1H, 15N and 13C resonance assignments for free and IEEVD peptide-bound forms of the tetratricopeptide repeat domain from the human E3 ubiquitin ligase CHIP

Huaqun Zhang,Matthew M Mannion,Richard C Page,Cameron Mcglone

doi:10.1007/s12104-016-9710-y

Abstract

The ubiquitin ligase CHIP catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90. CHIP interacts with Hsp70/Hsc70 and Hsp90 by binding of a C-terminal IEEVD motif found in Hsp70/Hsc70 and Hsp90 to the tetratricopeptide repeat (TPR) domain of CHIP. Although recruitment of heat shock proteins to CHIP via interaction with the CHIP-TPR domain is well established, alterations in structure and dynamics of CHIP upon binding are not well understood. In particular, the absence of a structure for CHIP-TPR in the free form presents a significant limitation upon studies seeking to rationally design inhibitors that may disrupt interactions between CHIP and heat shock proteins. Here we report the 1H, 13C, and 15N backbone and side chain chemical shift assignments for CHIP-TPR in the free form, and backbone chemical shift assignments for CHIP-TPR in the IEEVD-bound form. The NMR resonance assignments will enable further studies examining the roles of dynamics and structure in regulating interactions between CHIP and the heat shock proteins Hsp70/Hsc70 and Hsp90.

Highlights

The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90
CHIP binds to the C-terminal IEEVD motif of heat shock proteins through the tetratricopeptide repeat (TPR) domain (Zhang et al 2005, 2015), while the U-box domain is required for E3 ligase activity (Connell et al 2001)
A recent hydrogen/deuterium exchange mass spectrometry (HDXMS) study found that the TPR domain of CHIP was significantly more dynamic in the free form, whereas it was stabilized upon binding to full-length heat shock proteins or C-terminal IEEVD motif peptides (Graf et al 2010)

Summary

Biological context

The C-terminus of Hsc interacting protein (CHIP) is a ubiquitin ligase involved in protein degradation by the ubiquitin–proteasome pathway, as well as a co-chaperone. Backbone resonance assignments in the free form (Fig. 1) and the IEEVD-bound form (Fig. 2) are consistent with the X-ray crystal structure of the CHIP-TPR/Hsp70IEEVD complex (Fig. 3), as indicated by secondary structure predictions from TALOS? Interpreted in light of previous HDX-MS data, chemical shift perturbations between CHIP-TPR helices suggest that while secondary structure content remains unchanged, the relative positions of helices likely change between the free and IEEVD-bound states. The chemical shift perturbations and previous HDX-MS data suggest that the absence of a CHIP-TPR crystal structure in the free form may not be for lack of effort, but rather because conformational variability or dynamics of free form CHIP-TPR preclude the formation of diffraction quality crystals This dynamic behavior of CHIP-TPR suggests that solution NMR studies, enabled by the resonance assignments reported here, will be important for gaining structural insights for CHIP-TPR in the absence of an IEEVD peptide

Protein expression and purification

Assignment and data deposition