Molecular chaperones DnaK and DnaJ share predicted binding sites on most proteins in the E. coli proteome

Abstract

In Escherichia coli, the molecular chaperones DnaK and DnaJ cooperate to assist the folding of newly synthesized or unfolded polypeptides. DnaK and DnaJ bind to hydrophobic motifs in these proteins and they also bind to each other. Together, this system is thought to be sufficiently versatile to act on the entire proteome, which creates interesting challenges in understanding the interactions between DnaK, DnaJ and their thousands of potential substrates. To address this question, we computationally predicted the number and frequency of DnaK- and DnaJ-binding motifs in the E. coli proteome, guided by free energy-based binding consensus motifs. This analysis revealed that nearly every protein is predicted to contain multiple DnaK- and DnaJ-binding sites, with the DnaJ sites occurring approximately twice as often. Further, we found that an overwhelming majority of the DnaK sites partially or completely overlapped with the DnaJ-binding motifs. It is well known that high concentrations of DnaJ inhibit DnaK-DnaJ-mediated refolding. The observed overlapping binding sites suggest that this phenomenon may be explained by an important balance in the relative stoichiometry of DnaK and DnaJ. To test this idea, we measured the chaperone-assisted folding of two denatured substrates and found that the distribution of predicted DnaK- and DnaJ-binding sites was indeed a good predictor of the optimal stoichiometry required for folding. These studies provide insight into how DnaK and DnaJ might cooperate to maintain global protein homeostasis.

FIGURE 1. DNAK ATPASE CATALYTIC CYCLE

J-domain proteins recruit client proteins, dock to NBD of DnaK, and catalyze ATP hydrolysis. This converts DnaK from the ATP bound low affinity conformation to the ADP bound conformation with high affinity for client proteins. Nucleotide exchange factors (NEFs), such as GrpE, trigger ADP release. Binding of ATP completes the catalytic cycle by causing release of client protein and co-chaperones.

FIGURE 2. CONSENSUS BINDING SEQUENCES OF E. COLI MOECLULAR CHAPERONES

(A) DnaK: Hydrophobic residues, e.g. Leu, Ile, Gly, Val, and basic residues (Arg, Lys) are highly preferred, while acidic residues are disfavored. (B) DnaJ: Hydrophobic and aromatic residues are clearly preferred, but in a position-specific manner. Figure made using WebLogo Tool (Crooks 2004).

FIGURE 3. PROTEOME-WIDE MAPPING OF DNAK AND DNAJ BINDING SITES IN E. COLI

(A) Venn diagram of DnaK and DnaJ-binders. The majority of the proteome is bound by both chaperones, with only one peptide binding neither (UniProtID: P60995). (B) Average binding affinities for DnaK and DnaJ across the proteome. Values given as −ΔΔGbinding. (C) Chaperones binding site coverage. Values are given as percent coverage of individual protein lengths, averaged over the entire proteome.

FIGURE 4. MOST DNAK SITES ARE ALSO DNAJ SITES

(A) Chaperone binding site composition given as unique, overlapped (by corresponding chaperone), or identical, given as percent of total sites (DnaK: 95,558; DnaJ: 224,530). (B) Binding site affinity shows little to no variation across the three types of binding sites. Affinity values given as −ΔΔGbinding.

FIGURE 5. MODEL FOR THE ARRANGEMENT OF DNAK AND DNAJ BINDING SITES ON UNFOLDED SUBSTRATE PROTEINS

Unique DnaJ sites are intersperced between common DnaK and DnaJ binding targets.

FIGURE 6. GLASS-SLIDE PEPTIDE MICROARRAYS CONFIRM COMPUTATIONAL PREDICTION OF CHAPERONE BINDING SITES ON LUCIFERASE

(A) and (B). His-tagged DnaK and DnaJ,respectively, binding of luciferase confirm in silico predicitions (Figure 6). Results correlate well with previously published platforms (Rudiger 1997; Rudiger 2001). (C) Quantified binding site coverage for molecular chaperones on luciferase. Fluorescence is normalized to maximum values for each array.

FIGURE 7. DNAJ DIFFERENTIALLY INHIBITS LUCIFERASE AND MDH REFOLDING

(A) In vitro refolding assay shows DnaJ inhibiting refolding of luciferase at lower concentrations than for MDH. (B) and (C) Computational prediction of chaperone binding sites. Note luciferase contains many more DnaJ sites that bind at higher affinity than MDH.