title: Detection of Ligand-induced Conformational Changes in the Activation Loop of Aurora-A Kinase by PELDOR Spectroscopy.
abstract: The structure of protein kinases has been extensively studied by protein crystallography. Conformational movement of the kinase activation loop is thought to be crucial for regulation of activity; however, in many cases the position of the activation loop in solution is unknown. Protein kinases are an important class of therapeutic target and kinase inhibitors are classified by their effect on the activation loop. Here, we report the use of pulsed electron double resonance (PELDOR) and site-directed spin labeling to monitor conformational changes through the insertion of MTSL [S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1 H-pyrrol-3-yl)methyl methanesulfonothioate] on the dynamic activation loop and a stable site on the outer surface of the enzyme. The action of different ligands such as microtubule-associated protein (TPX2) and inhibitors could be discriminated as well as their ability to lock the activation loop in a fixed conformation. This study provides evidence for structural adaptations that could be used for drug design and a methodological approach that has potential to characterize inhibitors in development.
authors.affiliation: Astbury Centre for Structural and Molecular Biology Faculty of Biological Sciences University of Leeds Leeds LS2 9JT United Kingdom.
authors.forename: Selena G
authors.initials: SG
authors.lastname: Burgess
authors.affiliation: The Photon Science Institute and School of Chemistry University of Manchester Manchester M13 9PL United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Grazia Concilio
authors.affiliation: Astbury Centre for Structural and Molecular Biology Faculty of Biological Sciences University of Leeds Leeds LS2 9JT United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Bayliss
authors.affiliation: The Photon Science Institute and School of Chemistry University of Manchester Manchester M13 9PL United Kingdom.
authors.forename: Alistair J
authors.identifier: 0000-0002-4437-9791
authors.initials: AJ
authors.lastname: Fielding
keywords: PELDOR spectroscopy; drug design; electron paramagnetic resonance; inhibitor; protein kinases