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.

‖Bioinformatics Institute 30 Biopolis Street 07 01 Matrix Singapore 138671

§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

Department of Biological Sciences National University of Singapore 14 Science Drive 4 Singapore 117543

From the ‡IGMM University of Edinburgh Cancer Research Centre Cell Signalling Unit Crewe Road South Edinburgh EH4 2XR UK

School of Biological Sciences Nanyang Technological University 60 Nayang Drive Singapore 637551

Citace poskytuje Crossref.org