ClpS directs degradation of primary N-end rule substrates in Mycolicibacterium smegmatis

Drug-resistant tuberculosis infections are a major threat to global public health. The essential mycobacterial ClpC1P1P2 protease has received attention as a prospective target for novel antibacterial therapeutics. However, efforts to probe its function in cells are constrained by our limited knowledge of its physiological proteolytic repertoire. Here, we interrogate the role of mycobacterial ClpS in directing N-end rule proteolysis by ClpC1P1P2 in Mycolicibacterium smegmatis. Binding assays demonstrate that mycobacterial ClpS binds canonical primary N-degrons (Leu, Phe, Tyr, Trp) with moderate affinity. N-degron binding restricts the conformational flexibility of a loop adjacent to the ClpS N-degron binding pocket and strengthens ClpS*ClpC1 binding affinity [~]30-fold, providing a mechanism for cells to prioritize N-end rule proteolysis when substrates are abundant. Proteolytic reporter assays in M. smegmatis confirm degradation of substrates bearing primary N-degrons, but suggest that secondary N-degrons are absence in mycobacteria. This work expands our understanding of the mycobacterial N-end rule pathway and identifies ClpS as a critical component for substrate specificity, providing insights that may support the development of improved Clp protease inhibitors.