Mechanisms of Hsp100 chaperones

• Applicant: Privatdozent Dr. Axel Mogk
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2008 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 62379277

Hsp100 proteins are ring-forming and ATP-fueled machines that remodel and unfold substrate proteins. Hsp100s play crucial roles in bacterial physiology, virulence and stress resistance by linking ATP-dependent threading of substrates to degradation or refolding processes. Substrate selectivities and ATPase activities of Hsp100 chaperones are tightly controlled and loss of such control is deleterious for cells. Accordingly, deregulation of Hsp100s by small molecules is increasingly recognized as novel antibacterial strategy. Hsp100s are typically repressed in the ground state and require binding of adapters and substrates to reach an activated state with high ATPase and threading activity. The modes of Hsp100 activity control are diverse and activated Hsp100s also differ in ATPase activity and threading processivity. Control mechanisms are therefore tailored to the specific functional needs of a given Hsp100 in protein remodeling.We previously revealed several mechanistic aspects relevant to this proposal. First, we determined the basic control mechanisms of the disaggregase ClpB and the unfoldase ClpC, which are activated through binding of partner proteins (Hsp70, MecA) to regulatory M-domains (MDs). A detailed dissection of the ClpB ATP hydrolysis mechanism combined with cryo EM structural analysis suggests that activation induces a sequential mode of ATP hydrolysis, coupled to coordinated substrate threading. The processivity of substrate threading is lower for ClpB as compared to other Hsp100s pointing to a so far unknown mechanism that limits the time length of activation. Second, we showed that the kinase McsB and the antibacterial small molecule Cyclomarin A (CymA) activate ClpC in a MD-independent manner, indicating novel pathways for ClpC activation. Third, we identified ClpG as stand-alone disaggregase, which confers superior heat resistance to bacteria. ClpG itself selectively binds to protein aggregates via a unique N-terminal domain. We plan to further explore the diverse mechanisms of Hsp100 activity control and substrate selection that are underlying Hsp100 functions and concurrently protecting cells from deleterious Hsp100 activities. We will address the following major questions:- How do intra- and interring communications propel a sequential ATP hydrolysis mode upon ClpB activation? Which mechanism confines ClpB threading processivity to maximize disaggregase function? - How do CymA and McsB activate ClpC independent from MDs? What is the mechanistic basis of toxic ClpC-activation by CymA?- What is the structural basis for the unique ability of ClpG to independently and selectively recognize aggregated but not soluble misfoded proteins?With these approaches we are aiming at a comprehensive understanding of the discrete steps controlling Hsp100 activities: substrate targeting, ATPase activation by partners and substrates and the conversion of high ATP rates upon activation into a substrate processing mechanical force.

• DFG Programme: Research Grants