Woods, C. N. | Search bioRxiv

bioRxiv2025

Small heat shock protein HSPB5 uses disorder to bind zinc with high affinity.

Zinc is an essential metal that supports diverse cellular functions. Zinc exerts its biological activity through protein binding, serving as catalytic cofactors and structural stabilizers of many enzymes, transcription factors, and ubiquitin E3 ligases, among others. Despite total cellular zinc concentrations reaching hundreds of micromolar, free zinc levels are tightly buffered. Elevated free zinc promotes mismetalation and protein aggregation. While zinc is redox-inert, its cysteine-based protein ligands are readily oxidized. Oxidative modification of cysteines leads to zinc dissociation and a rapid increase in free zinc. With [~]3000 proteins in the human zinc proteome, uncontrolled zinc release could be highly deleterious. Metallothioneins buffer zinc under basal conditions, but their re-synthesis following oxidative inactivation occurs on the scale of hours, raising the question of how free zinc is managed in the interim. Histidine, the second most prevalent zinc-coordinating residue, is resistant to oxidative modification. We characterized zinc binding by the small heat shock protein HSPB5 (B-crystallin), a cysteine-free, histidine-rich protein chaperone that responds to cellular stress and found: (1) HSPB5 binds zinc with high affinity and rapid reversibility; (2) zinc binding requires the disordered HSPB5 N-terminal region; (3) zinc binding increases HSPB5 disorder; and (4) prolonged zinc exposure promotes formation of assemblies of oligomers cross-bridged by zinc. We propose that HSPB5 has evolved specialized zinc-dependent properties distinct among human sHSPs, enabling it to function not only as a protein chaperone but also as a conditional zinc reservoir under oxidative stress.

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↗