Figure 2. Superposition of the ATP binding sites for the typical snapshots of the Aurora–ADP simulation. 1.0 ns and 5.0 ns. For clarity, the key residue Lys–143 is shown in red line at 1.0 ns, while in sticks at 5.0 ns (cyan, carbon atoms; white, hydrogen atoms; blue, nitrogen atoms and red, oxygen atoms). After the first 1.6 ns, the hydrogen bond between Lys–143 (H) and ATP (O1B) is broken, but instead, the amino side chain of Lys–143 (HZ2) rotates to be contiguous to ATP, thus forming a new hydrogen bond (the dashed line) with the small molecular (O2B) in a ‘‘closed’’ state. The movement of Lys–143 is indicated by using the arrow. (b1), time evolutions of the distance between ADP (O1B) and Lys–143 (H and HZ2) in the Aurora–ADP simulation. doi:10.1371/journal.pone.0016757.g002 In the binary system, the free side chain of Arg-180 dramatically and transiently oscillates from 2180u to 180u as evidenced by its dihedral angle (C-CA-CB-CG), and stays at 180u in most of the simulated time (Fig. 4.d1). On this time scale, Arg-180 exhibits a relatively stable conformation (180u) and protrudes outside the ATP binding site in an ‘‘open’’ state (Fig. 4.D). In contrast to the large fluctuation of Arg-180, the activation segment only under- goes slightly changes (RMSF = 35 A˚). The slight in-outward movement of the activation segment leads to small-scale motions of the phosphate group of P?Thr-288 with its varied dihedral angel (O2P-P-OG-CB) between 2160u and 170u. Thus this residue can orient it in the outward direction so as to permit the accession of phosphatase such as PP1, with a consequence of loss of the phosphate group from this amino acid [19]. Given the ‘‘open’’ states of both Arg-180 and P?Thr-288 in the binary complex, clearly no H-bond is formed between them as their inter-residue distance is ,12 A˚ (Fig. 4.d4). However, once TPX2 binds to Aurora A, Arg-180 rotates downward (40u) and becomes a rigid, ‘‘closed’’ state (RMSF = 9.28 A˚) (Fig. 4.D). Meanwhile, the activation loop also significantly rotates inward to ATP (3 A˚), thus maintaining P?Thr-288 in an active conformation in the ternary complex (Fig. 4.D). In this ‘‘closed’’ state, a stable H-bond is formed between Arg-180 and P?Thr-288 and the H-bond length keeps 2.1 A˚ in Fig. 4.d4. Two TPX2-Dependent Switches We further find that the conformational transitions of Arg-180 and P?Thr-288 are respectively induced by two independent H- bond networks, i.e., 1) the upstream of TPX2 vs Helix aC (network-1); 2) the down stream of TPX2 vs activation loop (network-2). Network-1 includes H-bonds of Glu-175 (OE2) and Ser-7TPX2 (H3), Arg-179 (H) and Tyr-10TPX2 (OH), Glu-183 (OE2) and Asp-11TPX2 (H), and Arg-179 (HH21) and Glu-183 (OE2) (Fig. 4.d3). This network strengthens the contact between TPX2 and helix aC, which stabilizes this helix and also forces it to slightly descend by 0.3 A˚, thereby causing a strong H-bond (2 A˚) between Arg-180 and P?Thr-288 (Fig. 4. d3). And network-2 is composed of Ser-284 (HG) and Asn-43TPX2 (O), Ser-284 (HG) and Asn-43TPX2 (OXT), Arg-286 (HH12) and Glu-42TPX2 (OE2), as well as Thr-288 (O3P) and Arg-286 (HH21) (Fig. 4.d4). The network-2 makes the activation loop to rotate in a buried conformation as indicated by the black arrow in Fig. 4.D. The motion of this loop subsequently causes P?Thr-288 to flip inward by 100u from its outward state. Combined, the two networks triggered by the binding of TPX2 favors the inward flip of P?Thr-288. 3 Cross-correlation of the two switches As the residues involved in the switch-1 (Lys-143) and switch-2 (Arg-180) are structurally neighboring, we suppose that they might also functionally cooperate with each other. The dynamic cross- correlation map (DCCM) is then used to further investigate the correlations of switch-1 and switch-2. DCCM describes the global correlated motions among residues from the highly anticorrelated (blue) to the highly correlated (red). Fig. 5 shows that the fluctuations of Lys-143 and ADP are, on the average, positively correlated in both the binary and ternary systems. Interestingly, the TXP2 binding does not increase the correlation between the two residues with the correlation coefficient (R = 0.9) same as the binary simulation of 0.9. Similarly, strong positive correlations are also observed between Arg-180 and P?Thr-288 in the two systems, however, the R arises to 0.97 (ternary) from 0.83 (binary) due to TXP2 binding. In addition, we also find strong correlations between switch-1 and switch-2, with a correlation of R = 0.74 in the binary system, while 0.82 in the ternary system, meaning that inter-switch cooperation is also enhanced upon TPX2 binding. 4 Mutations of switch-1 and switch-2 To further map the functionality of the two switches, an additional MD simulation on K143A and R180A mutants is performed. Comparing K143A with the wild type Aurora A, the hydrogen bond interaction between the b-phosphate group of ADP and the backbone amino group of K143A is formed and lasts 1.5 ns due to the increased side chain fluctuation of K143A (RMSF is 158.44 A˚ for the mutant protein, and is 67.24 for the wild type protein). The formation of the hydrogen bond between ADP and K143A delays the release of ATP, resulting in the interruption of normal ATP cycle. Therefore, the evidence from the mutant ternary system demonstrates the function of swich 1 from another respect. While for the mutation of Arg-180 to Ala in Aurora A, this mutation actually interrupts the hydrogen bond interaction between Arg-180 and P?Thr-288 in the wild-type Aurora A, leading to the outward flip of P?Thr-288 in the activation loop (see Fig. S2). This is evidenced by the time evolution of the distance (10 A˚ ) between the phosphate group of Thr-288 and the backbone hydrogen of R180A, resulting in the stable open state of P?Thr-288 (Fig. S2). We thus reason that mutation of the two residues to other amino acids with short side chains might PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e16757