The C-terminus of Hsp70 Interacting Protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal M/IEEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating poly ubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proli... More
The C-terminus of Hsp70 Interacting Protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal M/IEEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating poly ubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ERAD system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose dependent manner. Optimal inhibition required both the TPR and U-box indicating cooperativity between the two domains. Neither wild-type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by presence of an Hsc70 client with a preference for the ADP bound state. Thus, the Hsp/c70 M/IEEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate binding- and C-terminal domains of Hsp/c70.
