Oligomeric state modulation of sugarcane Hsp101 by ionic strength: structural and functional implications

Molecular chaperones are proteins essential in ensuring proper protein folding in the cell, being directly related to the prevention of misfolding and aggregation issues. These problems are related to diverse pathologies such as neurodegenerative diseases in humans and even some types of cancer. Chaperones of the Hsp104 subfamily confer protection against various types of stress, and can even solubilize aggregates while also recovering client protein function, but interestingly, metazoans lack a cytosolic Hsp104. Our study focuses on the sugarcane Hsp101 (SsHsp101), a cytosolic Hsp104 homologous. By varying ionic strength we have observed changes in the oligomeric state of SsHsp101, with dissociation into smaller oligomers, in line with works observed for the E. coli homolog (ClpB). The molecular mass of the oligomers, observed by SEC-MALS and SAXS, is dislocated from the canonical hexamer to smaller oligomers by increasing ionic strength, indicating the disassembly of the structure. We have observed no significant changes in secondary structure nor in thermal unfolding profiles, which indicates structural modularity of SsHsp101 oligomers. On the other hand, SAXS measurements have indicated a structure that presents considerable plasticity when in solution, in line with previous Cryo-EM studies for the C. thermophila homolog, which has shown flexibility of the NTD regions as outer “fingers” on the structure. We have also compared both oligomers functionally, such as by ATPase activity, shedding more light into the biological role of the hexameric form. Our results are of special interest not only in advancing understanding of the Hsp104 subfamily of chaperones or of the sugarcane protein quality control, but also in searching for novel therapeutics for protein misfolding and aggregation diseases.

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 88887.816452/2022-00) and by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2017/26131-5).