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The E3 Ubiquitin Ligase CHIP and the Molecular Chaperone Hsc70 Form a Dynamic, Tethered Complex
journal contribution
posted on 2013-08-13, 00:00 authored by 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. ZuiderwegThe
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.