Activity control of standalone bacterial AAA+ disaggregases

• Applicant: Privatdozent Dr. Axel Mogk
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 556583451

The hexameric AAA+ proteins ClpG and ClpL function as superior protein disaggregases and provide extreme heat resistance to bacteria. They protect bacterial pathogens against temperature-based sterilization protocols, qualifying them as important persistence factors and potential drug target. Elucidating their regulatory and working mechanisms will provide important information for potential future drug development to inhibit or deregulate disaggregase activities. ClpG and ClpL work autonomously in contrast to canonical AAA+ disaggregases (e.g. ClpB), which require partner proteins for activation. Therefore they represent excellent model systems to study activity control of partner-independent AAA+ proteins. How is the activity of the disaggregases repressed in absence of substrate and how does substrate binding trigger AAA+ protein activation? Our unpublished work identified various layers of ClpG and ClpL activity control. AAA+ protein regulation involves the formation of alternative assembly states including interacting AAA+ rings and large AAA+ spirals. Furthermore, ATPase activity is additionally controlled in single hexameric rings. All layers are regulated by substrate binding and involve N-terminal substrate binding domains and additional extra domains including coiled-coil M-domains. We will explore the pathway of substrate-triggered disaggregase activation and how the different layers are functionally intertwined. We plan to: (1) Dissect how substrate binding converts inactive ClpG spirals into active hexameric assemblies. Substrate-induced structural changes will be determined by cryo EM and complementary XL-MS (crosslinking coupled to mass spectrometry) analysis. We will have a major focus on the regulatory functions of the substrate-binding N1 and N2 domains as they can directly link substrate binding to ClpG activation and analyze their role by extensive mutagenesis based on structural and crosslinking data. (2) Use a constitutively hexameric ClpG variant to specifically dissect the impact of substrate binding on ATPase stimulation. This will enable us to separately analyze the distinct activation steps of the AAA+ disaggregase. (3) Analyze the roles of the coiled-coil M-domain and the C-terminal disordered extension, which have opposing effects on ClpG assembly formation and dissect how their regulatory functions are intertwined. (4) Determine the dynamics of inactive ClpL and ClpG assemblies by a proximity-dependent fluorescence quenching assay reporting on interacting coiled-coil M-domains, which are crucial for assembly formation. We will test how substrate binding and regulatory domains modulate association and dissociation kinetics of the complexes.

• DFG Programme: Research Grants