Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy

Abstract

The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small-molecule inhibitors. By using single-molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora A kinase is in dynamic equilibrium between a DFG-in-like active T-loop conformation and a DFG-out-like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora A activating protein TPX2 shifts the equilibrium towards an active T-loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T-loop. We show that Aurora A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. Our approach will enable conformation-specific effects to be integrated into inhibitor discovery across the kinome, and we outline some immediate consequences for structure-based drug discovery.

Keywords: Aurora A; activation loop; drug design; kinases; single-molecule studies.

Figure 1

a) Current model of kinase activation: Phosphorylation on the activation loop (black line) locks the kinase in an active T‐loop conformation. b) Current equilibrium model of type II inhibitor binding: Active apo kinase is in equilibrium with inhibited kinase in an inactive T‐loop conformation. c) Proposed equilibrium model showing TMR‐labeled sites (pink stars) and the expected fluorescence signal. d) Conformational change and labeling sites in Aurora A. Active T‐loop: blue, inactive T‐loop: pink. Activation loop shown in cyan/bright pink. Labeling sites (K224 in orange and S283 in pink/cyan) shown as filled spheres. PDBs 1OL5 and 2WTV. e) Kinase activity assay for unlabeled pseudo‐wildtype Aurora A (□) and TMR‐labeled K224C/S283C (▴). Activity shown as the [ADP] produced over the course of a 1 h reaction. The difference in activity between pseudo‐wildtype and labeled protein cannot be accounted for by incomplete protein labeling (see the Supporting Information for labeling efficiency). f) Example trace of a double‐labeled K224C/S283C Aurora A single molecule showing the background‐subtracted fluorescence intensity over time.

Figure 2

Fluorescence intensity distribution for phosphorylated TMR‐labeled K224C/S283C Aurora A. a) Intensity histogram of unliganded Aurora A. b) Dwell time histogram of the quenched inactive T‐loop conformation. c–k) Fluorescence intensity distributions with c) 1 mm ATP, d) 3 mm kemptide, e) 1 mm AMP‐PNP, f) 1 mm AMP‐PNP and 3 mm kemptide, g) 5 μm TPX2, h) 5 μm TPX2, 1 mm AMPPNP, and 3 mm Kemptide, i) 10 μm MLN8054, j) 10 μm CD532, and k) 5 μm TPX2 and 10 μm MLN8054. l) Summary of the conformational preferences of Aurora A under different conditions. Error bars show propagated fitting errors.