Hsp90 dependence of a kinase is determined by its conformational landscape

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.

been observed in previous MD simulations 2 . The proportion of folded structures in c-Src decreases by ca. 10% during the 1 µs MD trajectory, while the protein folds remains stable in c-Src3MΔC. This difference may result from the strong interactions between the regulatory and kinase domain that imposes strain on the protein.

Extended analysis on how mutations induce electrostatic perturbations in active site dynamics
In order to quantify the difference in kinase dynamics induced by ATP-binding and phosphorylation in c-Src and in c-Src3MΔC, we analyzed the structure and electrostatic interactions of the central active site region (Supplementary Fig. S3 and Supplementary Table S5). We find that the active site residues form an extended electrostatic network, which may be involved in triggering the c-Src activation process. The C-helix has a large dipole moment, with E305/E310 at one end and K315/K316/R318 at the other end of the helix. The dynamics of this helix can thus be perturbed by surrounding charged residues, which are involved in mediating long-range couplings 3 . We find that the root-mean-square fluctuation (RMSF) of the C-helix is reduced upon ATP-binding ( Supplementary Fig. S4), which may result from the attraction between the ATP:Mg 2+ complex and conserved charged residues that form a tight ion paired network. In the ATP-bound state, E97 undergoes a significant movement and forms an ion pair with R409, while breaking the interaction with K315. This structural re-arrangement is further likely to stabilize the C-helix.
Upon phosphorylation of Y416, the strong ion pair between R409 and pY416 weakens the interaction between E310 and R409, which results in stabilization of the E310-K295 ion pair (Fig. 2b, main text). This in turn destabilizes the C-helix, as indicated by the increase in the RMSF of this region ( Supplementary Fig. S4). In addition, pY416 strongly attracts arginines in the A-loop and its immediate surroundings, forming tight ion pairs, consistent with recent findings by Meng and Roux 4 . This further leads to a partial unfolding of the A-loop.
In c-Src3MΔC, the R95W and R318Q mutations break the electrostatic network in the active site and decrease the dipole moment of the C-helix (Supplementary Fig. S3d and Supplementary Table S5), which contributes to the destabilization of the C-helix. This in turn weakens the interaction between K295 and D404, which may further contribute to the flickering of the E310-K295 ion pair.
In contrast to c-Src, ATP-binding in c-Src3MΔC stabilizes the E310-K295 ion pair and the C-helix, which results from a partially recovered ion paired network between ATP and the surrounding charged residues in the active center ( Supplementary Fig. S3e). The MD simulations further suggest that phosphorylation of Y416 in c-Src3MΔC recovers some of the electrostatic network observed in c-Src and partially compensates for the conformational perturbations caused by the mutations. Three arginines form tight ion pairs with pY416, which stabilizes the E305-R409 and D258-K315 interactions.  Table S1. Average interaction energies and standard deviations (±) between the protein and ATP:Mg 2+ complex obtained from 1 µs MD simulations.

c-Src3MΔC -ATP:Mg 2+
Total energy -442±20 Electrostatic interaction -429±22 Van der Waals interaction -13±7 Supplementary Table S2. pK a values for residues in the active site, calculated from structures obtained after 1 µs MD simulations for c-Src and c-Src3MΔC. P and D refer to residues that are protonated and deprotonated in the pH range 0-15, respectively.  Supplementary Table S5. Distances between ion pairs in the active site based on structures obtained after 1 µs MD simulations. The crystal structure (PDB ID: 1Y57) of the active state was also analyzed. The electrostatic network within this active site is perturbed due to the 3MΔC mutation, but ATP binding and pY416 recover to some extent the central interactions that stabilize the E310-K295 ion pair and the C-helix.