title: Pironetin Binds Covalently to αCys316 and Perturbs a Major Loop and Helix of α-Tubulin to Inhibit Microtubule Formation.
abstract: Microtubule-targeting agents are among the most powerful drugs used in chemotherapy to treat cancer patients. Pironetin is a natural product that displays promising anticancer properties by binding to and potently inhibiting tubulin assembly into microtubules; however, its molecular mechanism of action remained obscure. Here, we solved the crystal structure of the tubulin-pironetin complex and found that the compound covalently binds to Cys316 of α-tubulin. The structure further revealed that pironetin perturbs the T7 loop and helix H8 of α-tubulin. Since both these elements are essential for establishing longitudinal tubulin contacts in microtubules, this result explains how pironetin inhibits the formation of microtubules. Together, our data define the molecular details of the pironetin binding site on α-tubulin and thus offer a promising basis for the rational design of pironetin variants with improved activity profiles. They further extend our knowledge on strategies evolved by natural products to target and perturb the microtubule cytoskeleton.
authors.affiliation: Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland. Electronic address: andrea.prota@psi.ch.
authors.forename: Andrea E
authors.initials: AE
authors.lastname: Prota
authors.affiliation: Department of Protein Sciences, Seattle Genetics, Inc., Bothell, WA 98021, USA.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Setter
authors.affiliation: Department of Protein Sciences, Seattle Genetics, Inc., Bothell, WA 98021, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Waight
authors.affiliation: Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland.
authors.forename: Katja
authors.initials: K
authors.lastname: Bargsten
authors.affiliation: Depart. de Q. Inorgánica y Orgánica, Univ. Jaume I, 12071 Castellón, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Murga
authors.affiliation: Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas CIB-CSIC, 28040 Madrid, Spain.
authors.forename: José Fernando
authors.initials: JF
authors.lastname: Diaz
authors.affiliation: Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland. Electronic address: michel.steinmetz@psi.ch.
authors.forename: Michel O
authors.initials: MO
authors.lastname: Steinmetz
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D001665:Binding Sites; D001688:Biological Products / Q000494:pharmacology; D002417:Cattle; D008870:Microtubules / Q000378:metabolism*; D000072416:Protein Structural Elements; D011753:Pyrones / Q000494:pharmacology*; D012756:Sheep; D014404:Tubulin / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D001688:Biological Products; D011753:Pyrones; D014404:Tubulin; C088446:pironetin
keywords: X-ray crystallography; anticancer drug; microtubule-targeting agents; molecular mechanism of action; protein–ligand interactions