The E3 ubiquitin ligase CHIP and the molecular chaperone Hsc70 form a dynamic, tethered complex.

Journal Article (Journal Article)

The E3 ubiquitin ligase CHIP (C-terminus of Hsc70 Interacting Protein, a 70 kDa homodimer) binds to the molecular chaperone Hsc70 (a 70 kDa monomer), and this complex is important in both the ubiquitination of Hsc70 and the turnover of Hsc70-bound clients. Here we used NMR spectroscopy, biolayer interferometry, and fluorescence polarization to characterize the Hsc70-CHIP interaction. We found that CHIP binds tightly to two molecules of Hsc70 forming a 210 kDa complex, with a Kd of approximately 60 nM, and that the IEEVD motif at the C-terminus of Hsc70 (residues 642-646) is both necessary and sufficient for binding. Moreover, the same motif is required for CHIP-mediated ubiquitination of Hsc70 in vitro, highlighting its functional importance. Relaxation-based NMR experiments on the Hsc70-CHIP complex determined that the two partners move independently in solution, similar to "beads on a string". These results suggest that a dynamic C-terminal region of Hsc70 provides for flexibility between CHIP and the chaperone, allowing the ligase to "search" a large space and engage in productive interactions with a wide range of clients. In support of this suggestion, we find that deleting residues 623-641 of the C-terminal region, while retaining the IEEVD motif, caused a significant decrease in the efficiency of Hsc70 ubiquitination by CHIP.

Full Text

Duke Authors

Cited Authors

  • Smith, MC; Scaglione, KM; Assimon, VA; Patury, S; Thompson, AD; Dickey, CA; Southworth, DR; Paulson, HL; Gestwicki, JE; Zuiderweg, ERP

Published Date

  • August 13, 2013

Published In

Volume / Issue

  • 52 / 32

Start / End Page

  • 5354 - 5364

PubMed ID

  • 23865999

Pubmed Central ID

  • PMC3856692

Electronic International Standard Serial Number (EISSN)

  • 1520-4995

Digital Object Identifier (DOI)

  • 10.1021/bi4009209


  • eng

Conference Location

  • United States