Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble

Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. It is known to be activated by dissociation of the oligomer at heat shock temperatures. We wondered whether phosphorylation regulates its activity at physiological temperatures. In Hsp26, 9 phosphorylation sites which are located in different structural elements are known. Our analysis of phospho-mimetic mutations showed that phosphorylation activates Hsp26 at permissive temperature. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, the C-terminal extension, which also links subunits within the oligomer, contains several phosphorylation sites. The introduction of negative charges at these strategic positions makes regions in the N-terminal domain accessible for the binding of substrate proteins. Relieving the intrinsic inhibition via different phosphorylation sites allows the fine-tuning of chaperone activity in response to proteotoxic stresses independent of heat activation. The described weakening of domain interactions within and between subunits could be a general regulation principle of sHsps.