Using single-molecule spectroscopy to measure conformational changes in c-Met kinase with clinically arising resistance mutations Tamsin Wilcock, Charlotte Dodson, Liming Ying and Ian Eggleston University of Bath, UK Protein kinase enzymatic activity is governed by the conformation of a region of the kinase domain known as the activation loop, thought to exist in an equilibrium between active and inactive states. We have determined the dynamic behaviour of the activation loop of a receptor tyrosine kinase, c-Met, and of clinically arising mutants, which are resistant to current cancer drugs. We have successfully optimised expression of recombinant c-Met kinase domain in E. coli by coexpression with chaperone plasmid GroES-EL and labelled our samples with fluorescent dyes for single-molecule microscopy. Single molecule intensity frequency histograms show that the unphosphorylated kinase adopts both active and inactive conformations, with the majority of wildtype (79%), D1228V mutant (82%) and Y1230H mutant (84%) molecules occupying the inactive conformation. Crizotinib, a c-Met inhibitor currently in clinical use, shifts the equilibrium of wildtype c-Met towards almost 100% of molecules in an inactive conformation. In contrast, the addition of Crizotinib has minimal impact on the conformational equilibrium of the D1228V and Y1230H clinical mutants. c-Met conformational dynamics are currently being monitored bound to other ligands, including AMP-PNP, ADP and other small-molecule inhibitors Foretinib, BMS- 777607 and Savolitinib. We will use this data to model c-Met conformational behaviour and determine the effect of clinically arising mutations on inhibitor binding. Residues involved in c-Met clinical mutations are highly conserved among receptor tyrosine kinases regulated by double phosphorylation therefore, we expect our results to be applicable to related kinases, such as ALK, INSR, RET, VEGFR1/3, KDR and TRKA/B/C. References 1. This is unpublished work, previous work using this technique: 2. Gilburt, J. A. H., Sarkar, H., Sheldrake, P., Blagg, J., Ying, L., & Dodson, C. A. (2017). Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy. Angewandte Chemie - International Edition , 56 (38), 11409–11414. https://doi.org/10.1002/anie.201704654
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