Detection of Ligand‐induced Conformational Changes in the Activation Loop of Aurora‐A Kinase by PELDOR Spectroscopy

Selena G Burgess,Maria Grazia Concilio,Richard Bayliss,Alistair J Fielding

doi:10.1002/open.201600101

Selena G Burgess, Maria Grazia Concilio + Show 2 more

Open Access

https://doi.org/10.1002/open.201600101

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Journal: ChemistryOpen	Publication Date: Nov 11, 2016
Citations: 5	License type: CC BY 4.0

Affiliation: University of Leeds, University of Manchester

Abstract

The structure of protein kinases has been extensively studied by protein crystallography. Conformational movement of the kinase activation loop is thought to be crucial for regulation of activity; however, in many cases the position of the activation loop in solution is unknown. Protein kinases are an important class of therapeutic target and kinase inhibitors are classified by their effect on the activation loop. Here, we report the use of pulsed electron double resonance (PELDOR) and site‐directed spin labeling to monitor conformational changes through the insertion of MTSL [S‐(1‐oxyl‐2,2,5,5‐tetramethyl‐2,5‐dihydro‐1 H‐pyrrol‐3‐yl)methyl methanesulfonothioate] on the dynamic activation loop and a stable site on the outer surface of the enzyme. The action of different ligands such as microtubule‐associated protein (TPX2) and inhibitors could be discriminated as well as their ability to lock the activation loop in a fixed conformation. This study provides evidence for structural adaptations that could be used for drug design and a methodological approach that has potential to characterize inhibitors in development.

Highlights

The structure of protein kinases has been extensively studied by protein crystallography
Most protein kinase inhibitors[6] compete for the ATP binding site and are further classified as either Type I, which bind to the kinase in its active state, or Type II, which bind to an inactive DFG-out conformation of the kinase and occupy an additional hydrophobic pocket within the active site
Validation of the effect of inhibitors requires determination of the kinase-inhibitor structure by X-ray crystallography, and some compounds that would be classified as Type I based on their chemical structures induce DFG-flipped conformations of the activation loop that resemble Type II[7,8,9] inhibitors

Summary

Introduction

The structure of protein kinases has been extensively studied by protein crystallography. Methods for analyzing the activation loop conformation in solution would be invaluable in the development and characterization of kinase inhibitors, enabling classification in the absence of crystal structures.[10,11,12]

Results

Conclusion