The upstream stretch (residues 7–21) binds to the N-terminal lobe of Aurora A in a mostly extended conformation. The downstream stretch (residues 26–43) adopts an a-helical conformation that interacts with both the helix aC and the activation segment of Aurora A. Although the crystal structures of phosphorylated Aurora-A have been unveiled [16,17], several fundamental questions related to the regulation of Aurora A activity by TPX2 remain unclear. An unsolved question is how TPX2 binding is linked to efficient ATP cycle in Aurora, and how this cycle coupled with the binding of TPX2 affect the kinase activity. However, experimental evidence for the dynamics of ATP-, ADP- and TPX2-bond Aurora A is very hard to obtain. Much of the experimental information about protein dynamics has come from superposable crystal structures [15]. Therefore, molecular dynamics (MD) simulations, as a powerful method to study the mobility of proteins, is applied in this work to investigate the mechanism of activation modulation of the Aurora A conforma- tional dynamics at atomic resolution. Results and Discussion 1 Switch-1 in the ATP binding site Since the kinetic cycle of ATP is the essential step to make the kinase catalytically active [17], inhibition of the ATP cycle can induce a deactivation process of Aurora A, which makes the ATP binding site of Aurora-A a novel target for cancer therapy [18]. For Aurora-A, ATP is captured in the ATP binding site sandwiched between the C- and N-termini. Upon ATP hydrolysis, a conformational transition of the binding site is triggered by the binding of TPX2, which facilitates the transfer of phosphate group and the release of ADP [19]. Although structural and mutational studies [15,18] have identified several residues involved in Aurora A activity, it is still unclear which residues do play roles in the regulation of the ATP cycle, how the residues contact ATP/ADP, favoring the hydrolysis of ATP and the release of ADP, and whether these residues cooperate with each other to complete the cycle process. To explore the key residues to control the cycle of ATP, we have performed a total of 20 ns of all-atom MD simulations of three systems, i.e., Aurora-ATP, Aurora-ADP and Aurora-ADP-TPX2, in explicit water. Fig. 1.a2 shows that the RMSD of ATP inside the binding pocket vs. simulation time in the Aurora-ATP system. Interestingly, the RMSD of ATP remains ,0.5 A˚ during the first 5.2 ns, and it reaches up to ,0.9 A˚ and even more in the following 14.8 ns. Further analysis reveals that ATP switches between two conformations (upward and downward states) in its binding site, and the main difference between them lies in the state of phosphorous side chain (Fig. 1.a1), raising the question of how Aurora A regulate the conformational switching between the binding (upward) and the release (downward) states for ATP? The time evolution of the RMS fluctuation (RMSF) in Fig. 1.a5 shows that the binding of ATP leads to the stability of the ATP binding site (RMSF = 90,100 A˚) with the exception of residues 141–143 (RMSF = 160,320 A˚) in loop 1–2, indicating that loop 1–2 is extremely flexible. Loop 1–2 can swing up and down (,2 A˚), as indicated by the black arrow in Fig. 1.A. The movement of this loop makes Lys-143 on the top of ATP undergo significant conformational changes in the entire simulation time as evidenced by its large rotation of angle (CB-CG-CD-CE) from 2180u,180u (Fig. 1.a3). In the first 6.0 ns, a stable hydrogen bond (H-bond) is formed between Lys-143 (H) and ATP (O1B) with a H-bond length of ,2.5 A˚ (Fig. 1.a4). However after 6.0 ns, the interaction between them is suddenly broken, resulting in a Two TPX2-Dependent Switches significant increase of RMSF from 27 to 320 A˚, and this residue freely flips up and downward around the vertical plane for the loop 1–2 over 12 ns (Fig. 1.A). Interestingly, at the last 2 ns, Lys-143 rotates downward again (30u) and is weakly H-bounded to ATP (O1B) with the H-bond length of 3.560.5 A˚ (Fig. 1.a4). From this process, we can find that Lys-143 vividly captures the small molecule ATP in an intermittent manner, which is distinct from its motion in the Aurora-ADP-TPX2 system (Fig. 1. A). This formation and disruption of H-bond explains why the phospho- rous side chain of ATP flip up (H-bond formed) at the beginning but downward (H-bond broken) in the end. The upward conformation might indicate the intimate binding of ATP in the pocket, yet the downward mode might indicate a release trend of phosphate group from the protein. We thus conclude that Lys-143 constitutes a critical switch governing the binding or even release of ATP in the binding site. After ATP hydrolysis, Lys-143 further rotates into the ATP binding site and exhibits a ‘‘closed’’ conformation. (Fig. 2. B). Interestingly, in the first 1.6 ns, the backbone hydrogen of Lys-143 (H) generates an H-bond (H-1) with ADP (O1B) (Fig. 2. b1). However, in the subsequent time, the amino group of this residue undergoes a dramatic 60u clockwise rotation (Fig. 2. B), resulting in the disruption of the old H-1 and the simultaneous formation of a new H-bond (H-2) between the side chain of Lys-143 (HZ2) and ADP (O1B) (Fig. 2. b1). This finding suggests that Lys-143 can successfully ‘‘hook’’ the b-phosphate of ADP in this ‘‘closed’’ state (Fig. 2.B). However, Lys-143 still rotates up- and downward remarkably (35u) in the following 18.4 ns, leading to intermittent formation of H-2 in the Aurora-ADP complex (Fig. 2. b1). This result might imply that Lys-143 acts as a potential driving force to pull this ADP out of the binding pocket. As ADP release is the last step to complete kinase activity [20], we thus suggest that the conformational change of Lys-143 plays an important role in this dissociation process. In a contrast with the ‘‘closed’’ state for Lys-143 (switch-1), TPX2 binding makes this residue project out of the ATP binding site, in an ‘‘open’’ conformation (see Movie S1). In the ternary system, the binding of TPX2 initially forces loop 1–2 to undergo an upward movement (,1 A˚) and open the ATP binding site, causing Lys143 to rotate with a large fluctuation from 180u to 2180u. However, switch-1 still maintains its stable ‘‘open’’ configuration (,2100u) for about 60% of the simulation time (see its angle (CG-CB-CA-N) in Fig. 3.c1). In this case, Lys-143 does not form a H-bond with ADP as evidenced by the distance (,4 A˚) between them (data not shown), showing that ADP becomes more free when Lys-143 opens the ATP binding site. Further, compared with Aurora-ADP system, we observe that triggered by the binding of TPX2, the conformational state of Lys- 143 is shifted more stable in the ternary system, since RMSF of Lys-143 is 67.24 A˚ in the ternary complex, whereas is 144.76 in the binary complex. Such a transition shows a potential cooperation of Lys-143 and ADP, i.e., Lys-143 can swing ADP and favor its release from the binding pocket. Further analysis shows a H-bond network is responsible for the stable open-state of Lys-143, which is produced between TPX2 and b-sheet 4 close to loop 1-2 and helix aB, including H-bonds of Tyr-199 (OH) and Tyr-10TPX2 (H), Tyr-199 (H) and Pro-13TPX2 (O) (Fig. 3.c2). The binding of TPX2 forces b-sheet 4 to undergo a translation movement to ATP direction (,0.5 A˚), definitely reducing the space between helix aB and b-sheet 4 (Fig. 3.C), which results in the formation of H-bond between His-201 (ND1) and Lys-166 (HZ1) (Fig. 3.c2). In this process, helix aB rotates clockwise (,25u) and approaches more contiguous to loop 1-2 (,1 A˚). As a result, Gln-168 (OE1) in helix aB forms a H-bond PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e16757