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bioRxiv · 10.1101/369454

It takes a dimer to tango: Oligomeric small heat-shock proteins dissociate to capture substrate

Santhanagopalan, I.·Degiacomi, M. T.·Shepherd, D. A.·Hochberg, G. K. A.·Benesch, J. L. P.·Vierling, E.
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Abstract

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and their mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show that substructural dimers of plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and non-dimeric, to restrict how their assemblies can dissociate. When disulfide bonded at the non-dimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive, but when reduced had wild-type-like chaperone activity, demonstrating that dissociation at non-dimeric interfaces is essential for activity. In addition, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different than the Ta16.9 x-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) showed greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Size and secondary structure changes with temperature revealed that folded sHsp dimers interact with substrate, and support dimer stability as a determinant of chaperone efficiency. These data yield a model in which sHsp dimers capture substrate prior to assembly into larger, heterogeneous sHSP-substrate complexes for subsequent substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

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Santhanagopalan, I., Degiacomi, M. T., Shepherd, D. A., Hochberg, G. K. A., Benesch, J. L. P., Vierling, E.. 2018-07-14. It takes a dimer to tango: Oligomeric small heat-shock proteins dissociate to capture substrate. https://doi.org/10.1101/369454

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