Regulated structural transitions unleash the chaperone activity of αB-crystallin

Abstract

The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.olecular chaperones share the ability to bind nonnative, aggregation-prone polypeptides and assist their folding and assembly (1-3). Among these, the small heat shock protein (sHsp) αB-crystallin (also HspB5) is one of the major constituents of the vertebrate eye lens where it functions both as a chaperone and structural protein (4, 5). In nonlenticular tissues, αB-crystallin (αB) participates in sustaining cellular proteostasis. The involvement in neurodegenerative diseases (6, 7), multiple sclerosis (8), myopathies (9), as well as in cell cycle control, apoptosis, and cancer (10, 11) underlines its importance for cellular proteostasis.αB exhibits a tripartite organization (Fig. 1A) with a central α-crystallin domain (ACD) flanked by an N-terminal domain (NTD) and a short C-terminal extension (CTE) (12, 13). The ACD forms stable dimers (14-16) that further assemble into higher-order oligomers via interactions mediated by the NTD and CTE (17)(18)(19). αB forms dynamic populations of multimers with a variable number of subunits (20,21). Structural studies indicate that the variety of oligomeric states including a symmetric 24-mer (22) is created by addition of subunits to (or subtraction from) existing oligomers (17, 18). As for many other sHsps (23), the polydispersity of αB is coupled to spontaneous subunit exchange of yet undetermined units. αB quaternary dynamics was attributed to fluctuations of the intersubunit contacts mediated by the C-terminal IXI motif (24). However, given its involvement in oligomer formation, the NTD must also play a decisive role.In general, sHsps including αB recognize aggregation-prone, partially unfolded substrates (4, 25, 26) and keep them in a refolding-competent state (27, 28). The substrate binding sites of sHsps have not been defined yet. Recent studies suggest the involvement ...

Small heat-shock proteins: important players in regulating cellular proteostasis

“…Various studies have suggested that target protein binding is mediated by the N-terminal domain [95][96][97][98] or the ACD [99][100][101]. Indeed, with regards to the latter, we [18] and others [17,20,102] have shown that isolated ACDs exhibit chaperone function.…”

“…Phosphorylation reduces the average oligomer size, and increases oligomeric polydispersity and rate of subunit exchange of αBc [98,[132][133][134], whereas it leads to a dramatic decrease in the size of Hsp27 oligomers, such that the triply phosphorylated isoform is predominately dimeric in solution [135,136]. Thus, under stress conditions, Hsp27 is phosphorylated, triggering dissociation of the high molecular mass Hsp27 oligomers [117,135,137,138] and an increase in the amount of exposed hydrophobicity on the newly formed Hsp27 dimers [139].…”

“…Various factors may account for these apparent differences including the target protein used to assess the chaperone activity, the final concentration of sHsp in the assays (wild-type Hsp27 dissociates at low concentrations and therefore may be predominately dimeric, like the phosphorylated form, in some assays [136,138]), and buffer and temperature conditions of the assay, all of which have been shown to influence sHsp chaperone activity [133]. Overall, the consensus emerging from work with Bc and Hsp27 concerning the impact of phosphorylation on sHsp chaperone function is that phosphorylation boosts chaperone activity by (i) promoting dissociation of large oligomers into smaller species [98,132,133,135,136] and (ii) enhancing their affinity to bind destabilised target proteins [103,138]. Based on structural modelling of a full-length Bc into a 24-mer oligomer [154].…”

“…Based on structural modelling of a full-length Bc into a 24-mer oligomer [154]. Multiple molecular architectures of the eye lens chaperone B-crystallin elucidated by a triple hybrid approach), recent work has concluded that the negative charges introduced into the N-terminal domain of Bc by phosphorylation at S19, S45 and S59 result in oligomer destabilisation and dissociation due to the close proximity of adjacent N-terminal domains in the higher-order oligomeric structure [98]. Moreover, phosphorylation increases the rate of subunit exchange between oligomers and the flexibility of the N-terminal domain, leading to an increase in chaperone activity [98].…”

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

“…Human Cdc37 wild-type and Cdc37 mutants were expressed in the E. coli strain HB101 as a His6-tagged version using a pQE30 vector containing the respective gene. Hsp90, Cdc37, Hop, Hdj1, and Hsp70 were expressed and purified as described (62)(63)(64).…”