Two TPX2-Dependent Switches Figure 4. Superposition of the average structures of the Aurora–ADP (brown) and the Aurora–ADP–TPX2 (cyan) systems. Residues in the binary structure are shown in brown stick presentation, while residues in the ternary complex are shown in colored stick presentations: cyan for carbon, white for hydrogen, red for oxygen, and blue for nitrogen atoms. ADP is displayed in the red line. Upon the binding of TPX2, Arg–180 (HH12) forms a hydrogen bond with P?Thr–288 (O2P) to pull the latter residue in an inward conformation. The movement of P?Thr–288 is indicated by using an arrow. (d1), time–dependent rotation of Arg–180 about the (C–CA–CB–CG) dihedral angle in the binary simulation. (d2), side view of the binding region between helix aC and the upstream of TPX2. The Aurora A and the TPX2 fragment are respectively colored cyan and gray. The first H–bond network is formed between 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) (the dashed lines), thus constraining the fluctuation of Arg–180. The second hydrogen bond network is formed between Ser–284 (HG) and Asn–43TPX2 (O), Ser–284 (HG) and Asn–43TPX2 (OXT), Arg–286 (HH12) and Glu–42TPX2 (OE2), and Thr–288 (O3P) and Arg–286 (HH21) (the dashed lines), thus constraining the fluctuation of Thr–288. (d3), time evolutions of the distance between Arg–180 and P?Thr–288 in the binary and the ternary simulations. (d4), time evolutions of the distance between Glu–175 and Ser–7TPX2, Arg–179 and Tyr–10TPX2, Glu–183 and Asp–11TPX2, Arg–179 and Glu–183, and Arg–251 and Glu–37 TPX2 in the ternary simulation. (d5). time evolutions of the distance between Ser–284 and Asn–43TPX2, Ser–284 and Asn–43TPX2, Arg–286 and Glu–42TPX2, and Thr–288 and Arg–286 in the ternary simulation. doi:10.1371/journal.pone.0016757.g004 Aurora, this apo simulation was also generated from the crystal structure of Aurora A in complex with ADP and TPX2 (1ol5) by removing the ligand and TPX2 from the active site and the phosphate groups of phosphorylated Thr287 and Thr288. The Amber03 force field [22] was used with xLeap in Amber10 [23] for system setup, and AMBER force parameters for ATP, ADP [24] and Thr with unprotonated phosphate group [25] were derived from AMBER parameter database (www.pharmacy. manchester.ac.uk/bryce/amber). A box of TIP3P water molecules [26] that extended at least 12.0 A˚ outside the protein was added to solvate to each system (74.98 A˚690.58 A˚690.20 A˚ for apo- Aurora, 73.10 A˚668.40 A˚675.76 A˚ for Aurora-ATP, 78.38 A˚6 Figure 5. Cross correlation matrices of fluctuations of Aurora A atoms from their average values during the last 10 ns of the binary (e1) and ternary (e2) simulations. doi:10.1371/journal.pone.0016757.g005 PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e16757