.initials: C
authors.lastname: Phillips
authors.affiliation: the School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, LS2 9JT Leeds, West Yorkshire, United Kingdom, and.
authors.forename: John
authors.initials: J
authors.lastname: Trinick
authors.affiliation: From the School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT Leeds, West Yorkshire, United Kingdom.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Harrison
authors.affiliation: Institut National de la Recherche Agronomique, Institut National des Sciences Appliquées-Lyon, Université de Lyon, IFR 41, UMR203 BF2I, Biologie Fonctionnelle Insectes et Interactions, Batiment Louis-Pasteur 20, avenue Albert Einstein, F-69621 Villeurbanne, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Gressent
authors.affiliation: Abteilung Tierphysiologie, Fachbereich Biologie/Chemie Universität Osnabrück, 49069 Osnabrück, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Huss
mesh_terms: D000418:Albumins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001483:Base Sequence; D001577:Benzophenones / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D017931:DNA Primers; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D018532:Pisum sativum / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000418:Albumins; D001577:Benzophenones; D017931:DNA Primers; D007306:Insecticides; D019920:Photoaffinity Labels; D001710:Biotin; C047723:benzophenone; D025262:Vacuolar Proton-Translocating ATPases
keywords: ATPase; Drug Action; Electron Microscopy (EM); Membrane Protein; PA1b; Vacuolar ATPase