Abstract
Overexpression of Aurora A, a Ser/Thr kinase, underlies many human cancers. Aurora A’s activity is regulated by phosphorylation on the activation segment and/or binding of activator TPX2 in the PIF pocket of Aurora A. Despite more than a decade of studies on this important oncoprotein, the molecular mechanism of TPX2 binding and activation of Aurora A are still poorly understood. Particularly, pinpointing the allosteric network within Aurora A that is responsive to activation via TPX2, is as of yet, elusive.\r We used Ancestral Sequence Reconstruction and a number of biophysical techniques to search for the necessary and sufficient residues on Aurora A needed to respond to the effect of TPX2. TPX2 is evolutionarily younger than Aurora A. Aurora A ancestors were reconstructed from eras pre-dating (AurANC1 and AurANC2) and post-dating TPX2 (AurANC3, AurANC4 and Aurora A human). The kinase activity of ancestral and modern Aurora species, binding affinity to TPX2 and activation by TPX2 was initially studied. Using site-directed mutagenesis, HPLC, ATP/NADH-coupled assays and ITC, we found that changes in Aurora sequences in the course of evolution serve to accommodate TPX2 binding and mediate allosteric activation. Moreover, a network of 15 amino acid were shown to be necessary for eliciting the full allosteric activation response by TPX2. \r In parallel, to determine whether the allosteric binding site of TPX2 (the PIF-pocket) could be used in drug design, we screened for monobodies that could elicit the opposite effect to TPX2 and thus inhibit Aurora A activity. This work led to the identification of several inhibitors and an activator of Aurora A kinase. \r In conclusion, we show that a network of 15-residues is responsible for allosterically responding to TPX2 and that binding of other activators and inhibitors in the PIF-pocket (where TPX2 binds), favors either activation or inhibition of Aurora A kinase. Use of the PIF pocket as a drug-design hotspot could allow for synthesis of more specific, less toxic inhibitors of Aurora A kinase.