The protein kinase Aurora A controls the process of mitotic spindle assembly through two different signaling pathways operating at the centrosomes of the cell and on the spindle microtubules. Activation of Aurora A in these two pathways occurs through different molecular mechanisms, involving either site-specific phosphorylation on the regulatory activation loop near the active site of the kinase, or interaction with a protein partner called Tpx2. Using a combination of vibrational spectroscopy, double electron-electron resonance spectroscopy and Forster resonance energy transfer to track movements of different structural elements of the kinase in solution, we have shown that the two activation mechanisms of Aurora A have strikingly different effects on the dynamics of the kinase domain. While binding to Tpx2 locks the kinase into a single structural state, when activated by phosphorylation on the activation loop the kinase domain instead remains dynamic and continues to sample the inactive conformational state. By profiling the effects of kinase inhibitor binding on Aurora A structure and dynamics, we also showed that the distinct dynamic properties of the two activation states of Aurora A result in entirely differential interaction profiles with inhibitors. So-called DFG-out inhibitors, which recogize an inactive state of Aurora A, are strongly selective for the phosphorylation-activated form of the kinase, while inhibitors recognizing the active DFG-in state are selective for the Tpx2-activated form. These insights highlight the critical role of conformational effects in molecular recognition, and point to the importance of considering inhibitor conformational preferences for targeting specific biochemical forms of Aurora A implicated in cancer pathogenesis.

Harnessing allostery for selective targeting of Aurora kinase A in cancer

2019 Fall National Meeting

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