Mechanisms of Hsp100 chaperones

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Bacterial Hsp100 chaperones are central components of protein quality control systems. They form hexameric ring structures and thread protein substrates in an ATP-fueled process through their central channel. Hsp100s come in two flavors. Some (e.g. ClpC, ClpE) associate with peptidases (e.g. ClpP) to form proteolytic complexes targeting misfolded substrates to degradation. Others (e.g. ClpB, ClpG) function as disaggregases reactivating aggregated proteins. The ATPase and threading activities of Hsp100 are tightly controlled to protect cells from deleterious unfolding events. This can be achieved by partner proteins, which target Hsp100s to their substrates while concurrently stimulating ATPase activities. We previously dissected the regulation of the partner-dependent ClpB and ClpC chaperones. M-domains (MDs) function as molecular switches, repressing ClpB/ClpC ATPase activities in absence of partners, while also enabling for activation by additionally serving as partner binding sites. In the former funding period we identified ClpG, ClpL and ClpE as partner-independent Hsp100 family members, raising the question how their ATPase and threading activities are regulated. We observe that these Hsp100s form diverse, large assemblies consisting of individual rings. Ring interactions are mediated via MDs. MD mutants that only form hexamers exhibit toxicity in vivo, while mutants that enhance assembly formation exhibit reduced activity. These findings underline the functional relevance of ring assemblies and point to a novel mode of Hsp100 regulation. We plan to:- Determine the dynamics of ring interactions and study how interventions that stabilize or destabilize the assemblies affect Hsp100 activities- Dissect how the assembly pathway is altered by substrate proteins- Determine the architecture of Hsp100 ring assemblies by cryo EM tomographyWe additionally identified ClpG and ClpL as standalone disaggregases providing enhanced resistance to Gram-negative (ClpG) and Gram-positive bacteria (ClpL), against thermal-based sterilization protocols applied in food industry and hospitals. Both disaggregases are encoded on plasmids or mobile genomic islands allowing for spreading of these persistence factors among bacterial communities. ClpG and ClpL target aggregated but not soluble misfolded proteins, protecting nascent polypeptides from the potent unfoldases. This specificity is provided by distinct N-terminal domains (NDs), which are essential and sufficient for aggregate binding. We plan to:- Determine the structures of the aggregate-binding NDs of ClpG and ClpL and identify and validate substrate binding sites- Determine how many NDs must be present in a ring to provide selectivity for protein aggregates- Study how both disaggregases cooperate with sHsp chaperones that organize protein aggregation- Transfer enhanced heat resistance to higher eukaryotes (e.g. plants) by heterologous expression of clpG and clpL genes