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.

The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small-molecule inhibitors.Byusing single-molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora Ak inase is in dynamic equilibrium between aD FG-in-like active T-loop conformation and aD FG-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.Weshow that Aurora Ab inds TPX2 and MLN8054 simultaneously and provideanew model for kinase conformational behavior.Our approach will enable conformation-specific effects to be integrated into inhibitor discovery across the kinome,a nd we outline some immediate consequences for structure-based drug discovery.Protein kinases are essential for the regulation and signaling of eukaryotic cells and are important drug targets in cancer and inflammatory disease.[1] Many kinases are regulated by phosphorylation of ar egulatory Ser/Thr/Tyr residue on ar egion of the kinase known as the activation loop or T-loop.T he influence of phosphorylation and interactions with small-molecule inhibitors on kinase conformation can be summarized by two models.I nt he first model, phosphorylation achieves activation by "locking" the activation loop in ac onformation where the catalytic residues are aligned ( Figure 1a). [2] In the second model, an inactive-conformation kinase bound to at ype II inhibitor (an inhibitor whose binding site extends into aspecific allosteric pocket adjacent to the ATP-binding site) is in equilibrium with the ligand-free kinase in an active conformation (Figure 1b). In the context of these models,t he active conformation is typified by the activation loop being oriented to form the protein substrate binding site and the aspartic acid of the conserved DFG motif at the beginning of this loop pointing into the ATPb inding site to coordinate Mg 2+