Relating protein interaction networks to physiology by systematic mutant analyses

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Hsp90 is an essential eukaryotic chaperone that helps to produce and maintain the active state of a select set of substrates or clients that are disproportionately linked to signaling processes including many that promote cancer and the emergence of drug resistance in fungi. For these reasons, inhibitor strategies to manipulate the Hsp90 chaperone system promise many potential therapeutic benefits. Inhibitors targeting the ATPase site of Hsp90 effectively limit the emergence of drug resistance in fungi and show promise as anti-cancer agents. However, ATPase inhibitors block all known functions of Hsp90, leading to undesirable side effects. In our previous work, we generated a comprehensive library of all possible mutations at each of the 709 positions in Hsp90 and quantified the effects of each variant on yeast growth rate as a readout of overall network function integrated over all client proteins. We will build on this work to investigate Hsp90-client interaction mechanism. We will determine how four specific clients are impacted by Hsp90 mutations, which will identify sites on Hsp90 that could be targeted to inhibit specific clients. We will perform deep mutational scanning on specific client proteins to identify the biophysical features that determine Hsp90 dependence. In addition, we have developed a novel approach to quantify the dominant effects of Hsp90 mutations, which we are using as a powerful new route to understand its mechanism of action. Our studies will provide important mechanistic insights into a protein-protein interaction network that is biologically and medically important.