Abstract
HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function.
Highlights
Acting as ATP-independent molecular chaperones, small heat shock proteins play an important role in protein housekeeping [1,2,3]
Expression and purification of HSPB6 deletion mutants The N-terminal domain (NTD) of vertebrate small heat shock protein (sHSP) has previously been described as a determinant of chaperoning activity
With insulin as the smallest substrate used (5.8 kDa), we investigated the size of the resulting solution species 90 min after addition of DTT, with and without HSPB6, using dynamic light scattering (DLS) (Table 3)
Summary
Acting as ATP-independent molecular chaperones, small heat shock proteins (sHSPs) play an important role in protein housekeeping [1,2,3]. A number of studies have been conducted to pinpoint the sequence-specific epitopes that define sHSP chaperone activity, and the majority support a central role for the N-terminal domain (NTD) These analyses have primarily focused on the canonical members of the sHSP-family: the a-crystallins and HSPB1 [12,13,19,20,21]. The protein shows the propensity to transiently selfassociate, even at high concentrations the formation of a higherorder oligomeric assembly has not been observed [25,27,28] This inability to form oligomers occurs despite the NTD showing similar sequence length and properties to the a-crystallins (Fig. 1A and Fig. S1). HSPB6 is an excellent candidate for studies on elucidating the role of the NTD in defining chaperone-like activity
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