Alderson, T. R. | bioRxiv Science

bioRxiv2018

Local unfolding of the HSP27 monomer regulates chaperone activity

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved -crystallin domain, and provide the first structural characterization of a small heat-shock protein monomer. While HSP27 assembles into oligomers, we show that the transiently populated monomers released upon reduction are highly active chaperones in vitro, but are kinetically unstable and susceptible to uncontrolled aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance spectroscopy, we reveal that the pair of {beta}-strands that mediate dimerization become partially disordered in the monomer. Strikingly, we note that numerous HSP27 mutations associated with inherited neuropathies cluster to this unstructured region. The high degree of sequence conservation in the -crystallin domain amongst mammalian sHSPs suggests that partially unfolded monomers may be a general, functional feature of these molecular chaperones.

biophysics↗

bioRxiv2018

Phosphorylation of HspB1 regulates its mechanosensitive molecular chaperone interaction with native filamin C

Small heat-shock proteins (sHsps; HspBs) are molecular chaperones involved in the cellular stress response and a range of basal functions. Despite a multitude of targets, sHsp interactions are not well understood due their heterogeneous structures and weak binding affinities. The most widely expressed human sHsp, HspB1, is prevalent in striated muscle, where the actin cross-linker filamin C (FLNC, {gamma}-filamin, ABP-L) is a putative binding partner. Musculoskeletal HspB1 is phosphorylated in response to a variety of cues, including mechanical stress, which promotes oligomer disassembly and association with myoarchitectural elements. Here, we report the up-regulation and interaction of both proteins in the hearts of a mouse model of heart failure, with HspB1 being phosphorylated and FLNC increasingly associated with the sarcomeric Z-disc. We used a combination of structural approaches to reveal that phosphorylation of HspB1 results in increased availability of the residues surrounding the phosphosite, facilitating their interaction with folded FLNC domains equivalent to a force-sensing region in the paralog filamin A. By employing native mass spectrometry, we show that domains 18 to 21 of FLNC are extensible under conditions mimicking force, with phosphorylated HspB1 stabilising an intermediate from further unfolding. These findings report on conformations accessible during the cycles of mechanical extension central to filamin function, and are consistent with an interaction between the chaperone and a native target that is strengthened upon the application of force. This may represent a new mode of molecular chaperone activity, allowing HspB1 to protect FLNC from over-extension during mechanical stress.

biophysics↗