Two TPX2-Dependent Switches Figure 3. Superposition of the average structures of the Aurora–ADP (brown) and the Aurora–ADP–TPX2 (cyan) simulations. Residues in the binary structure are shown in brown line presentation, while residues in the ternary complex and ADP are shown in stick presentations: cyan for carbon, white for hydrogen, red for oxygen, and blue for nitrogen atoms. The TPX2 fragment is displayed in the gray ribbon. The position of the binding region among helix aB, b–sheet 4 and the upstream of TPX2 is circled in dashed lines. After TPX2 binding, Tyr–199 respectively forms a H–bonds with Tyr–10TPX2 (H) and Pro–13TPX2(O) (the dashed lines) to pull b–sheet 4 to be more contiguous to helix aB. Due to the decreased distance between b–sheet 4 and helix aB, His–201 (ND1) interacts with Lys–166 (HZ1) to force helix aB to undergo a rotation movement (,1 A˚ ). This process causes the formation of a H–bond between Gln–168 (OE1) in helix aB and Gly–145 (H) in loop 1–2. As a result, the constrained loop 1–2 leads to a stable, ‘‘open’’ state of Lys–143. (c1), time–dependent rotation of Lys–143 about the (CG–CB–CA–N) dihedral angle in the ternary simulation. (c2), the view is rotated 90u about the y axis relative to (C). (c3), time evolutions of the distance between Lys–166 (HZ1) and His–201 (ND1), Gln–168 and Gly–145 (H), Tyr–199 (OH) and Tyr–10TPX2 (H), and Tyr–199 (H) and Pro–13TPX2 (O) in the ternary simulation. doi:10.1371/journal.pone.0016757.g003 respectively prevent the normal cycle of ATP and phosphory- lation of Aurora A, therefore blocking activation of the protein. Conclusions The kinase Aurora A is overexpressed in many tumors, and has become an effective target for anticancer drug. There are two routes to inhibiting its function: 1) interfere with its ability to interact with its binding partners, such as TPX2; 2) occupy the catalytic ATP binding site. Targeting protein–protein interactions with small-molecule inhibitors and selectively targeting the enzymatic activity of kinase with small molecules have proved to be great challenges since the key residues involved in the regulating of ATP cycle and TPX2 binding are still not unveiled. In this study, we present two functional correlated TPX2-dependent switches important for Aurora A activation, which have not been investigated so far. Swith-1 composed of Lys-143 plays an essential role in kinetic cycle of ATP binding, nucleotide hydrolysis, and release of ADP, thus regulating the activity of Aurora A. Switch-2 includes Arg-180, the open and closed states of which restricts the conformational movement of P?Thr-288, thus completing the cell cycle- dependent feedback of phosphorylation/dephosphorylation of Aurora A. Our in-depth study have uncovered that the molecular basis of Aurora A activation is largely determined by the two novel switches, which is important in designing the compounds for treating tumors. Methods 1 Structural preparation Initial coordinate of Aurora A was taken from the human X-ray crystal structure [15] (PDB entries 1ol5, 1ol6 and 1ol7). Molecular dynamics (MD) simulations in explicit water have been indepen- dently carried out on a simulation time scale of 20 ns for (1) the unbound apo form of the Aurora A in solution (apo-Aurora), (2) Aurora A complexes with the natural substrate ATP (Aurora- ATP), (3) Aurora A complexes with the substrate ADP (Aurora- ADP), (4) Aurora A-ADP complexes with the coactivator TPX2 (Aurora-ADP-TPX2) and (5) Aurora-ADP-TPX2 with the K143A and R180A mutants. The missing residues (Leu22-Gln29 of the TPX2 segment (1ol5), and Gly142, Gly173 and Val174 of Aurora A (1ol6)) were repaired by using the loop search method in the Swiss-PdbViewer (aka DeepView, http://spdbv.vital-it.ch/). After removal of the phosphate group from the phosphorylated Thr287, the crystal structure of Aurora A in complex with ADP and TPX2 (1ol5), was used directly in the simulation of Aurora-ADP-TPX2 complex, and was also applied to generate the starting structure of the Lys143-Ala and Arg180-Ala mutants. This Ala was selected for the reason that no H-bond could be formed in the mutants for this residue. By analyzing the Lys143Ala and Arg180Ala trajectory, we observed that the mutated Aurora A could retain the basic native structure. Similarly, the simulations of Aurora-ATP and Aurora- ADP complexes were respectively generated from the crystal structures 1ol6 and 1ol7. In the absence of crystal structure of apo- PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e16757