Dynamic Equilibrium of the Aurora?A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy.

ABSTRACT: The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small-molecule inhibitors. By using single-molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora?A kinase is in dynamic equilibrium between a DFG-in-like active T-loop conformation and a DFG-out-like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora?A activating protein TPX2 shifts the equilibrium towards an active T-loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T-loop. We show that Aurora?A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. 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.

SUBMITTER: Gilburt JAH 

PROVIDER: S-EPMC5601181 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature