PES inhibits human-inducible Hsp70 by covalent targeting of cysteine residues in the substrate-binding domain

Abstract

Hsp70 proteins are a family of ancient and conserved chaperones. They play important roles in vital cellular processes, such as protein quality control and the stress response. Hsp70 proteins are a potential drug target for treatment of disease, particularly cancer. PES (2-phenylethynesulfonamide or pifithrin-μ) has been reported to be an inhibitor of Hsp70. However, the mechanism of PES inhibition is still unclear. In this study we found that PES can undergo a Michael addition reaction with Cys-574 and Cys-603 in the SBDα of human HspA1A (hHsp70), resulting in covalent attachment of a PES molecule to each Cys residue. We previously showed that glutathionylation of Cys-574 and Cys-603 affects the structure and function of hHsp70. In this study, PES modification showed similar structural and functional effects on hHsp70 to glutathionylation. Further, we found that susceptibility to PES modification is influenced by changes in the conformational dynamics of the SBDα, such as are induced by interaction with adjacent domains, allosteric changes, and mutations. This study provides new avenues for development of covalent inhibitors of hHsp70.

Keywords: Hsp70; PES; covalent inhibitor; cysteine modification.

Figure 1

Schematic diagram of the Michael addition reaction between cysteine residues in hHsp70 and PES. hHsp70, human HspA1A.

Figure 2

Covalent binding of PES to hHsp70 detected by mass spectrometry.A–D, the molecular weight of full-length hHsp70 (A–B) and the SBD of hHsp70 (C–D) were detected by Q-TOF mass spectrometry before (A and C) and after (B and D) 24-h incubation with PES. After incubation with PES, both the molecular weight of full-length hHsp70 and the SBD of hHsp70 increased by ∼362 Da, which is the molecular weight of two PES molecules (181 Da). E–F, detection by mass spectrometry of PES modification at Cys-574 (E) and Cys-603 (F) of WT hHsp70 treated with PES in the presence of ADP. NanoLC-LTQ-Orbitrap XL analysis confirmed the presence of the PES-modified peptide VLDKC∗(574)QEVISWLDANTLAEK (E) and the PES-modified peptide RKELEQVC∗(603)NPIISGLYQGAGGPGPGGFGAQGPK (F) after trypsin digestion. The detected peaks (main panel) correspond to the predicted peptides (inset), where red corresponds to observed N-terminal peptide fragments and blue corresponds to observed C-terminal peptide fragments. C∗ indicates Cys-574 and Cys-603, which undergo PES modification. G–H, the molecular weight of the SBDα of hHsp70 was detected by Q-TOF mass spectrometry before (G) and after (H) 24-h incubation with PES. After incubation with PES, the molecular weight of most hHsp70 SBDα is unchanged, and only a small proportion shows an increased molecular weight. hHsp70, human HspA1A.

Figure 3

Covalent attachment of PES to hHsp70 results in structural changes in the SBD similar to those induced by glutathionylation of Cys residues. Conformation and secondary structure of untreated control (black) and PES-treated (red) SBD(385–641) were compared by SEC (A), intrinsic tryptophan fluorescence (after excitation at 295 nm) (B) and far-UV CD (C). D, far-UV CD spectra of control (black) and PES-treated (red) SBD(385–641)-AA were compared. E, ¹H-¹⁵N HSQC spectra of untreated control (black), PES-treated (red), and glutathionylated (-G, blue) SBD(385–641) were compared. F, ¹H-¹⁵N HSQC spectra of untreated control (black) and PES-treated (red) SBD(385–641)-AA were compared. hHsp70, human HspA1A.

