Project Summary Ubiquitous and conserved molecular chaperones Hsp70s and Hsp110s form one of the most essential chaperone machineries in maintaining protein homeostasis (proteostasis). However, the molecular mechanisms and exact role of Hsp110s in this chaperone machinery remain largely unclear. As distant homologs of Hsp70s, Hsp110s are both independent chaperones and co-chaperones for Hsp70s. As independent chaperones, Hsp110s demonstrate a unique high chaperone activity in preventing aggregation of denatured proteins, the “holdase” activity. As co-chaperones, Hsp110s function as the major nucleotide-exchange factors (NEFs) for Hsp70s. Although the importance and mechanism of the NEF activity is well-characterized, the function and involvement of the holdase activity is almost completely unknown primarily due to the lack of any available approach to disrupt this holdase activity without affecting the NEF activity. Msi3 is the sole and essential Hsp110 in Candida albicans, the most prevalent cause of fungal infections in human. Our preliminary studies have identified a novel inhibitor for Msi3, named 2H. Excitingly, 2H specifically abolishes the holdase activity while leaving the NEF activity largely intact. Importantly, 2H was seen to reduce protein folding both in vitro and in vivo, providing the first direct evidence to support the importance of the holdase activity of an Hsp110 in the Hsp70/Hsp110 chaperone machinery. In addition, as the first inhibitor for fungal Hsp110s, 2H effectively eliminates the growth of Candida albicans with limited toxicity in human cells, supporting that Hsp110s are an important target for designing novel and potent therapeutics for fungal infections and 2H may represent a promising lead compound for a new class of antifungals for future medicinal chemistry efforts. Taking advantage of the unique selectivity of 2H, the overall objective of this proposal is two-fold: 1) to characterize the elusive molecular mechanism and biological function of the holdase activity of Hsp110s, and 2) to increase mechanistic understanding of Hsp110s as a new therapeutic target for fungal infections. Accordingly, we propose two Specific Aims. Aim 1: Characterize the in vivo function of Hsp110’s holdase activity in the proteostasis using 2H. Taking advantage of the powerful and facile genetics available to the yeast Saccharomyces cerevisiae, we aim to directly analyze the conserved functions of the Hsp110s’ holdase activity in two essential in vivo processes in proteostasis: protein folding and import into endoplasmic reticulum. We expect to identify and characterize endogenous substrates for Hsp110s for the first time. Aim 2: Elucidate the inhibitory mechanism via structural characterization of 2H in complex with Msi3. We aim to solve the structures of Msi3, both alone and complexed with 2H, which will reveal the specific binding interactions between 2H and Msi3 and the mechanism of 2H’s inhibition of Msi3. We expect that our innovat...