Abstract
Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.
Highlights
Small heat shock proteins form a large family of chaperones expressed in all kingdoms of life including viruses, bacteria, fungi, plants, and animals [1,2]
The largest impact of the mutation has been observed in human HspB8 which is important due to disease-related mutations that were discovered in this protein and that correlate with several neurodegenerative and neuromuscular diseases [30,31,32]
Of the five proteins studied here, this is only the case in HspB1, HspB4, and HspB5. This motif plays an important role in the interaction of small heat shock proteins (sHsp) dimers within the large oligomers, stability of these large oligomers appears to be mostly dependent on the structure and properties of their respective N-terminal domain (NTD)
Summary
Small heat shock proteins (sHsps) form a large family of chaperones expressed in all kingdoms of life including viruses, bacteria, fungi, plants, and animals [1,2]. The ACD is flanked by a poorly ordered N-terminal domain (NTD) and a comparatively shorter C-terminal domain (CTD). The latter can be further divided into the conserved (I/V/L)-X-(I/V/L) tripeptide (IXI) or C-terminal anchoring motif (CAM) [6], and the highly flexible and polar C-terminal extension (CTE) [5,7,8]. SHsp monomers were shown to participate in the dynamic association of larger homo- and heterooligomers [8]. Oligomerization plays an important role in the regulation of chaperone-like activity and interaction of sHsp with their protein targets [12,13]
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