Objective

James A H Gilburt (1), Hajrah Sarkar (1), Peter Sheldrake (2), Julian Blagg (2), Liming Ying (1), Charlotte A Dodson (1) (1) National Heart & Lung Institute, Imperial College London, London SW7 2AZ, UK (2) CRUK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK c.dodson@imperial.ac.uk The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small molecule inhibitors. We have used single molecule fluorescence spectroscopy to monitor the movement of the T-loop (activation loop) of Aurora-A kinase between two major conformations. Phosphorylated Aurora-A is in dynamic equilibrium between a DFG-in-like active T-loop conformation and a DFG-out-like inactive T-loop conformation and we have measured the rate constants of interconversion. We have directly measured the equilibrium between the two conformations and determined the free energy difference between them to be 0.7 ± 0.1 kcal mol-1. Addition of activating protein (TPX2) shifts the equilibrium towards the active T-loop conformation, whereas addition of the inhibitors MLN8054 and CD532 favours an inactive T-loop conformation. Notably, 36% of Aurora-A still occupies an active T-loop conformation in the presence of saturating CD532. We show that TPX2 and MLN8054, whose binding modes are thought to be mutually exclusive, bind Aurora-A simultaneously. Our data is inconsistent with standard models of conformational change as part of an induced fit or conformational selection mechanism and we provide a new model for kinase conformational behaviour. Our approach will enable conformation-specific effects to be integrated into inhibitor discovery across the kinome and we outline some immediate consequences for structure-based drug discovery. Keywords. Aurora-A, protein kinase, activation loop, single molecule fluorescence, dye quenching, drug development, DFG-in, DFG-out