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.