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

James Gilburt, Hajrah Sarkar, Peter Sheldrake, Julian Blagg, Liming Ying, Charlotte Dodson

Research output: Contribution to journalArticle

9 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)11409-11414
Number of pages6
JournalAngewandte Chemie-International Edition
Volume56
Issue number38
Early online date7 Aug 2017
DOIs
Publication statusPublished - 6 Sep 2017

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Aurora Kinase A
Fluorescence Spectrometry
Drug Discovery
Protein Kinases
Phosphotransferases
Proteins
Single Molecule Imaging
MLN8054

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Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy. / Gilburt, James; Sarkar, Hajrah; Sheldrake, Peter; Blagg, Julian; Ying, Liming; Dodson, Charlotte.

In: Angewandte Chemie-International Edition, Vol. 56, No. 38, 06.09.2017, p. 11409-11414.

Research output: Contribution to journalArticle

Gilburt, James ; Sarkar, Hajrah ; Sheldrake, Peter ; Blagg, Julian ; Ying, Liming ; Dodson, Charlotte. / Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy. In: Angewandte Chemie-International Edition. 2017 ; Vol. 56, No. 38. pp. 11409-11414.
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