Summary The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the TPR domain of CHIP in complex with the α-helical “lid” subdomain and unstructured “tail” of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting a novel mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Post-translational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a new bipartite mode of interaction between TPR domains and their binding partners.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.

Heat-shock protein 90 (Hsp90) is one of the most important chaperones involved in multiple cellular processes. The chaperoning function of Hsp90 is intimately coupled to the ATPase activity presented by its N-terminal domain. However, the molecular mechanism for the ATP-dependent working cycle of Hsp90 is still not fully understood. In this study, we use NMR techniques to investigate the structural characteristics and dynamic behaviors of Hsp90 N-terminal domain in its free and AMPPCP (ATP analogue) or ADP-bound states. We demonstrated that although AMPPCP and ADP bind to almost the same region of Hsp90, significantly different effects on the dynamics behaviors of the key structural elements were observed. AMPPCP binding favors the formation of the active homodimer of Hsp90 by enhancing the slow-motion featured conformational exchanges of those residues (A117–A141) within the lid segment (A111–G135) and around region, while ADP binding keeps Hsp90 staying at the inactive state by increasing the conformational rigidity of the lid segment and around region. Based on our findings, a dynamic working model for the ATP-dependent functioning cycle of Hsp90 was proposed.