The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity

Abstract

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-β peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.X-ray crystallography | ion mobility mass spectrometry | nuclear magnetic resonance spectroscopy

Small Heat-shock Proteins Prevent α-Synuclein Aggregation via Transient Interactions and Their Efficacy Is Affected by the Rate of Aggregation

“…Notably, the ␣B-c core domain inhibited the aggregation of ␣-syn with similar efficacy to the wild-type (full-length) protein, demonstrating that the sites required to inhibit ␣-syn aggregation are present in the core domain of these sHsps. The finding that the core domain of ␣B-c is sufficient to prevent ␣-syn aggregation is consistent with previous studies showing that this region is capable of preventing other target proteins from forming amorphous or fibrillar aggregates (47,57).…”

“…2A), variant forms of these sHsps that either mimic phosphorylation (Hsp27 3D ) or consist of only the core ␣-crystallin domain (␣B-c core ) were selected for use in these experiments. This is because these variants exist predominately as either monomers or dimers (46,47) and therefore their molecular masses more closely match that of ␣-syn (an important factor in these absorbance-based AUC experiments as it ensures that both species sediment at similar rates). We first established that both variant forms also inhibit the fibrillar aggregation of ␣-syn at similar levels to the wild-type proteins (Fig.…”

pH-dependent structural modulation is conserved in the human small heat shock protein HSBP1

“…HSPB1 expression is upregulated in response to certain stressors but little is known about how oligomeric HSPB1 structure and dynamics are affected by changing conditions. Recent structures of the truncated α-crystallin domain (ACD) of HSPB1 (Baranova et al 2011;Hochberg et al 2014;Rajagopal et al 2015a) provide a platform on which to probe effects of environmental changes and disease-associated mutations at residue-level resolution in the ACD.…”

“…NMR and crystal structures of truncated and full-length mammalian sHSPs, including HSPB1, 4, 5, and 6, (Bagneris et al 2009;Baranova et al 2011;Hochberg et al 2014;Jehle et al 2010Jehle et al , 2011Laganowsky et al 2010;Rajagopal et al 2015a, b;Weeks et al 2013) all have a conserved β-sandwich IgG-like fold. In almost all cases, two ACDs form a homodimer via antiparallel alignment of their β6+7 strands.…”

The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility

“…The ACDs dimerize and assemble to form a polydisperse ensemble of oligomers, largely through dynamic interactions mediated by the terminal regions (6). The isolated ACD has been shown to have potent chaperone activity in vitro (7), and it is hypothesized that it might become exposed within the context of the wild-type protein in a manner regulated by phosphorylation (8) or cellular stress directly (9). The HSPB5 ACD contains two hydrophobic grooves, one between the ␤4 and ␤8 strands and the other at the dimer interface, both of which serve as putative binding sites for chaperone action (10).…”