The preferential heterodimerization of human small heat shock proteins HSPB1 and HSPB6 is dictated by the N-terminal domain

Abstract

Small heat shock proteins are ATP-independent molecular chaperones. Their function is to bind partially unfolded proteins under stress conditions. In vivo, members of this chaperone family are known to preferentially assemble together forming large, polydisperse heterooligomers. The exact molecular mechanisms that drive specific heteroassociation are currently unknown. Here we study the oligomers formed between human HSPB1 and HSPB6.Using small-angle X-ray scattering we could characterize two distinct heterooligomeric species present in solution. By employing native mass spectrometry we show that such assemblies are formed purely from heterodimeric building blocks, in line with earlier crosslinking studies. Crucially, a detailed analysis of truncation variants reveals that the preferential association between these two sHSPs is solely mediated by their disordered Nterminal domains.

Specific sequences in the N-terminal domain of human small heat-shock protein HSPB6 dictate preferential hetero-oligomerization with the orthologue HSPB1

“…The equimolar mixture of WT HSPB1 and HSPB6 prepared at 37 o C yielded a broad elution profile on analytical SEC, composed of two peaks with maxima corresponding to a molecular weight of 508 and 160 kDa, in line with previous studies (35). The corresponding eluted fractions contained equimolar amounts of both sHSPs when analysed by SDS-PAGE (Fig.…”

“…Assuming both sHSPs can freely exchange subunits, random protomer turnover would be expected to result in oligomers containing both homo and heterodimers. Recently, using native mass spectrometry, we have observed that although both sHSPs alone can exchange subunits in such a stochastic fashion, the heterooligomeric complex is composed solely of heterodimers (35). This result, in good agreement with earlier residue specific cross-linking studies (21), supports a heterooligomer model where HSPB1 and HSPB6 preferentially associate at the core dimer level.…”

“…This result, in good agreement with earlier residue specific cross-linking studies (21), supports a heterooligomer model where HSPB1 and HSPB6 preferentially associate at the core dimer level. Curiously this preferred association, which is mediated by the ACD, requires the presence of the highly flexible NTDs (35). This latter region of human HSPB1 is 90 residues long and has low sequence homology with the 72 residue NTD of HSPB6, with the most prominent exception being a conserved RLFDQxFG motif close to the center of this domain in the two proteins.…”

“…The exposure time per frame was 750 ms, with a dead time of 1500 ms between frames. Data processing was done as described previously (35). …”

The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins

“…Furthermore, specific interactions of the N-terminal region in intact αBc were inferred by solid-state NMR through chemical shift changes and alterations in dynamics of resonances in the N-terminal region (Mainz et al 2015). Similarly, the N-terminal region of Hsp20 (HspB6) has multiple sites of interaction with a target protein as well as a role in regulating chaperone activity ) whilst also being important in the formation of a hetero-oligomer with Hsp27 (Heirbaut et al 2016). There is evidence from interactome studies that the Nterminal region of plant sHsps is involved in interacting with amorphously aggregating target proteins (Jaya et al 2009).…”