Hsp90 dependence of a kinase is determined by its conformational landscape

Qi Luo,Ville R I Kaila,Qi Wang,Johannes Buchner,Edgar E Boczek

doi:10.1038/srep43996

Abstract

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone, involved in the folding and activation of 60% of the human kinome. The oncogenic tyrosine kinase v-Src is one of the most stringent client proteins of Hsp90, whereas its almost identical homolog c-Src is only weakly affected by the chaperone. Here, we perform atomistic molecular simulations and in vitro kinase assays to explore the mechanistic differences in the activation of v-Src and c-Src. While activation in c-Src is strictly controlled by ATP-binding and phosphorylation, we find that activating conformational transitions are spontaneously sampled in Hsp90-dependent Src mutants. Phosphorylation results in an enrichment of the active conformation and in an increased affinity for Hsp90. Thus, the conformational landscape of the mutated kinase is reshaped by a broken “control switch”, resulting in perturbations of long-range electrostatics, higher activity and increased Hsp90-dependence.

Highlights

The cellular Src kinase, c-Src, is a tyrosine kinase that plays a central role in cell survival and proliferation, and its increased activity has been linked to cancer development[1,2,3]
To probe the effects of ATP-binding and Y416 phosphorylation on Src kinase dynamics, we performed microsecond molecular dynamics simulations of c-Src and c-Src3MΔC initiated from the active state
Upon phosphorylation of Y416, we find that E310-K295 is stabilized in comparison to the ATP-bound state, while the activation loop (A-loop) samples partially unfolded states, as indicated by large values (Fig. 2b)

Summary

Introduction

The cellular Src kinase, c-Src, is a tyrosine kinase that plays a central role in cell survival and proliferation, and its increased activity has been linked to cancer development[1,2,3]. C-Src contains another central phosphorylation site that is important for kinase activity[24], the conserved Y416 residue, which is located within the activation loop (A-loop). After dephosphorylation of Y527 has taken place, the c-Src activation process is further driven by ATP-binding and phosphorylation of Y416, which trigger large conformational changes in c-Src (Boczek et al, unpublished data). ATP-binding near the C-helix triggers the activation of c-Src by large conformational changes[27]. We showed that three single point mutations (R95W, D117N, and R318Q; “3M”) and a deletion of the C-terminal stretch (“ΔC”), significantly increase the activity of c-Src, transforming the kinase into a strong client protein of Hsp[90] that mimics the oncogenic v-Src[17]. It was suggested that the perturbation of central interactions near the active site causes the kinase to flicker between activation states, but the detailed underlying molecular principles still remained enigmatic

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Conclusion