Structural and Functional Impacts of the Altered Hydrophobicity in the Dimer Interface of Tpv HSP 14.3

Abstract

Small heat shock proteins (sHSPs) are the ATP-independent molecular chaperones that prevent protein aggregation in the cell by forming stable complexes with unfolded and misfolded proteins. Their distinctive structural characteristics are their low molecular weight (from 14 to 43 kDa) and a tripartite domain architecture. The highly conserved Alpha Crystallin Domain (ACD) plays a central role in the dimerization of sHSPs and acts as the structural building block for oligomerization. The point mutations in the ACD of the human sHSPs that interfere with the dimer integrity are linked to several diseases, including cataracts, desmin-related myopathy, cardiomyopathy, and distal hereditary motor neuropathy. In the present study, we investigated the functional and structural implications of amino acid changes at two putative dimer interface residues, L33 and Y34. These residues are located on the β2 strand of Tpv HSP 14.3, which is implicated in ACD dimerization via strand exchange. Effects of the substitutions were evaluated by performing chaperone assays using the client proteins pig heart Citrate Synthase (phCS) and Alcohol Dehydrogenase (ADH) and through in silico molecular bond and structure analyses of the wild type and generated mutant proteins. Our results indicated that an excess amount of WT and the mutant proteins are required to maintain phCS activity to a level comparable to or even higher than the positive control. At a lower substrate/sHSP ratio, the Y34F mutant protected the phCS activity more effectively than the WT and L33S mutant sHSPs. Also, the Y34F mutant sHSP afforded the highest protection of ADH enzyme from heat inactivation. It is likely that increased hydrophobicity by Y34F substitution contributed to the formation of a hydrophobic surface that may capture aggregation-prone substrates. According to molecular bond analysis, the loss of intermolecular hydrophobic interactions between leucine 33 on the β2 strand and tyrosine 77 and isoleucine 78 on the β6 strand can be critical for the reduced structural/thermodynamic stability of the L33S mutant protein.