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

Vikram Narayan,Vivien Landré,Jia Ning,Lenka Hernychova,Petr Muller,Chandra Verma,Malcolm D Walkinshaw,Elizabeth A Blackburn,Kathryn L Ball

doi:10.1074/mcp.m115.051169

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.

Highlights

CHIP-K30A has an Intrinsic Defect in E3-Ubiquitin Ligase Activity—The data presented above demonstrate that binding of an Hsp70-based peptide ligand to the Tetratricopeptide repeats (TPR)-domain of CHIP was sufficient to modulate its docking-dependent E3-ligase activity
From the ‡IGMM, University of Edinburgh Cancer Research Centre, Cell Signalling Unit, Crewe Road South, Edinburgh EH4 2XR, UK; §CTCB, Institute of Structural and Molecular Biology, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK; ¶Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic; ʈBioinformatics Institute (A*STAR), 30 Biopolis Street, 07-01 Matrix, Singapore 138671; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543; School of Biological Sciences, Nanyang Technological University, 60 Nayang Drive, Singapore 637551
In the canonical pathway, where CHIP acts as a link between the molecular chaperones and the ubiquitin proteasome system, the Tetratricopeptide repeats (TPR)-domain binds to a well-defined consensus motif in the C terminus of Hsp70 or Hsp90, facilitating the ubiquitination of client proteins [13]

Summary

Introduction

CHIP-K30A has an Intrinsic Defect in E3-Ubiquitin Ligase Activity—The data presented above demonstrate that binding of an Hsp70-based peptide ligand to the TPR-domain of CHIP was sufficient to modulate its docking-dependent E3-ligase activity. We noted that CHIP-K30A did not undergo auto-ubiquitination (Fig 2C; Ub-CHIP), suggestive of differences in its intrinsic activity in a way which, as predicted, might reflect a stabilization of the TPR-domain structure by Ala. To determine if CHIP’s intrinsic E3-ligase activity was affected by the TPR-domain mutation (K30A), the mutant protein was assayed alongside the wild-type.

Results

Conclusion