Two TPX2-Dependent Switches Figure 1. Superposition of the ATP binding sites for the typical snapshots of the Aurora–ATP simulation. 4.35 ns and 8.35 ns. For clarity, only the key region of loop 1–2 is colored in gray (4.35 ns) and blue (8.35 ns). Lys–143 at 4.35 ns is shown in blue line presentation, while residues and ADP at 8.35 ns are shown in stick presentations: cyan for carbon, white for hydrogen, red for oxygen, and blue for nitrogen atoms. As indicated by the black arrow, loop1–2 moves upward, accompanied by the formation and disruption of the hydrogen bond (the dashed line) between Lys–143 (H) and ATP (O1B) in an ‘‘open’’ state. (a1), superposition of ATP focusing on the different conformations of the triphosphate side chain (upward and downward states). (a2), time–dependence of RMSD of ATP. The dashed line depicts the upward and downward conformations of ATP. (a3), time–dependent rotation of Lys–143 about the (CB–CG–CD–CE) dihedral angle. (a4), time evolution of the distance between Lys–143 (H) and ATP (O1B). (a5), time evolution of the RMSF in the Aurora–ATP simulation. The red lines depict residues involved in the ATP binding site: Leu139– Arg–151, Glu–211–Thr–217 and Arg–255–Gly276. The result of the RMSF analysis indicates that in the binary simulation there are significant fluctuations centered around residues 141–143 (loop 1–2). doi:10.1371/journal.pone.0016757.g001 with Gly-145 (H) in loop 1–2 as evidenced by the H-bond distance (,3.5 A˚) (Fig. 3. c3). This H-bond interaction exerts a constraint force on loop 1-2, thereby leading to conformational stable Lys- 143. This analysis thus suggests that the stability of secondary structure in the loop 1–2 that facilitates the swing and open conformation of Lys-143 is regulated by a considerable concom- itant consolidation of the TPX2 fragment. 2 Switch-2 in the activation loop Aurora A kinase activity depends on autophosphorylation of Thr-288 in the activation loop. TPX2 binding locks the catalytic domain of the kinase into an active configuration to keep the phosphorylated Thr-288 into a phosphatase-inaccessible confor- mation [21]. The X-ray crystal structure has released that the binding site of TPX2 is more than 20 A˚ away from P?Thr288, implying that Aurora A should exhibit certain conformational transition in its N-terminal lobe upon binding of TPX2, and such a global alteration in kinase conformation might be associated with certain allosteric characteristics of the activation loop where Thr288 is located. However, how does the binding of TPX2 influence P?Thr288? Whether there exist certain actions from helix aC, which regulates P?Thr288 in an animate (inward) or catalyzed (outward) state, since the downstream stretch (residues 26–43) of TPX2 directly interacts with the helix aC of Aurora A [16]? To address these questions, we compare the motion of the activation loop and its vicinity over the 20 ns trajectories of Aurora-ADP and Aurora-ADP-TPX2. The RMSF analysis has shown that the flexibility of Arg-180 in helix aC significantly differs in the binary and ternary simulations (94.19 A˚ for Aurora- ADP, and 9.28 A˚ for Aurora-ADP-TPX2), which may indicate that this residue is functionally associated with Aurora A activity. PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e16757