The E3 Ubiquitin Ligase CHIP and the Molecular Chaperone Hsc70 Form a Dynamic, Tethered Complex

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• 作者：Matthew C. Smith ; K. Matthew Scaglione ; Victoria A. Assimon ; Srikanth Patury ; Andrea D. Thompson ; Chad A. Dickey ; Daniel R. Southworth ; Henry L. Paulson ; Jason E. Gestwicki ; Erik R. P. Zuiderweg
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：August 13, 2013
• 年：2013
• 卷：52
• 期：32
• 页码：5354-5364
• 全文大小：559K
• 年卷期：v.52,no.32(August 13, 2013)
• ISSN：1520-4995

文摘

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鈥揅HIP interaction. We found that CHIP binds tightly to two molecules of Hsc70 forming a 210 kDa complex, with a K_d of approximately 60 nM, and that the IEEVD motif at the C-terminus of Hsc70 (residues 642鈥?46) 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鈥揅HIP complex determined that the two partners move independently in solution, similar to 鈥渂eads on a string鈥? These results suggest that a dynamic C-terminal region of Hsc70 provides for flexibility between CHIP and the chaperone, allowing the ligase to 鈥渟earch鈥?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鈥?41 of the C-terminal region, while retaining the IEEVD motif, caused a significant decrease in the efficiency of Hsc70 ubiquitination by CHIP.