ClpS Directs Degradation of N‐Degron Substrates With Primary Destabilizing Residues in Mycolicibacterium smegmatis

ABSTRACT 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‐degron pathway proteolysis by ClpC1P1P2 in Mycolicibacterium smegmatis . Binding assays demonstrate that mycobacterial ClpS binds canonical primary destabilizing residues (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‐degron 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 absent in mycobacteria. This work expands our understanding of the mycobacterial N‐degron pathway and identifies ClpS as a critical component for substrate specificity, providing insights that may support the development of improved Clp protease inhibitors.