title: EPR Studies of V-ATPase with Spin-Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit c.
abstract: Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N'-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.
authors.affiliation: Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Str. 1, 53121, Bonn, Germany.
authors.forename: Jan Philipp
authors.identifier: http://orcid.org/0000-0003-0023-3413
authors.initials: JP
authors.lastname: Gölz
authors.affiliation: Fachbereich Biologie/Chemie, Universität Osnabrück, 49069, Osnabrück, Germany.
authors.forename: Svenja
authors.initials: S
authors.lastname: Bockelmann
authors.affiliation: Institut für Organische Chemie, Ruprecht-Karls-Universität Heidelberg, INF 270, 69120, Heidelberg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Mayer
authors.affiliation: Fachbereich Physik, Universität Osnabrück, 49069, Osnabrück, Germany.
authors.forename: Heinz-Jürgen
authors.initials: HJ
authors.lastname: Steinhoff
authors.affiliation: Fachbereich Biologie/Chemie, Universität Osnabrück, 49069, Osnabrück, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Wieczorek
authors.affiliation: Fachbereich Biologie/Chemie, Universität Osnabrück, 49069, Osnabrück, Germany. markus.huss@osnanet.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Huss
authors.affiliation: Fachbereich Physik, Universität Osnabrück, 49069, Osnabrück, Germany. jklare@uos.de.
authors.forename: Johann P
authors.initials: JP
authors.lastname: Klare
authors.affiliation: Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Str. 1, 53121, Bonn, Germany. dirk.menche@uni-bonn.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Menche
mesh_terms: D000818:Animals; D001665:Binding Sites; D003497:Cyclic N-Oxides / Q000737:chemistry; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D018942:Macrolides / Q000737:chemistry / Q000494:pharmacology*; D018402:Manduca / Q000187:drug effects / Q000201:enzymology*; D008958:Models, Molecular; D013113:Spin Labels; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003497:Cyclic N-Oxides; D004791:Enzyme Inhibitors; D018942:Macrolides; D013113:Spin Labels; D013844:Thiazoles; C519727:archazolid A; D004024:Dicyclohexylcarbodiimide; D025262:Vacuolar Proton-Translocating ATPases; C003959:TEMPO
keywords: EPR spectroscopy; V-ATPase; archazolid; natural products; spin labels