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. 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. Tetratricopeptide repeats (TPR)1 are versatile structural modules conserved from E. coli to man, which function in fundamental processes such as transcriptional control, kinase signaling, protein folding and immunity (1.Cerveny L. Straskova A. Dankova V. Hartlova A. Ceckova M. Staud F. Stulik J. Tetratricopeptide repeat motifs in the world of bacterial pathogens; role in virulence mechanisms.Infection Immunity. 2012; 81: 629-635Crossref PubMed Scopus (103) Google Scholar, 2.D'Andrea L.D. Regan L. TPR proteins: The versatile helix.Trends Biochem. Sci. 2003; 28: 655-662Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar, 3.Zeytuni N. Zarivach R. Structural and functional discussion of the tetra-trico-peptide repeat, a protein interaction module.Structure. 2012; 20: 397-405Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). TPR-domains are composed of two antiparallel α-helices (containing a total of 34 amino acids) packed in tandem arrays to create a characteristic fold and binding cleft. Cleft formation facilitates protein–protein interactions and underpins the role of TPR-domains as molecular scaffolds for the assembly of multi-protein complexes (4.Andrade M.A. Perez-Iratxeta C. Ponting C.P. Protein repeats: structures, functions, and evolution.J. Struct. Biol. 2001; 134: 117-131Crossref PubMed Scopus (470) Google Scholar, 5.Smith D.F. Tetratricopeptide repeat cochaperones in steroid receptor complexes.Cell Stress Chaperones. 2004; 9: 109-121Crossref PubMed Scopus (123) Google Scholar). Althou