Exploring the Conformational Landscape and Stability of Aurora A Using Ion-Mobility Mass Spectrometry and Molecular Modeling

Abstract

Protein kinase inhibitors are highly effective in treating diseases driven by aberrant kinase signaling and as chemical tools to help dissect the cellular roles of kinase signaling complexes. Evaluating the effects of binding of small molecule inhibitors on kinase conformational dynamics can assist in understanding both inhibition and resistance mechanisms. Using gas-phase ion-mobility mass spectrometry (IM-MS), we characterize changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Aided by molecular modeling, we establish three major conformations, the relative abundances of which were dependent on the Aur A activation status: one highly populated compact conformer similar to that observed in most crystal structures, a second highly populated conformer possessing a more open structure infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank. Notably, inhibitor binding induces more compact configurations of Aur A, as adopted by the unbound enzyme, with both IM-MS and modeling revealing inhibitor-mediated stabilization of active Aur A.

Figure 1

Wild-type (WT) phosphorylated Aur A (122–403) is less thermodynamically stable than a catalytically inactive nonphosphorylated D274N Aur A (122–403) variant. (A) DSF thermal stability assay with 5 μM Aur A (black), in the presence of 1 mM ATP (blue), 10 mM MgCl₂ (green), or 1 mM ATP + 10 mM MgCl₂ (red). (B) Difference in melting temperature (ΔT_m) compared with buffer control is presented for both WT and D274N Aur A (122–403).

Figure 2

Active phosphorylated Aur A (122–403) is more conformationally compact than inactive nonphosphorylated Aur A. Native ESI mass spectrum of phosphorylated active WT (A) or nonphosphorylated inactive D274N (B) Aur A (122–403). (C–E) ^TWCCS_N2→He for the [M + 11H]¹¹⁺ species of WT (C) or D274N (D) Aur A (122–403). The red line is the average of three independent replicates. Black error bars representing the S.D. Gaussian fitting was performed using the Fit Peaks Pro function in Origin (Version 2021b), with R² values listed. (E) Overlaid ^TWCCS_N2→He for WT (red), D274N (blue) Aur A, and an overall distribution from all-atom simulations (black) and Go̅-model (gray). (F) Percentage area of the four different conformational states (as determined by Gaussian fitting in C,D): I (blue), II (red), III (green), IV (yellow) for WT and D274N Aur A (122–403). Average % area presented from three individual experiments. (G) Zoomed-in view showing the position of the Phe side-chain in select example crystal structures: DFG-in (black, 1OL7), DFG-up (green, 5L8K), DFG-out (blue, 6HJK). The ATP-binding site is marked by an ADP molecule (orange) from 1OL7; this highlights the clash with the DFG-out Phe. (H) Overlay of crystal structures from 1OL7 (black) and 4C3P (red). Each 4C3P Aur A monomer exhibits a displaced A-loop and αEF helix (colored blue) compared to other Aur A crystal structures.

Figure 3

Aur-A-activating TPX2 peptide alters the conformational landscape of both phosphorylated and nonphosphorylated Aur A (122–403). (A) In vitro peptide-based Aur A kinase assays using 5 μM WT or D274N Aur A in the presence of the minimal activating TPX2 peptide at the indicated concentrations. (B,C) ^TWCCS_N2→He of the [M + 11H]¹¹⁺ species of WT phosphorylated active (B) or D274N nonphosphorylated inactive (C) Aur A in the presence of a 10 M excess of the minimal TPX2 peptide. The red line is the average of three independent replicates. Black error bars representing the S.D. Gaussian fitting was performed using the Fit Peaks Pro function in Origin (Version 2021b), with R² values listed.

Figure 4

Active Aur A (122–403) is less kinetically stable than inactive Aur A. Collision-induced unfolding profiles for the isolated 11+ charge state of WT (A) and D274N (B) Aur A (122–403) (or overlaid in (C)). Stepped collision energy was applied between 16 and 34 V in 2 V intervals. Data analysis was carried out in MassLynx 4.1, (A,B) generating heat-maps using CIUSuite 2 and (C) mountain plots using Origin (Version 2016 64Bit). Presented are data from an average of three independent experiments.

Figure 5

IM-MS of inhibitor-bound Aurora A (122–403). ^TWCCS_N2→He of the [M + 11H]¹¹⁺ species of WT phosphorylated active (left) or D274N nonphosphorylated inactive (right) Aur A (122–403) in the presence of a 10-fold M excess of (A) MLN8237, (B) VX-680, (C) ENMD-2076, (D) MK-8745, or (E) staurosporine. The red line is the average of three independent replicates. Black error bars representing the S.D. Gaussian fitting was performed using the Fit Peaks Pro function in Origin (Version 2021b), with R² values listed. (F) Percentage area of the four different conformational states (as determined by Gaussian fitting): I (blue), II (red), III (green), IV (yellow) for WT (left) and D274N (right) Aur A (122–403) in DMSO control or in the presence of the different inhibitors as indicated. Average % area presented from three individual experiments.

Figure 6

Collision-induced unfolding profiles of inhibitor-bound Aur A. The isolated 11+ charge state of (A) WT (left) and D274N (right) Aur A (122–403) in the presence of a 10 M excess of (B) MLN8237, (C) VX-680, (D) ENMD-2076, (E) MK-8745, or (F) staurosporine was subject to CIU using a stepped collision energy between 16 and 34 V (2 V intervals). Data analysis was carried out in MassLynx 4.1 (generating heat-maps using CIUSuite 2). Presented are data from a single experiment, representative of the data from independent triplicate analyses.

Figure 7

Inhibitor-induced complexation stabilizes both catalytically active and inactive Aur A. (A) DSF thermal stability assay with 5 μM Aur A + 4% DMSO (black), in the presence of 40 μM of each inhibitor. (B) Difference in melting temperature (ΔT_m) relative to 4% DMSO control is presented for both WT and D274N Aur A (122–403).