Site-Directed Mutagenesis Study of the Alpha-Crystallin Domain in Human Small Heat Shock Protein HspB5

Abstract

Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that exhibit “holdases” functions. Under cellular stresses including changes in temperatures, pH, or oxidation-reduction states, proteins can expose their hydrophobic regions and become partially unfolded. sHsps bind to these nonnative proteins to prevent aggregation, facilitate refolding, and maintain proteostasis. Flanked by the N- and C-terminal regions, the -crystallin domain (ACD) is conserved across different sHsps. It predominantly consists of antiparallel -sheets and contributes significantly to dimer formation. Although sHsps are known to populate at various oligomeric states, it is unclear how their dynamic conformations relate to chaperone activities. Therefore, it is important to gain more insight into the structural features of sHsps, in an effort to understand their mechanisms of action.

This study focuses on one of the sHsps, HspB5, otherwise known as B-crystalline. It is one of the major eye lens proteins, but is also found in other parts of the body such as muscles. This mutagenesis study of the full-length HspB5 aims to elucidate the key amino acid residues in the ACD, specifically on the  strand at the dimer interface. The study includes some disease mutations such as D109A and R120G which are found in patients with myofibrillar myopathy and cataracts respectively. Other point mutations, namely F113Y, R116C, and R120C, introduce the possibilities of additional hydrogen bonding or disulfide bonds. Results from in vitro chaperone-like light-scattering assays reveal loss of chaperone functions of the mutants compared to that of the wild-type HspB5 and suggest changes in substrate-sHsp interactions. The extent of the reductions in chaperone activities are different among mutants and specific to the substrate proteins.