Engineering topics

Zhang, Huaqun

Publications and source records attributed to Zhang, Huaqun.

2 records

DOE OSTI2018

Shifting Sands: Modulating Interactions and Outputs of the CHIP‐Hsp70/HOP‐Hsp70 Protein Quality Control Complexes

The 70‐kilodalton heat shock protein (Hsp70) interacts with tetratricopeptide repeat (TPR) domains of CHIP (C‐terminus of Hsp70 Interacting Protein) and HOP (Hsp Organizing Protein) using a two‐carboxylate clamp binding mechanism. Differential affinity for the TPR domains of CHIP and HOP are achieved through interactions with the six residues preceding the carboxy‐terminal Asp 641 of Hsp70. In particular, phosphorylation of Hsp70 Thr 636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Given that CHIP‐Hsp70 complexes promote ubiquitin‐mediated degradation of Hsp70‐chaperoned clients while HOP‐Hsp70 complexes promote refolding of Hsp70‐chaperoned clients, phosphorylation of Hsp70 Thr 636 acts as a switch that directs the output and function of cellular protein quality control. Herein, through biophysical assays and new structures of CHIP‐Hsp70 and Hop‐Hsp70 we identify the molecular mechanism that enables phosphorylation‐driven switching that directs interactions within the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes. We also present a structure‐guided mutation of Hsp70 that bypasses Thr 636 phosphorylation‐driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP. This structure‐guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes toward chaperoned ubiquitination via the CHIP‐Hsp70 complex. Support or Funding Information The authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Page, Richard C.↗

DOE OSTI2018

Interactions and Dynamics Within CHIP/Hsp70 Complexes

Interactions between the E3 ubiquitin ligase CHIP (C‐terminus of Hsp70 interacting protein) and the chaperone Hsp70 represent the physical intersection of two competing protein quality control efforts: refolding, and degradation. Interaction with CHIP allows for the formation of a productive encounter complex that facilitates ubiquitination of Hsp70‐bound substrates, thereby targeting those proteins for proteasomal degradation. Central to the CHIP/Hsp70 interaction are changes in structure and dynamics that occur upon binding. Although recruitment of Hsp70 to CHIP via interaction with the CHIP tetratricopeptide repeat (TPR) domain is well established, alterations in structure and dynamics of CHIP and Hsp70 upon binding are not well understood. Using NMR we describe residue‐level changes in dynamics to the CHIP‐TPR domain that occur upon binding Hsp70. We also utilize small angle X‐ray scattering (SAXS) and electron paramagnetic resonance (EPR) to identify motions with the CHIP/Hsp70 complex. Independent SAXS and EPR experiments both point to a mobile Hsp70 substrate binding domain that is only partially constrained through interactions with the CHIP‐TPR. Building upon static models of the full‐length CHIP/Hsp70 complex, afforded by our recent crystal structure of the CHIP‐TPR/Hsp70‐lid‐tail complex, we have assembled a dynamic model of the CHIP‐Hsp70 complex. This dynamic model sheds light on the function of the CHIP/Hsp70 chaperoned ubiquitination complex and how CHIP harnesses dynamics of Hsp70 to overcome significant distance separations identified in earlier static models of the CHIP/Hsp70 complex. Support or Funding Information The authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Mannion, Matthew M.↗