Stone, N. L. | Search bioRxiv

bioRxiv2025

Disorder with consequence: Phosphorylation sites in HSPB5 yield distinct structural outcomes

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, {gamma}D-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.

biophysics↗

bioRxiv2022

HSPB5 disease-associated mutations have long-range effects on structure and dynamics through networks of quasi-ordered interactions

Found from bacteria to humans, small heat shock proteins (sHSPs) are the least understood protein chaperones. HSPB5 (or B-crystallin) is among the most widely expressed of the ten human sHSPs, including in muscle, brain, and eye lens where it is constitutively present at very high levels and carries out a myriad of functions. A high content of disorder in HSPB5 has stymied efforts to uncover how its structure gives rise to function. To uncover its mechanisms of action, we compared human HSPB5 and two disease-associated mutants, R120G and D109H. Expecting to learn how the mutations lead to loss of function, we found instead that the mutants are constitutively activated chaperones while wild-type HSPB5 can transition reversibly between non-activated (low activity) and activated (high activity) states in response to changing conditions. Techniques that provide information regarding interactions and accessibility of disordered regions revealed that the disordered N-terminal regions (NTR) that are required for chaperone activity exist in a complicated interaction network within HSPB5 oligomers and are sequestered from solvent in non-activated states. Either mutation or an activating pH change cause rearrangements in the network that expose parts of the NTR, making them more available to bind an aggregating client. While beneficial in the short-term, failure of the mutants to adopt a state with lower activity and lower NTR accessibility leads to increased co-aggregation propensity and, presumably, early cataract. The results support a model where chaperone activity and solubility are modulated through the quasi-ordered NTR and its multiple competing interactions. SignificanceSmall heat shock proteins (sHSPs) are the oldest known protein chaperones, but how they recognize misfolding proteins in early stages of aggregation is unknown. Disordered regions within sHSPs are critical to their function, raising the question of how disorder recognizes disorder. We investigated a human sHSP (HSPB5) and two disease mutants using approaches that provide residue-level information in regions of disorder. The findings reveal that large, heterogeneous oligomers of HSPB5 contain complicated networks of interactions involving their disordered regions that protect them from solvent. Conditions associated with stress or disease mutations cause a rearrangement of the interaction network and enhancement of chaperone activity. The study provides new information regarding how disordered regions are modulated in a network of competing interactions.

biophysics↗