Protein-Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)

Abstract

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control.

Fig. 1.

Hsp70 differentially modulates CHIP-dependent ubiquitination. (A, C) Immunoblot of in vitro ubiquitination reactions assembled using ATP, ubiquitin, UBE1, UbcH5a, His-CHIP and GST-IRF-1 in the presence of a titration of Hsp70 with or without Hsp40 (A) or Hsp90 (C) at either a 1:1 or 1:2 molar ratio of Hsp70/Hsp90 with CHIP. (B, E) Immunoblot of in vitro ubiquitination assays assembled as in (A) except using untagged p53 (B) or GST-BAG-1s (E) as substrate, in the presence of Hsp70 and Hsp40. (D) Snapshot of the crystal structure of mCHIP dimer (protomers in shades of gray) in complex with Hsp90 peptide (yellow sticks; PDB code 2C2L) generated using PyMOL v1.4.1. Lys³⁰ is highlighted in blue. (F, G) Immunoblot of in vitro ubiquitination reactions assembled using ATP, ubiquitin, UBE1, UbcH5a, His-CHIP and His-IRF-1 (F) or untagged p53 (G) in the presence of a titration of Hsp70 (wt: GPTIEEVD; mut: GAAAEEVD) or Hsp90 (DTSRMEEVD) peptide as indicated. A carrier only control (DMSO) was included. (H) As above, except that GST-BAG-1s was used as the substrate and both full-length Hsp70/Hsp40 as well as Hsp70 wt peptide were included in the assay as indicated. (I) As in (G) except using GST-Mdm2 as the E3 ligase.

Fig. 2.

CHIP-K30A is intrinsically defective in E3-ligase activity. (A) Close-up of the Hsp90 binding site on CHIP extracted from the crystal structure of mCHIP dimer (protomers in shades of gray; also see Fig 1D) in complex with Hsp90 peptide (yellow sticks; PDB code 2C2L) generated using PyMOL v1.4.1. Lys³⁰ on CHIP and Asp⁷³² on Hsp90 are highlighted in blue and green respectively. (B) An AlphaScreen assay was set up (see cartoon) to measure binding dynamics of His-CHIP wt or K30A mutant with biotin-tagged Hsp70 peptide (GPTIEEVD) in solution. (C) Ubiquitination of exogenous IRF-1 in H1299 cells transiently transfected with plasmids encoding CHIP wt or K30A mutant and His-tagged ubiquitin. Immunoblots show ubiquitinated protein (His-pulldown) and total protein (Direct lysis). (D, E) In vitro ubiquitination assays were assembled using ATP, ubiquitin, UBE1, UbcH5a, untagged CHIP wt or K30A, and His-IRF-1 (D) or untagged p53 (E) as substrate. Reactions were analyzed by 4–12% NuPAGE/immunoblot. (F) Immunoblot of in vitro ubiquitination assays assembled as above except in the absence of substrate to study auto-ubiquitination of untagged CHIP wt or K30A proteins over time.

Fig. 3.

CHIP-K30A and Hsp70-bound CHIP are conformationally distinct from the wild-type protein. (A) Graph showing the unfolding of His-CHIP wt pre-incubated with the indicated peptides based on Hsp70 (left panel) or His-CHIP wt or K30A mutant (right panel) as a function of temperature change measured by the uptake of the fluorescent dye SYPRO Orange. Shown is the means ± S.E. of mean of 3 experiments. (B) Table listing the mid-point temperature of phase transition (T_m) of each sample in (A) that was calculated by plotting the gradient of protein unfolding against the temperature gradient [-d(RFU)/dT]. (C) InstantBlue stained gel of untagged CHIP wt or K30A (left panel) digested with the protease Glu-C. FL is the full-length protein and band 1 is a cleavage product that persists in the K30A mutant. Band 2 is only observed in digests of the wt protein. Also shown is a Glu-C digest of His-CHIP wt protein in complex with wt or mutant Hsp70 peptides (right panel).

Fig. 4.

CHIP-K30A and Hsp70-bound CHIP have similar equilibrium structures. (A-D) Images were generated using PyMOL v.1.4.1. (A) Crystal structure of murine CHIP dimer (monomers in shades of blue) in complex with Hsp90 peptide (pink sticks; adapted from PDB 2C2L). (B) Overlay of the CHIP dimer before (blue ribbon) and after (gray mesh) 20 ns MD simulations for unliganded CHIP wt (upper left), CHIP wt in complex with Hsp90 peptide (upper right) and CHIP-K30A (bottom). (C) Overlaid snapshots of the CHIP dimer in apo and liganded forms and with Lys³⁰ mutated to Ala after 20 ns MD simulations (from (B)). (D) Root mean square fluctuation (RMSF) of Cα obtained from the trajectories of the 20 ns simulations of CHIP wt ± peptide and the CHIP-K30A mutant. The score of the positional fluctuation analysis averaged over amino acid were color coded and indicated on the crystal structure. (E) For differential scanning calorimetry, protein and buffer controls were heated at a rate of 60 °C/hour from 5 to 85 °C. The thermal transition mid-point (T_m) and specific heat capacities (C_p) were determined using the instrument software (Origin, version 7.0).

