Using ancestral proteins to unravel the molecular mechanism and druggability of Aurora A kinase Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biochemistry Dorothee Kern, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Biochemistry by Yuejiao Zheng May 2016 I Copyright by Yuejiao Zheng © 2016 II ABSTRACT Using ancestral proteins to unravel the molecular mechanism and druggability of Aurora A kinase A thesis presented to the Department of Biochemistry Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Yuejiao Zheng 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. 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 (Aur and Aur ) and post-dating TPX2 (Aur , Aur and Aurora ANC1 ANC2 ANC3 ANC4 A ). The kinase activity of ancestral and modern Aurora species, binding affinity to TPX2 human 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 III network of 15 amino acid were shown to be necessary for eliciting the full allosteric activation response by TPX2. 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. 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. IV Table of Contents Abstract...........................................................................................................................................iii Table of Contents.............................................................................................................................v List of Figures................................................................................................................................vii List of Tables................................................................................................................................ix Introduction......................................................................................................................................1 Aurora and TPX2......................................................................................................................1 PIF binding pocket (PIF pocket).............................................................................................. 3 Ancestral Sequence Reconstruction..........................................................................................4 Monobodies.............................................................................................................................. 4 Results and Discussions...................................................................................................................6 Ancestral Aurora Kinase Activity and TPX2 Activation.........................................................6 Monitoring TPX2 Binding and Activation in Ancestral Aurora using Site-Directed Mutagenesis.....................................................................................................................................8 Attempts at crystallization of Aur , the youngest ancestral Aurora A species..............19 ANC4 Monobodies.............................................................................................................................22 V Conclusion.....................................................................................................................................26 Material and Methods....................................................................................................................28 References......................................................................................................................................33 VI List of Figures Figure 1. TPX2 activates Aurora A in vivo......................................................................................... 2 Figure 2. The front and back view of TPX2 1-45 bound to Aurora A 122-403 in cartoon modern representation ....................................................................................................................................... 3 Figure 3 Ancestral sequence reconstruction of Aurora A and TPX2...................................................................................................................................................... 6 Figure 4. Aurora A evolves to respond to TPX2 activation.................................................................. 7 Figure 5. Sequence alignment of Aurora ancestors................................................................................................................................................ 8 Figure 6. A structural comparison of wild type and Y199H Aurora A ................................... 9 modern Figure 7. TPX2 binds to Aurora A and activates Aurora A in vitro. ...............................................10 Figure 8. ITC measurements for Y199H Aurora A and H199Y Aur .............................11 modern ANC2 Figure 9. ATP/NADH-coupled assay for Y199H Aurora A and H199Y Aur ................12 modern ANC2 Figure 10. A structural comparison of wild type and H187N Aurora A . ..............................13 modern Figure 11. ITC measurements for H187N Aurora A ...............................................................13 modern Figure 12. ATP/NADH-coupled assay for H187N Aurora A and N187H Aur ..............15 modern ANC2 Figure 13. A structural comparison of wild type and E211H R232T Aurora A . ....................................................................................................................................................16 modern Figure 14. HPLC assay for wild type and E211H R232T Aurora A ......................................17 modern Figure 15. ATP/NADH-coupled assay for Aur andAur +15aa...........................................18 ANC2 ANC2 Figure 16. A structural comparison of wild type and Aur +15aa. ............................................19 ANC2 VII Figure 17. Structural representation of Long Construct Aur 122-403..........................................21 ANC4 Figure 18. Preliminary screening for Aur 122-403 crystal............................................................21 ANC4 Figure 19. Structural representation of T288V Avi-AurA-TPX2 chimera.....................................22 Figure 20. HPLC assay for T288V Avi-AurA-TPX2 chimera.........................................................23 Figure 21. ITC measurements for Aurora A bound to sGFP Mb2.........................................24 modern Figure 22. HPLC assay for Mb1, Mb2, and sGFP Mb2...................................................................25 Figure 23. Scheme of the coupled assay............................................................................................31 VIII List of Tables Table1. Testing Conditions for Aur Crystallization................................................................. 20 ANC4 IX Introduction Aurora A and TPX2 Aurora A, a serine/threonine kinase, has become an attractive target cancer therapeutics, given its important role in cell cycle regulation and thus cell proliferation (Aliagas-Martin et al. 2009; Bebbington et al. 2009; Cheok et al. 2010). During mitosis, Aurora A regulates centrosome separation and bipolar spindle assembly (Glover 2003; Giubettini et al. 2010). However, over-expression of Aurora A has been found in different mammalian tumor cell lines, where cells are show centrosomal abnormalities and chromosomal instability (Kallioniemi et al., 1994; Zhou et al., 1998; Jeng et al., 2004). Aurora A is known to regulate spindle formation by phosphorylating pole-area kinetochore to ensure correct chromosome alignment and kinetochore-microtubule connections (Ye et al. 2015). TPX2, Aurora A’s specific binding protein, stabilizes Aurora A by forming a TPX2- Aurora A complex to prevent Aurora A from proteasome-dependent degradation (Giubettini et al. 2010) TPX2 also localizes Aurora A to the spindle microtubules during mitosis, which is a necessary step in Aurora A’s regulation of spindle formation (Carmena, Earnshaw 2003; Kufer et al. 2002)(Figure 1). It is previously reported that autophosphorylation of Aurora A’s T288 residue is correlated with increased catalytic activity (Littlepage et al., 2002). By directly measuring kinase activity using HPLC-based assays, Zorba et al proposed two activation 1