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March 16, 2026Open Access

Crystal structures reveal phosphorylation-dependent disruption of the HSP70-CHIP interface; a compensatory G132N variant restores binding affinity

Key Points

This research aims to elucidate the structural basis of phosphorylation-dependent disruption of the HSP70-CHIP interface and explore compensatory mechanisms.
Compared crystal structures of unphosphorylated and phosphorylated HSP70 C-terminal peptides with CHIP
Used biolayer interferometry and fluorescence polarization to measure binding affinities
Conducted molecular dynamics simulations to analyze stability and interactions between the proteins
Engineered a G132N variant in CHIP to assess its effect on binding affinity
Phosphorylation at Thr636 decreases HSP70-CHIP binding affinity by over 10-fold
Steric clashes and electrostatic repulsion were identified as key factors resulting from phosphorylation
The G132N substitution in CHIP restores binding affinity for phosphorylated HSP70 peptides in isolated domains
Full-length CHIP shows partial recovery of transient interactions but not stable binding due to additional constraints

Abstract

Heat shock protein 70 (HSP70) and its E3 ligase co-chaperone CHIP ( STUB1 ) form a critical quality-control complex that directs client proteins toward folding or degradation. Phosphorylation of HSP70 at a conserved threonine in the C-terminal tail influences the fate of clients during cellular stress, yet the structural basis for this regulation remains unclear. Here, we present crystal structures of the CHIP tetratricopeptide repeat (TPR) domain bound to unphosphorylated and phosphorylated HSP70 C-terminal peptides at 1.6–1.9 Å resolution. Phosphate occupancy at Thr636 (HSPA1A numbering) causes steric clashes and electrostatic repulsion within the TPR-binding groove, decreasing affinity by more than 10-fold, as shown by biolayer interferometry and fluorescence polarization. Molecular dynamics simulations confirm destabilization of key hydrogen bonds. A structure-guided G132N substitution in CHIP introduces new hydrogen bonds to the phosphate group, restoring affinity for phosphorylated peptides in isolated TPR domains without losing native ubiquitination activity. However, in full-length CHIP, interface modifications do not restore phosphorylation-impaired stable binding but yield only partial recovery of transient interactions in cells, indicating additional context-dependent constraints on HSP70–CHIP regulation. These findings reveal the atomic mechanism by which phosphorylation impairs HSP70-CHIP interaction during stress and demonstrate that targeted interface engineering can compensate for post-translational changes in isolated domains. Overall, the results explain how cells switch chaperone-mediated triage pathways and offer a framework for understanding how proteostasis becomes dysregulated in neurodegenerative diseases and cancer. • Crystal structures reveal how Thr636 phosphorylation disrupts HSP70–CHIP binding • Phosphate clashes eliminate two key hydrogen bonds at the TPR interface • Engineered G132N CHIP restores affinity for phosphorylated HSP70 in isolated TPR • Targeted interface engineering overrides phosphorylation control of chaperone triage

Crystal structures reveal phosphorylation-dependent disruption of the HSP70-CHIP interface; a compensatory G132N variant restores binding affinity

Key Points

Abstract

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