Fig. 5.

Coordinated movements between the TPR and U-box regulate CHIP activity. (A) Dynamic cross-correlation map (left panel) of Cα atoms for the un-liganded wt CHIP dimer. Correlated motions are represented above the diagonal in blue and anticorrelated below in red. Correlated movements of the CHIP U-boxes are indicated by a blue box. Anticorrelated movements of the TPR domain (right panel in brown) with both U-boxes (right panel in green) are indicated with red boxes. Cartoon of CHIP dimer (right panel) was generated using PyMOL v.1.4.1. (B) As above except the dynamic cross-correlation maps of Cα atoms are for Hsp90 peptide bound wt CHIP dimer (left panel) and the K30A mutant CHIP dimer (right panel). (C) Snapshot of the crystal structure of zebrafish CHIP-Ubox in complex with UbcH5 (from PDB 2OXQ) superimposed onto the crystal structure of mouse CHIP (from PDB 2C2L). The image, showing a single CHIP monomer, was generated using PyMOL v1.4.1. Blue ribbon: CHIP; red ribbon: UbcH5. (D) His-UbcH5a was charged with ubiquitin (Ub∼E2; thiolester linkage) by incubating with UBE1 and ubiquitin in the presence of ATP, following which ubiquitin discharge from the E2 by His-CHIP wt or K30A mutant was monitored. The E2-binding-defective mutant H260Q was included as a control. Shown is an immunoblot probed for CHIP and the E2. (E) An AlphaScreen assay was set up (see cartoon) to measure binding dynamics of untagged CHIP wt or K30A mutant anchored on protein A acceptor beads with His-tagged UbcH5a captured on Nickel-chelate donor beads in solution. (F-G) GST alone controls showed negligible binding and are therefore not indicated on the graphs. (F) Binding assay with fixed amounts of GST-IRF-1 immobilized on microtitre wells. Fixed amounts of His-CHIP wt together with a carrier control (DMSO) or a titration of Hsp70 wt or mutant peptide was added in the mobile phase. CHIP binding to IRF-1 was measured on a luminometer using an anti-CHIP antibody. (G) Upper panel: Binding assay as in (F) except that a titration of His-CHIP wt or K30A mutant was added in the mobile phase. Lower panel: Binding assay with fixed amounts of His-CHIP wt or K30A mutant coated on microtitre wells and a titration of GST-IRF-1 added in the mobile phase. (H) Binding assay as in (G) (lower panel) except that a titration of Hsp70 wt peptide was added in the mobile phase. Binding was detected using streptavidin-HRP.

Fig. 6.

Mapping conformational changes using HDX-MS. (A) Sequence of human CHIP showing the distribution of the 67 peptides identified in the HDX-MS analysis. (B) The % deuteration of a given peptide from the 60s analysis was mapped onto the crystal structure of mCHIP (PDB 2C2L). Shown is the data for wt CHIP (left) and K30A CHIP (right). (C) Graph showing the average deuteration (%) of single amino acids of CHIP wt (blue) or K30A (green) at the 60s time point calculated as described in (32). (D) Graphs showing the kinetics of deuteration for selected TPR-domain peptides from wt (blue) and K30A (green) CHIP. Amino acid number is given in the bottom right hand side of the individual graphs. (E) As in (D) except that the selected peptides were from the U-box. Amino acid numbers are shown at the top left hand corner. (F) Structural representation of CHIP dimer in complex with Ubc13 (black) and ubiquitin (white), showing the predicted orientation of the U-box in the activated E2 complex. Shown is the structure of nearly full-length dimeric CHIP (PDB 2C2L) aligned with the CHIP U-box and Ubc13 complex (E2; PDB 2C2V), followed by alignment with ubiquitin from the complex of RING domain dimer Rnf4, E2 conjugating enzyme Ubch5a and ubiquitin (PDB 4AP4). U-box core residues (red) and N-terminal helix (salmon pink) that have reduced flexibility in the K30A-CHIP mutant are indicated. The U-boxes form the main dimer interface between CHIP protomers and also function as scaffolds for loops known to be involved in protein-protein interactions.