Two TPX2-dependent switches control the activity of Aurora A

Abstract

Aurora A is an important oncogenic kinase for mitotic spindle assembly and a potentially attractive target for human cancers. Its activation could be regulated by ATP cycle and its activator TPX2. To understand the activation mechanism of Aurora A, a series of 20 ns molecular dynamics (MD) simulations were performed on both the wild-type kinase and its mutants. Analyzing the three dynamic trajectories (Aurora A-ATP, Aurora A-ADP, and Aurora A-ADP-TPX2) at the residue level, for the first time we find two TPX2-dependent switches, i.e., switch-1 (Lys-143) and switch-2 (Arg-180), which are tightly associated with Aurora A activation. In the absence of TPX2, Lys-143 exhibits a "closed" state, and becomes hydrogen-bonded to ADP. Once TPX2 binding occurs, switch-1 is forced to "open" the binding site, thus pulling ADP away from Aurora A. Without facilitation of TPX2, switch-2 exits in an "open" conformation which accompanies the outward-flipping movement of P·Thr288 (in an inactive conformation), leading to the crucial phosphothreonine exposed and accessible for deactivation. However, with the binding of TPX2, switch-2 is forced to undergo a "closed" movement, thus capturing P·Thr288 into a buried position and locking its active conformation. Analysis of two Aurora A (K143A and R180A) mutants for the two switches further verifies their functionality and reliability in controlling Aurora activity. Our systems therefore suggest two switches determining Aurora A activation, which are important for the development of aurora kinase inhibitors.

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).

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.

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 αB, β–sheet 4 and the upstream of TPX2 is circled in dashed lines. After TPX2 binding, Tyr–199 respectively forms a H–bonds with Tyr–10^TPX2 (H) and Pro–13^TPX2(O) (the dashed lines) to pull β–sheet 4 to be more contiguous to helix αB. Due to the decreased distance between β–sheet 4 and helix αB, His–201 (ND1) interacts with Lys–166 (HZ1) to force helix αB to undergo a rotation movement (∼1 Å). This process causes the formation of a H–bond between Gln–168 (OE1) in helix αB 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 90° 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–10^TPX2 (H), and Tyr–199 (H) and Pro–13^TPX2 (O) in the ternary simulation.

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 αC 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–7^TPX2 (H3), Arg–179 (H) and Tyr–10^TPX2 (OH), Glu–183 (OE2) and Asp–11^TPX2 (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–43^TPX2 (O), Ser–284 (HG) and Asn–43^TPX2 (OXT), Arg–286 (HH12) and Glu–42^TPX2 (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–7^TPX2, Arg–179 and Tyr–10^TPX2, Glu–183 and Asp–11^TPX2, 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–43^TPX2, Ser–284 and Asn–43^TPX2, Arg–286 and Glu–42^TPX2, and Thr–288 and Arg–286 in the ternary simulation.

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.