Tania J. Lupoli

Functional interactions between the molecular chaperone DnaK and cofactor J‐proteins (DnaJs), as well as their homologs, are crucial to the maintenance of proteostasis across cell types. In the bacterial pathogen Mycobacterium tuberculosis , DnaK–DnaJ interactions are essential for cell growth and represent potential targets for antibiotic or adjuvant development. While the N‐terminal J‐domains of J‐proteins are known to form important contacts with DnaK, C‐terminal domains have varied roles. Here, we have studied the effect of adding C‐terminal tags to N‐terminal J‐domain truncations of mycobacterial DnaJ1 and DnaJ2 to promote additional interactions with DnaK. We found that His 6 tags uniquely promote binding to additional sites in the substrate binding domain at the C‐terminus of DnaK. Other C‐terminal tags attached to J‐domains, even peptides known to interact with DnaK, do not produce the same effects. Expression of C‐terminally modified DnaJ1 or DnaJ2 J‐domains in mycobacterial cells suppresses chaperone activity following proteotoxic stress, which is exaggerated in the presence of a small‐molecule DnaK inhibitor. Hence, this work uncovers genetically encodable J‐protein variants that may be used to study chaperone–cofactor interactions in other organisms.