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

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 His6 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.

Complementary protocols to evaluate inhibitors against the DnaK chaperone network.

Bacterial DnaK belongs to the Hsp70 chaperone family, which plays a critical role in maintaining proteostasis by catalyzing protein folding, and is a proposed antibacterial target in the pathogen Mycobacterium tuberculosis. Here, we describe an experimental toolbox for evaluating inhibitors against the mycobacterial DnaK chaperone network: a coupled-enzymatic assay to monitor ATPase activity, a proteolytic cleavage assay to study DnaK conformational changes upon ligand addition, as well as a protein renaturation assay to assess chaperone function. For complete details on the use and execution of this protocol, please refer to Hosfelt et al. (2021).

Protein Cofactor Mimics Disrupt Essential Chaperone Function in Stressed Mycobacteria.

Bacterial DnaK is an ATP-dependent molecular chaperone important for maintaining cellular proteostasis in concert with cofactor proteins. The cofactor DnaJ delivers non-native client proteins to DnaK and activates its ATPase activity, which is required for protein folding. In the bacterial pathogen Mycobacterium tuberculosis, DnaK is assisted by two DnaJs, DnaJ1 and DnaJ2. Functional protein-protein interactions (PPIs) between DnaK and at least one DnaJ are essential for survival of mycobacteria; hence, these PPIs represent untapped antibacterial targets. Here, we synthesize peptide-based mimetics of DnaJ1 and DnaJ2 N-terminal domains as rational inhibitors of DnaK-cofactor interactions. We find that covalently stabilized DnaJ mimetics are capable of disrupting DnaK-cofactor activity in vitro and prevent mycobacterial recovery from proteotoxic stress in vivo, leading to cell death. Since chaperones and cofactors are highly conserved, we anticipate these results will inform the design of other mimetics to modulate chaperone function across cell types.

An allosteric inhibitor of bacterial Hsp70 chaperone potentiates antibiotics and mitigates resistance.

DnaK is the bacterial homolog of Hsp70, an ATP-dependent chaperone that helps cofactor proteins to catalyze nascent protein folding and salvage misfolded proteins. In the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), DnaK and its cofactors are proposed antimycobacterial targets, yet few small-molecule inhibitors or probes exist for these families of proteins. Here, we describe the repurposing of a drug called telaprevir that is able to allosterically inhibit the ATPase activity of DnaK and to prevent chaperone function by mimicking peptide substrates. In mycobacterial cells, telaprevir disrupts DnaK- and cofactor-mediated cellular proteostasis, resulting in enhanced efficacy of aminoglycoside antibiotics and reduced resistance to the frontline TB drug rifampin. Hence, this work contributes to a small but growing collection of protein chaperone inhibitors, and it demonstrates that these molecules disrupt bacterial mechanisms of survival in the presence of different antibiotic classes.

Nonredundant functions of Mycobacterium tuberculosis chaperones promote survival under stress.

Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.