title: The E3 ubiquitin ligase CHIP and the molecular chaperone Hsc70 form a dynamic, tethered complex.
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.
authors.affiliation: Departments of Pathology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Smith
authors.forename: K Matthew
authors.initials: KM
authors.lastname: Scaglione
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Assimon
authors.forename: Srikanth
authors.initials: S
authors.lastname: Patury
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Thompson
authors.forename: Chad A
authors.initials: CA
authors.lastname: Dickey
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Southworth
authors.forename: Henry L
authors.initials: HL
authors.lastname: Paulson
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gestwicki
authors.forename: Erik R P
authors.initials: ER
authors.lastname: Zuiderweg
mesh_terms: D001665:Binding Sites; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases