Abstract
HSPB5, a member of the small heat shock protein family, acts as a first responder to cellular stress. One proposed mechanism of stress activation is phosphorylation. HSPB5 is phosphorylated at three sites-serine residues at positions 19, 45, and 59-located within its disordered N-terminal region (NTR). The extent of phosphorylation of the different sites leads to different cellular outcomes. HSPB5 forms polydisperse oligomers, where the NTR regions can either be exposed to the solvent or buried within the oligomer, forming internal contacts. We assessed the effect of single and triple phospho-mimicry on HSPB5 oligomeric properties. Our findings indicate that single phosphorylation causes localized and subtle changes in oligomer size, subunit exchange, hydrogen-deuterium protection patterns, and ability to delay aggregation of a known eye lens client, γD-crystallin. In contrast, the triple phosphomimic shows substantial structural and functional alterations. We provide a rationale for the increased chaperone activity observed in the S45D phosphomimic. Taken together, our results offer structural insights into how different phosphorylation events lead to distinct cellular outcomes.