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.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal “Periscope Proteins” as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this “rule,” suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Activation loop phosphorylation changes the position of equilibrium between DFG-in-like and DFG-out-like conformations but not the conformational preference of inhibitors.

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+