Protein-Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP).
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.
From the ‡IGMM, University of Edinburgh Cancer Research Centre, Cell Signalling Unit, Crewe Road South, Edinburgh EH4 2XR, UK;
Vikram
V
Narayan
From the ‡IGMM, University of Edinburgh Cancer Research Centre, Cell Signalling Unit, Crewe Road South, Edinburgh EH4 2XR, UK;
Vivien
V
Landré
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;
Jia
J
Ning
¶Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic;
Lenka
L
Hernychova
¶Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic;
Petr
P
Muller
‖Bioinformatics 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.
Chandra
C
Verma
§CTCB, Institute of Structural and Molecular Biology, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK;
Malcolm D
MD
Walkinshaw
§CTCB, Institute of Structural and Molecular Biology, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK;
Elizabeth A
EA
Blackburn
From the ‡IGMM, University of Edinburgh Cancer Research Centre, Cell Signalling Unit, Crewe Road South, Edinburgh EH4 2XR, UK; kathryn.ball@ed.ac.uk.
Kathryn L
KL
Ball
D000494:Allosteric Regulation; D001665:Binding Sites; D045744:Cell Line, Tumor; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
D018840:HSP70 Heat-Shock Proteins; D050856:Interferon Regulatory Factor-1; D016159:Tumor Suppressor Protein p53; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex