UCSF

ClpAP is a bacterial AAA+/protease complex responsible for regulated protein degradation of various substrates. ClpA undergoes large conformational changes coupled to ATP hydrolysis to unfold substrates targeted for degradation. The unfolded substrates are fed into the enclosed ClpP proteolytic cavity where they are degraded. This system in some drug resistant bacteria such as Mycobacterium tuberculosis is a compelling target for new antibiotics, as our current stock are rendered useless by ever evolving bacterial strains. In order to understand these systems, my thesis started with an investigation into the mechanisms of how Escherichia coli ClpAP couples large conformational changes with proteolysis. With cryoEM structures of the ClpAP complex bound to a native substrate, I propose a model where processive substrate translocation by ClpA results in a rotation relative to ClpP. Furthermore, I explore the interactions of the adaptor protein ClpS that delivers N-degron substrates to ClpAP. Again, cryoEM provides useful insight into how ClpS interacts with ClpA and how substrate is transferred between the two proteins. Finally, I examine the M. tuberculosis ClpP1P2 protease bound to a small molecule activator derived from a class of natural product inhibitor molecules. This activator stimulates ClpXP1P2 activity by mimicking a peptide agonist and binds in the active site, which aligns the catalytic residues. From my thesis work, I uncovered the mechanism for how bacterial Clp proteins process various substrates, how adaptor proteins alter these processes, and propose a novel stimulation mechanism for AAA/protease complexes.