Targeting ClpP proteases: From dynamics and function to allosteric modulation by drugs – ProteaseInAction

Biological cells perform their fundamental tasks through the orchestrated action of numerous proteins and cellular assemblies. Large molecular assemblies synthesize natural products, metabolites, and proteins, assist protein folding or unfolding, or allow the communication within cells and with their environment. Understanding how these "nanomachines" work on the atomic scale has stimulated decades of research. A particularly fascinating aspect of the action of such machineries is that they often function through the concerted of different parts of the molecule, often through poorly understood long-range couplings. In this project we focus on the ClpP protease, a 300 kDa large protein assembly which features some of the hallmarks of such allosteric coupling and concerted mechanisms. ClpP is able to degrade proteins which enter the central enzymatic cavity through axial pores, and cooperates with its cognate AAA+ chaperone, which delivers unfolded polypeptide chains to the protease. The caseinolytic protease (Clp) system is a paradigmatic case of a protease machinery, present in bacteria and most eukaryotes. Clp plays an active role in survival and virulence of pathogenic bacteria. Therefore, the development of drugs targeting ClpPs has recently emerged as a promising strategy to address multi-resistant bacteria. Furthermore, indications that mitochondrial ClpP expression is induced in response to cellular stress, e.g. as a consequence of tumor genesis, stresses the importance of ClpP proteases in eukaryotic protein homeostasis. While structures are available, the mechanism of the interaction between ClpP and it co-chaperone and the gate opening of the protease remain unclear. Dysrfunction of the Clp system can cause major physiological defects in bacteria. In particular, acyldepsipeptide (ADEP), a natural peptide-based compound, has been shown to activate ClpP, converting it from a highly-regulated peptidase that can degrade proteins only with the aid of its partner AAA+ to an independent and unregulated protease. ADEP treatment was shown successful to be effective antibiotics for bacteria resistant to other treatments. Furthermore, a novel class of antibiotics is able to activate ClpP, via a not fully resolved allosteric gate opening. Here we integrate information from multiple experimental techniques, centered around solution and magic-angle-spinning NMR, SAXS and EM, with advanced in silico simulation methods to elucidate the mechanisms of conformational exchange and allosteric coupling in ClpP. We investigate the effects that activators or co-chaperones have on ClpP structure and dynamics to gain insight into the mechanisms that exceed the information that any single method alone may obtain. We further address the effect of drugs on the allosteric transition leading to impaired activation of ClpP. In addition to providing mechanistic insights into protease machineries, this project pushes back the frontiers of both NMR – with new site-specific NMR dynamics experiments collected in a system that exceeds by far standard NMR applications – and MD simulations, developing original strategies for enhanced ergodic sampling.