The heat shock protein 70 (Hsp70) family aids in the maintenance of protein homeostasis, or proteostasis, across species. In bacteria, the major Hsp70, DnaK, is known as a central hub of the molecular chaperone network [1], as it collaborates with chaperonins (GroEL) [1,2], disaggregases (ClpB) [3], and other chaperones (HtpG) [4, 5, 6], as well as proteolytic machinery (Figure 1) [7]. DnaK plays an integral role in nascent protein folding [1], especially for multidomain proteins [8] and those with complex tertiary structures [1], and responds to stress by aiding in the resolution of misfolded and aggregated proteins [8,9]. Due to its varied roles, loss of dnaK can have pleiotropic effects. In Escherichia coli, ΔdnaK cells exhibit increased heat sensitivity [10], a hallmark of abrogated chaperone function due to proteome instability at higher temperatures. In the pathogen Mycobacterium tuberculosis, dnaK is essential for growth, as it is required for folding of proteins departing the ribosome [11]. In fact, mutation of dnaK affects the virulence of several pathogenic species [12,13], and impacts the evolution of antibiotic resistance mechanisms and antibiotic sensitivity [14, 15, 16, 17]. Similar to the search for inhibitors of eukaryotic Hsp70s that are implicated in cancers, there has been a recent focus on the discovery and design of molecules that can disrupt bacterial DnaK activity due to its importance in survival and pathogenesis [18]. Inhibitors of bacterial DnaK hold promise not only as antibiotics, but also as adjuvants that promote the bioactivity of existing antibiotics or counteract resistance mechanisms [19]. Finally, probes for DnaK would help to distinguish its seemingly redundant functions with other chaperones.

Targeting DnaK requires an understanding of its catalytic cycle, which is best studied in E. coli. DnaK is an ATP-dependent chaperone that cooperates with two co-chaperone or cofactor proteins, DnaJ, a J-domain protein, and GrpE, a nucleotide exchange factor [1,20, 21, 22]. DnaK is composed of a nucleotide binding domain (NBD) and substrate binding domain (SBD) connected by a flexible linker domain (Figure 1a). DnaJ binds non-native protein substrates and delivers them to DnaK in its ATP-bound “open” state, stimulating its ATPase activity and leading to formation of the ADP-bound “closed” state, which has up to 20-fold higher affinity for substrate than the open state (Figure 1b) [23, 24, 25, 26]. It should be noted that DnaJ alone can interact with DnaK to stimulate its ATP hydrolysis activity [27,28]. GrpE then exchanges ADP for ATP, leading to substrate release. The cycle repeats to facilitate preferred folding trajectories and promote unfolding of misfolded states [8].

The SBDs of eukaryotic and bacterial Hsp70s show a preference for 5–7 amino acid sequence motifs with hydrophobic residues flanked by basic residues [29]. The heptameric DnaK/Hsp70 ligand NRLLLTG [30] was first discovered in peptide binding assays, and has since been studied in complex with various Hsp70 family members along with structurally similar peptides (Figure 1c). Structural work indicates that the peptide binding cleft in the SBD of Hsp70s is similar in bacteria and eukaryotes. SBDs make key contacts with five consecutive amino acid residues in protein substrates, as well as other peptide-based ligands [30, 31, 32•].

While several recent reviews have highlighted current strategies for small molecule inhibition of bacterial and eukaryotic Hsp70s [18,33,34], here we focus on reports of peptide-based molecules that target different regions of DnaK. Many of these peptides have been inspired by primary sequences that are known to bind DnaK in nature, which we will discuss first. Based on growing structural information on DnaK, we highlight opportunities for Chemical Biologists to design new ligands that may represent antibiotic adjuvants or enable chemical genetic experiments to understand chaperone function across microbes.