75.31 A˚673.09 A˚ for ADP-Aurora, 70.09 A˚686.58 A˚690.20 A˚ for Aurora- ADP-TPX2, and 73.60 A˚673.96 A˚669.47 A˚ for mutant Aurora-ADP-TPX2). To neutralize the system, Na+ and Cl2 counter ions were added. Ions were placed on the basis of the electrostatic energy of the water oxygens. Finally, the five systems were minimized respectively, usually for 5000 steepest-descent steps followed by 5000 conjugate-gradient steps. 2 Simulation Protocol All simulations were run by the sander module in Amber 10 [25]. MD simulations started with heating the system from 0 to 300 K in 100 ps followed by a 50 ps pressure-constant period to raise the density while still keeping the complex atoms constrained, after which 0.5 ns of equilibration was conducted. The following 20 ns were considered as further equilibration time and analysis was done on the following parts of trajectories. Finally, the production phase was run for 20 ns, considering that each trajectory is long enough to ensure sufficient sampling of the protein’s configuration space and the ligand, i.e., allowing the systems to cross the barrier between folded and misfolded free energy basins more than once. MD simulations were run in the NPT ensemble, using periodic boundary conditions, a 2 fs timestep, and SHAKE algorithm [27] to constrain all bonds to hydrogens. The temperature was kept at 300 K using Langevin dynamics [28] with a collision frequency of 1 ps21. The cutoff distance was kept to 8 A˚ to compute the nonbonded interactions. All the simulations were performed under periodic boundary conditions, and long-range electrostatics was treated by using the particle-mesh-Ewald (PME) method [29]. Snapshots were col- lected from the stable structures during the last 10 ns of the four simulations for analysis (1snapshot/10 ps). References 1. Fu J, Bian M, Jiang Q, Zhang C (2007) Roles of Aurora Kinases in Mitosis and Tumorigenesis. Mol Cancer Res 5: 1–10. 2. Marumoto T, Honda S, Hara T, Nitta M, Hirota T, et al. (2003) Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa cells. J Biol Chem 278: 51786–51795. 3. Vader G, Lens S (2008) The Aurora kinase family in cell division and cancer. Biochim Biophys Acta BBA 1786: 60–72. 4. Gautschi O, Heighway J, Mack PC, Purnell PR, Lara PN, Jr., et al. (2008) Aurora Kinases as Anticancer Drug Targets. Clin Cancer Res 14: 1639–1648. 5. Zhou H, Kuang J, Zhong L, Kuo WL, Gray J, et al. (1998) Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet 20: 189–193. 6. Giet R, Petretti C, Prigent C (2005) Aurora kinases, aneuploidy and cancer, a coincidence or a real link? Trends Cell Biol 15: 241–250. 7. Marumoto T, Zhang D, Saya H (2005) Aurora-A–A guardian of poles. Nat Rev Cancer 5: 42–50. 8. Moyer ML, Gilbert SP, Johnson KA (1998) Pathway of ATP hydrolysis by monomeric and dimeric kinesin. Biochemistry 37: 800–813. 9. Goodsell DS, Cassier P (1996) Our molecular nature: The body’s motors, machines and messages. Heidelberg: SpringerLink Press. 183 p. 10. Hirota T, Kunitoku N, Sasayama T, Marumoto T, Zhang D, et al. (2003) Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell 114: 585–598. 11. Satinover DL, Leach CA, Stukenberg PT, Brautigan DL (2004) Activation of Aurora–A kinase by protein phosphatase inhibitor–2, a bifunctional signaling protein. Proc Natl Acad Sci USA 101: 8625–8630. 12. Pugacheva EN, Golemis EA (2005) The focal adhesion scaffolding protein HEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome. Nat Cell Biol 7: 937–946. 13. Hutterer A, Berdnik D, Wirtz–Peitz F, Zigman M, Schleiffer A, et al. (2006) Mitotic activation of the kinase Aurora-A requires its binding partner Bora. Dev Cell 11: 147–157. 14. Bibby RA, Tang C, Faisal A, Drosopoulos K, Lubbe S, et al. (2009) A cancer– associated Aurora A mutant is mislocalized and misregulated due to loss of interaction with TPX2. J Biol Chem 284: 33177–33184. 15. Bayliss R, Sardon T, Vernos I, Conti E (2003) Structural basis of Aurora-A activation by TPX2 at the mitotic spindle. Mol Cell 12: 851–862. 16. Zhao B, Smallwood A, Yang J, Koretke K, Nurse K, et al. (2008) Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallography Two TPX2-Dependent Switches Supporting Information Figure S1 Ribbon diagram of the Aurora A structure (cyan) oriented to show the relative positions of the C- and N-terminal lobes. TPX2 is colored in red ribbon, and ADP and Thr-288 are shown in blue sticks. (tif) Figure S2 The average structure of the mutant Aurora- ADP-TPX2 (cyan) simulation. Residues and ADP in the ternary structure are shown in stick presentation: cyan for carbon, white for hydrogen, red for oxygen, and blue for nitrogen atoms. TPX2 is displayed in the gray ribbon. The dashed lines show the distance between K143A (H) and ADP (O1B), and between R180A (H) and Thr288 (O2P). (a) the time evolution of distance between K143A (H) and ADP (O1B), and between R180A (H) and Thr288 (O2P) in the ternary simulation. (tif) Movie S1 The open and closed motions of switch-1 (Lys- 143) and switch-2 (Arg-180) in the Aurora A-ATP, Aurora A-ADP, and Aurora A-ADP-TPX2 simulations. Aurora A is shown in gray ribbon, Lys-143 and ATP/ADP are shown in stick representation, and Arg-180 and Thr-288 are shown in ball and line presentation. (AVI) Author Contributions Conceived and designed the experiments: XX YW. Performed the experiments: XX XW. Analyzed the data: XX XW ZX. Contributed reagents/materials/analysis tools: YL. studies on Aurora A interactions with VX–680 and with TPX2. Protein Sci 17: 1791–1797. 17. Agnese V, Bazan V, Fiorentino FP, Fanale D, Badalamenti G, et al. (2007) The role of Aurora-A inhibitors in cancer therapy. Ann Oncol 18: 47–52. 18. Nowakowski J, Cronin CN, McRee DE, Knuth MW, Nelson CG, et al. (2002) Structures of the cancer-related Aurora-A, FAK, and EphA2 protein kinases from nanovolume crystallography. Structure 10: 1659–1667. 19. Groen AC, Needleman D, Brangwynne C, Gradinaru C, Fowler B, et al. (2008) A novel small-molecule inhibitor reveals a possible role of kinesin–5 in anastral spindle–pole assembly. J Cell Sci 121: 2293–2300. 20. Lu B, Wong CF, McCammon JA (2005) Release of ADP from the catalytic subunit of protein kinase A: A molecular dynamics simulation study. Protein Sci 14: 159–168. 21. Eyers PA, Maller JL (2003) Regulation of Xenopus Aurora A activation by TPX2. J Bio Chem 279: 9008–9015. 22. 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