The E3 Ubiquitin Ligase CHIP and the Molecular Chaperone Hsc70 Form a Dynamic, Tethered Complex
Digital Object Identifier (DOI)
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.
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Citation / Publisher Attribution
Biochemistry, v. 52, issue 32, p. 5354-5364
Scholar Commons Citation
Smith, Matthew C.; Scaglione, K. Matthew; Assimon, Victoria A.; Patury, Srikanth; Thompson, Andrea D.; Dickey, Chad A.; Southworth, Daniel R.; Paulson, Henry L.; Gestwicki, Jason E.; and Zuiderweg, Erik R. P., "The E3 Ubiquitin Ligase CHIP and the Molecular Chaperone Hsc70 Form a Dynamic, Tethered Complex" (2013). Molecular Medicine Faculty Publications. 58.