title: Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET.
abstract: Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK.|Department of Chemistry, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Louis
authors.initials: L
authors.lastname: Perdios
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK.|London Centre for Nanotechnology, 17-19 Gower St, London, WC1H 0AH, UK.|Division of Biosciences, Birkbeck College, Malet St, London, WC1E 7HX, UK.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Lowe
authors.affiliation: Institute of Structural and Molecular Biology, Department of Chemistry, University College London, Gower St, London WC1E 6BT, UK.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Saladino
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK.
authors.forename: Tom D
authors.initials: TD
authors.lastname: Bunney
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK.
authors.forename: Nethaji
authors.initials: N
authors.lastname: Thiyagarajan
authors.affiliation: Department of Physics, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Alexandrov
authors.affiliation: Department of Physics, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Dunsby
authors.affiliation: Department of Physics, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
authors.affiliation: Institute of Structural and Molecular Biology, Department of Chemistry, University College London, Gower St, London WC1E 6BT, UK.
authors.forename: Francesco Luigi
authors.initials: FL
authors.lastname: Gervasio
authors.affiliation: Department of Chemistry, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK.
authors.forename: Matilda
authors.initials: M
authors.lastname: Katan
mesh_terms: D019943:Amino Acid Substitution; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D051496:Receptor, Fibroblast Growth Factor, Type 1