A Co‐Chaperone Mimetic Perturbs Cellular Nitrogen Metabolism in Mycobacteria

Protein quality control in bacteria relies on dynamic chaperone networks that rapidly respond to environmental stresses. In mycobacteria, a central Hsp70 chaperone, DnaK, and its co‐chaperones, J‐domain proteins (JDPs), play essential roles in the cellular response to stress and antibiotics. Conserved “J‐domain” sequences of JDPs are known to mediate transient interactions with Hsp70s and other protein partners. We have previously shown that a rationally designed proteomimetic of a J‐domain is toxic to stressed mycobacterial cells. Here, we aimed to trap J‐domain mimetic interaction partners to potentially reveal vulnerabilities in cellular stress‐response pathways. To do so, we modified a J‐domain proteomimetic of the mycobacterial JDP DnaJ1 (J1C) with a photoaffinity label moiety, resulting in J1C–PAL. In heat‐shocked mycobacteria, J1C–PAL crosslinked to DnaK in cells, confirming retention of canonical Hsp70‐binding activity under proteotoxic stress. Chemoproteomic profiling revealed additional cellular targets, namely a conditionally essential mycobacterial protein, glutamine synthetase (GS). Biochemical assays demonstrated that J1C–PAL bound GS and inhibited its enzymatic activity. Cellular studies validated that J1C perturbed nitrogen metabolism mediated by GS in mycobacteria. Together, these findings establish J1C–PAL as a cell‐permeable chemoproteomic tool for mapping protein targets and uncover a surprising link between a co‐chaperone mimetic and bacterial metabolism.