Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism

Abstract

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.

Figure 1. Screening plant extracts against the DnaK-DnaJ system reveals epicatechin-3-gallate as the major inhibitor in white tea

(A) Schematic of the DnaK chaperone, with the nuceotide-binding domain (NBD), substrate-binding domain (SBD) and the NBD subdomains (IA, IIA, IB, IIB) highlighted. The co-chaperones DnaJ and GrpE are shown near their approximate binding sites on DnaK’s NBD. (B) Results of the screen of natural product extracts (40 μg/mL) against the ATPase activity of the DnaK (0.6 μM) and DnaJ (1 μM). Each extract was screened in duplicate and the range is shown relative to a solvent control. The active compounds (> 25% inhibition) are shown in open symbols (Table S1). (C) Chemical structure of ECG and its dose dependent inhibition of DnaK-DnaJ. The active component of white tea was identified by bio-assay guided fractionation and the structure confirmed by comparing the NMR spectra to an authentic sample (Figure S1). The dose dependence experiment was performed against DnaK-DnaJ in triplicate and the error bars represent standard error of the mean.

Figure 2. The structure activity relationship of flavonoid-based inhibitors of DnaK-DnaJ

(A) Approximately 80 flavonoids were chosen based on their structural similarity to EGC (Figure S2) and screened against the DnaK-DnaJ ATPase activity. The activities of select examples are shown in relation to a DMSO solvent control. The ATPase assays were performed in triplicate and the error bars represent standard error of the mean. (DnaK, 0.6 μM; DnaJ, 1 μM) (B) A summary of the structure-activity relationships, highlighting the key structural features of the flavonoid scaffold important for activity against DnaK-DnaJ. (C) Summary of the functional group decoration and activity of select flavonoids. These examples were selected to highlight key SAR observations.

Figure 3. Myricetin binds to the IB and IIB subdomains of DnaK NBD and does not compete with ATP

(A) Mapping of the chemical shifts above 0.02 ppm (pink; Figure S3A) onto DnaK suggests that myricetin binds to the upper nucleotide cleft, between the IB and IIB subdomains. (PDB: 1DKG). The approximate position of the nucleotide is shown for orientation and the cartoon schematic is used to identify the subdomains. (B) Saturating myricetin (100 μM) did not block tryptophan fluorescence (excitation 295 nm) in response to ATP. DnaK = 5 μM, ATP = 100 μM. Results are representative of experiments performed in triplicate. (C) ATP (2 mM) enhanced the apparent affinity of myricetin for DnaK, as measured by tryptophan fluorescence (Ex 295/Em. 342 nm). DnaK = 5 μM. Results are the average of triplicates and the error bars represent standard error of the mean. Error bars are often smaller than the symbols. (D) A representative conformation of myricetin in DnaK, determined by LD simulations of the myrecitin+ADP+P_i DnaK NBD (see the Methods section for details). Contacts observed are in good agreement with observed NMR shifts and SAR data. (E) Point mutations in K55A and D233A (highlighted in purple) significantly reduce binding of myricetin to DnaK, as measured by tryptophan fluorescence, compared to wild type and a control mutant (K106A). Results are the average of triplicate experiments and the error is standard error of the mean.

Figure 4. Myricetin specifically blocked DnaJ cochaperone activities

(A) In ATPase assays, myricetin blocked DnaJ-mediated stimulation. In all the ATPase experiments, the results are the average of triplicates and the error bars represent standard error of the mean. (B) Even at the highest concentrations, myricetin did not impact GrpE-mediated stimulation of ATP turnover. (C) Similarly, myricetin was unable to block stimulation by a model substrate peptide. Additional tests of this idea can be found in Figure S4.

Figure 5. GrpE enhanced myricetin activity on DnaK-DnaJ

(A) GrpE (2 μM) decreased the IC₅₀ of myricetin for the DnaK-DnaJ complex. (B) Dose dependence of GrpE action on the IC₅₀ value of myricetin. DnaK = 0.6 μM and DnaJ = 1 μM. Results are the average of triplicates and the error bars represent standard error of the mean. (C) Model for how GrpE might impact myricetin binding. Only the NBD is shown for clarity. Myricetin does not block GrpE-mediated stimulation, so it either dissociates prior to the next cycle or otherwise does not interfere with GrpE function.

Figure 6. Myricetin blocked DnaJ-mediated enhancement of DnaK’s binding to substrate and interfered with the DnaK-DnaJ interaction

(A) Myricetin inhibited the DnaJ-mediated stimulation of DnaK binding to partially-digested firefly luciferase. Each data point is the average of triplicates and the error bars represent the standard error of the mean. (B) Labeled DnaJ was titrated into fluorescent DnaK and the apparent binding affinity (K_app) measured by FRET. Myricetin, but not the control compound (catechin), partially blocked binding. The results are the average of triplicate and the error bars represent standard error of the mean. Further results are shown in Figure S5. (C) Model for the allosteric mechanism of myricetin. By impacting the clam-like motions of the subdomains, myricetin might impact DnaJ binding at a distal site. The hydrophobic cleft between subdomains IA and IIA is shown in green.