Allosteric mechanisms of AAA+ machines: the case of the central bacterial protease ClpC/ClpP

• Applicant: Privatdozent Dr. Axel Mogk
• Subject Area: Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 497321492

Hsp100 chaperones are ring-forming ATP-fueled protein quality machines that play crucial roles in bacterial physiology and virulence. They use mechanical work to thread substrates through their central channel, leading to disassembly or unfolding of cellular targets. Many Hsp100 proteins (e.g. ClpC) associate with peptidases (e.g. ClpP) to form bacterial proteasomes. The degradation activities of proteases are potentially deleterious; substrate selection and ATPase activities of Hsp100 proteins are therefore tightly controlled. This is achieved by specific partner proteins (adaptors) that provide substrate specificity and strongly increase Hsp100 ATPase activity upon substrate transfer. Persistent, adaptor-independent activation of Hsp100 proteins is highly toxic to cells and therefore represents a useful anti-bacterial strategy. There are multiple conundrums related to the mechanism of operation of Hsp100 proteins. Here we propose to use ClpC/ClpP, the central protease of the important pathogen Staphylococcus aureus, as a model that will allow us to tackle many of these open questions. ClpC is composed of two ATPase domains that form two distinct rings in the hexameric assembly and additional domains that mediate binding to diverse adaptors. It combines with the heptameric peptidase ClpP to form an effective ATP-driven protein degradation machine. We would like to understand: How is the activity of the machine regulated by various binding effectors? How does the hexameric ClpC cooperate with the heptameric peptidase ClpP during function? And how is ATP hydrolysis coupled to substrate transport through the protein’s pore? We plan to tackle these key questions by combining structural, biochemical and single-molecule protein dynamics studies, which rely on the strong complementary expertise of the two participating groups. We base our plans on our vast experience in the Hsp100 field and on the expertise of Gilad Haran’s lab in applying single-molecule FRET (smFRET) spectroscopy to study the dynamics of complex molecular machines. In particular, we will (i) define the structural basis of the specific regulatory circuits controlling ClpC function by a combination of cryo-EM structure determination of Hsp100 hexamers in diverse activity states with smFRET studies of machine dynamics. We will further (ii) study machine mechanics by directly observing substrate threading through the central pore in real time, and exploring (potentially rotational) motions at the symmetry mismatched interface of the ClpC hexamer and the ClpP heptamer. The project will culminate with (iii) a search for small-molecule activators of ClpC that can serve as potential novel antibiotics, thus bringing the biophysical-biochemical knowledge acquired here close to medical application.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professor Dr. Gilad Haran