Bacterial J‐Domains with C‐Terminal Tags Contact the Substrate Binding Domain of DnaK and Sequester Chaperone Activity

Abstract

Functional interactions between the molecular chaperone DnaK with cofactor J‐proteins (DnaJs), and 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 study 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 find that His6 tags uniquely promote binding to additional sites in the substrate binding domain on 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‐terminal‐modified DnaJ1 or DnaJ2 J‐domains in mycobacterial cells leads to suppression of 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.