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

Biochemistry. 2013 Aug 13;52(32):5354-64. doi: 10.1021/bi4009209. Epub 2013 Aug 2.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • HSC70 Heat-Shock Proteins / chemistry*
  • HSC70 Heat-Shock Proteins / metabolism
  • Humans
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Structure, Tertiary
  • Surface Plasmon Resonance
  • Ubiquitin-Protein Ligases / chemistry*
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination

Substances

  • HSC70 Heat-Shock Proteins
  • HSPA8 protein, human
  • STUB1 protein, human
  • Ubiquitin-Protein Ligases