A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins

Abstract

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.

Figure 1. Structure of the CHIP-TPR/Hsc70-lid-tail Complex

(A) Arrangement of domains within CHIP and Hsc70. (B) Cartoon view of the Hsc70-lid-tail (Hsc70_541-646Δ_626-638) in complex with the CHIP-TPR domain (CHIP_21-154). Hsc70-lid-tail and CHIP-TPR domains are colored orange and grey respectively. Specific CHIP-TPR residues that interact with Hsc70-lid, Hsc70-tail, or both domains are colored green, purple and yellow respectively. (C) Alignment of human, murine and bovine Hsc70- and Hsp70-lid-tail sequences with secondary structure overlay. Lid and tail residues that interact with the CHIP-TPR are colored orange. See also Figures S1, S2 and S3.

Figure 2. Residues that mediate CHIP-TPR/Hsc70-lid interactions modulate CHIP-mediated ubiquitination

(A) Western blot with anti-GST antibody to detect ubiquitination of GST-Hsc70_395-646 by wild type (WT) and mutant CHIP_1-303. (B) Western blot with anti-CHIP antibody to detect autoubiquitination of wild type (WT) and mutant CHIP_1-303. (C) Pull-down of GST~Hsc70_395-646 (GST~Hsc70-SBDβ-lid-tail) by wild type (WT) and mutant His₆-CHIP_1-303 bound to Ni²⁺ mag-sepharose beads analyzed by SDS PAGE with Coomassie staining. Lanes indicate GST~Hsc70_395-646 and CHIP_1-303 components utilized (INPUT), the final wash fraction (Wash) and the elution fraction (Elute). See also Figure S4.

Figure 3. Hsc70 residues that mediate CHIP-TPR/Hsc70-lid interactions modulate CHIP-mediated ubiquitination

(A) Western blot with anti-GST antibody to detect ubiquitination of wild type (WT) and mutant GST~Hsc70_395-646 (GST~Hsc70-SBDβ-lid-tail) constructs by wild type CHIP_1-303. (B) Pull-down of wild type (WT) and mutant GST~Hsc70_395-646 constructs by wild type His₆-CHIP_1-303 bound to Ni²⁺ mag-sepharose beads analyzed by SDS PAGE with Coomassie staining. Lanes indicate the GST~Hsc70_395-646 and CHIP_1-303 components utilized (INPUT), the final wash fraction (Wash) and the elution fraction (Elute).

Figure 4. CHIP-mediated Chaperoned Ubiquitination in vitro and ex vivo

(A) CHIP mediated ubiquitination of firefly luciferase was monitored by western blot using an anti-luciferase antibody. Control reactions were carried out with all reagents present except Hsp70, UbcH5b or CHIP respectively. (B) iNOS expression, induced by LPS and IFN-γ treatment, was monitored for 36 hours in Raw264.7 macrophages transfected with wild-type CHIP and CHIP mutants in which interactions between the U-box and E2 enzymes are disrupted (I235A, R272A) or the Hsp70-lid:TPR interaction is disrupted (V59D/L129D). Protein levels were monitored by western blot with anti-iNOS, anti-CHIP and anti-GAPDH (loading control). (C) HEK293 cells were transfected with ^GFPiNOS and CHIP or CHIP mutants that disrupt E2:U-box interactions (I235A or R272A) or the Hsp70-lid:TPR interaction (V59D/L129D). Cells were incubated for 48 hours prior to imaging. (D) Cells from (C) were then collected, lysed and analyzed by western blot with anti-GFP, anti-CHIP and anti-β-actin (loading control) (E) Western blots from (D) were repeated six times and band intensities were quantified. Data represent mean±SD. *P<0.05. **P<0.01. ***P<0.001.

Figure 5. Posttranslational modifications of Hsc70 residues modulate CHIP-mediated ubiquitination

(A) Western blot with anti-GST antibody to detect ubiquitination of GST~Hsc70_395-646Y611E phosphomimic, GST~Hsc70_395-646K561R -dimethyl-mimetic in comparison to wild type (WT) GST~Hsc70_395-646 by wild type CHIP_1-303. (B) Pull-down of GST~Hsc70_395-646 Y611E and K561R mutant constructs by wild type His₆-CHIP_1-303 bound to Ni²⁺ mag-sepharose beads, analyzed by SDS PAGE with Coomassie staining. Lanes indicate the GST~Hsc70_395-646 and CHIP_1-303 components utilized (INPUT), the final wash fraction (Wash) and the elution fraction (Elute). See also Figure S5.

Figure 6. Models of Chaperoned Ubiquitination Complexes in ADP- and ATP-Bound States

(A) A model of Hsp70, based on the structure of ADP-bound E. coli DnaK (Bertelsen et al., 2009) is fully compatible with binding to the TPR domain of CHIP protomer with an occluded U-box. CHIP, CHIP-TPR, UbcH5, Ub, Hsc70-Lid, Hsc70-Tail, Hsc70-SBDβ and Hsc70-NBD are colored white, grey, purple, green, orange, black, wheat and blue, respectively. (B) ADP-bound Hsp70 is also compatible with binding to TPR domain of CHIP protomer with accessible U-box. (C) A model of ATP-bound Hsp70, based on the structure of DnaK in the ATP-bound form (Qi et al., 2013), is compatible with binding to CHIP via the TPR domain of the protomer with an occluded U-box. (D) The CHIP-TPR from a protomer with an accessible U-box is also compatible with binding to ATP-bound Hsp70.