Modulation of Hsp70 Interactions with CHIP and HOP Co‐chaperones via Phosphorylation
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Hsp70 plays a vital role in protein homeostasis. It associates with co-chaperones HOP and CHIP to promote protein refolding or degradative pathways, respectively. Phosphorylation of an Hsp70 threonine residue near the C-terminus modulates the binding affinity of Hsp70 towards co- chaperones HOP and CHIP. Upon Hsp70 C-terminal phosphorylation, Hsp70 preferentially binds with HOP as binding to CHIP is attenuated. Hsp70 is mainly found in the phosphorylated state in highly proliferating cells such as cancer, indicating upregulation of protein refolding over degradation. Unfortunately, the field lacks structural knowledge on how and why the affinity of Hsp70 for co-chaperones changes upon phosphorylation of the C-terminus. Here, we present structural and biophysical insights into the modulation of Hsp70 interactions with CHIP and HOP co-chaperones with phosphorylation at the C-terminus. Our data suggests that a rearrangement of the phosphorylated Hsp70 peptide and conformational changes induced in CHIP-TPR collectively lead to the disruption of an inter-molecular hydrogen bond in the complex of CHIP-TPR/pT-Hsp70 peptide. We built upon this structural knowledge to design novel CHIP-TPR variants to promote interaction of the CHIP-TPR with phosphorylated Hsp70. Our affinity data demonstrated that the novel variants exhibit higher affinity towards phosphorylated Hsp70 peptide compared to wild- type CHIP-TPR. We conducted molecular dynamics (MD) simulation studies to confirm effectiveness of the novel variants and suggest a mechanism by which the affinity is modulated. Our MD simulation data, in accordance with the crystallography data and the observed hydrogen bond network, explain the observed changes in binding affinity. Overall, this chapter explains the structural insights for modulation of the CHIP/Hsp70 interaction by phosphorylation status and the feasibility of designing biologically important CHIP variants to promote CHIP interactions with phosphorylated Hsp70.
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