Figure 4

Covalent attachment of PES to hHsp70 results in functional changes of hHsp70.A, the effect of PES and PES-Cl on the ATPase activity of hHsp70 and hHsc70 was detected and compared with glutathionylated hHsp70. B, peptide binding ability of untreated control (black), PES-treated (red), and glutathionylated (-G, blue) full-length hHsp70 (residues 1–641) in the presence of 0.5 mM ADP in Buffer B was compared. Fluorescence polarization (FP) at 520 nm after excitation at 485 nm was used to monitor the binding of 20 nM FITC-labeled ALLLSAPRR (FAR) peptide to different concentrations of hHsp70 or its mutants, as indicated. C, the stimulatory effects of cochaperones Hdj1 (2 μM) and Bag1 (0.5 μM) on ATPase activity of PES and PES-Cl modified hHsp70 and hHsc70 (1 μM) were compared. D–E, the effects of PES and PES-Cl on luciferase refolding activity of hHsp70 and hHsc70 were measured. The stimulatory effects of the cochaperone Hdj1 (0.5 μM) on luciferase refolding activity of PES and PES-Cl modified hHsp70 and hHsc70 (1 μM) were measured after 2-h refolding at 37 °C (D), and the time course of luciferase refolding in the presence of Hdj1 (0.5 μM) and PES and PES-Cl-modified or unmodified hHsp70 and hHsc70 (1 μM) was also compared as indicated (E). PES and PES-Cl-treated hHsp70 and hHsc70 were prepared by incubating hHsp70 or hHsc70 in the presence of 1 mM PES or PES-Cl and 1 mM ADP at 37 °C for 24 h followed by dialysis at 4 °C to remove PES, PES-Cl, and ADP. The data shown are the mean of three individual experiments and the error bars represent the standard error of the mean. hHSc70, human HspA8; hHsp70, human HspA1A.

Figure 5

Covalent binding of PES to the SBDα of hHsp70 is affected by domain communication and allostery of hHsp70. The time course of conformational changes accompanying PES modification of 10 μM hHsp70 or its mutants was recorded by monitoring the CSM of the intrinsic fluorescence spectrum. PES modification was induced by 1 mM PES. A, PES modification kinetics of full-length hHsp70, hHsp70 NBD(1–385), hHsp70 SBD(385–641), and hHsp70 SBDα(511–641) in the presence of 0.5 mM ADP were compared. B, PES modification kinetics of hHsp70 T204 in the presence of 0.5 mM ADP or ATP were compared. C, PES modification kinetics of hHsp70 SBD(385–641) in the absence or presence of 1 mM AR peptide (ALLLSAPRR) were compared. D, PES modification kinetics of full-length hHsp70 in the presence of 0.5 mM ADP and in the absence or presence of 1 mM AR peptide were compared. hHsp70, human HspA1A.

Figure 6

Covalent attachment of PES to the SBDα of hHsp70 is affected by the conformational dynamics of the SBDα. Measurement of PES modification kinetics was performed as in Figure 5. A, PES modification kinetics of hHsp70 SBDα(537–610) and hHsp70 SBDα(511–641) were compared. B, PES modification kinetics of full-length hHsp70, hHsp70-CSSCA and hHsp70-CSSAC in the presence of 0.5 mM ADP were compared. C, the crystal structure of the SBD of hHsp70 (PDB code 4PO2, in blue) and the NMR structure of the isolated SBDα(537–610) of hHs70 (PDB code 2LMG, in violet) were aligned. The arrow indicates the possible PES binding site in hHsp70. D, thermal denaturation of hHsp70 SBDα(537–610), SBDα(537–641), SBDα(525–641), and SBDα(511–641) was monitored by the CD signal at 222 nm. hHsp70, human HspA1A.

Figure 7

Difference between hHsp70 and hHsc70 in covalent binding of PES. Measurement of PES modification kinetics was performed as in Figure 5. A, alignment of the SBDs of hHsp70 and hHsc70, with Cys-574 and Cys-603 indicated by boxes, and Trp-580 indicated with an asterisk. B, PES modification kinetics of hHsp70, hHsc70, and the chimeric hHsp70-hHsc70(α) (see Table 1) in the presence of 0.5 mM ADP were compared. C, PES modification kinetics of hHsp70 SBD(385–641) and hHsc70 SBD(385–646) were compared. hHsc70, human HspA8.

Figure 8

Difference between PES and PES-Cl in covalent binding to hHsp70. Measurement of modification kinetics was performed as in Figure 5, in the presence of 0.5 mM ADP. hHsp70, human HspA1A.