Structural and functional impact of the p.R163C mutation in the conserved palindromic motif within the C-terminal domain of human αB-crystallin.

Human αB-crystallin is a small heat shock protein that functions as a chaperone and anti-apoptotic protein to maintain cellular protein integrity. A specific mutation (p.R163C) in the C-terminal domain has been linked to dilated cardiomyopathy (DCM). However, the impact of this mutation on the protein's structure, activity, stability, and amyloidogenic properties remains unclear. Here, we introduced the mutation, expressed and purified the protein, and used spectroscopic and microscopic techniques to conduct a comprehensive investigation of the mutant protein. The p.R163C mutation in αB-crystallin induces subtle changes in its secondary and tertiary structures, resulting in a slight increase in the distance and angle between monomer units within the dimer. The mutation causes the protein to form larger oligomers with increased chaperone activity, which may protect against cell death but could also lead to excessive client protein sequestration or coaggregation, potentially causing cytotoxicity. Accompanied by these alterations, the chemical and thermal stability of the mutant protein decrease, the resistance of the protein to enzymatic digestion increases, and finally, the propensity of the p.R163C mutated protein to form amyloid fibrils elevates. The substitution of the conserved arginine at position 163 with cysteine likely impacts the ability of the mutated protein to interact with cardiac muscle proteins. Collectively, these structural and functional modifications in the mutated protein may perturb cellular homeostasis and contribute to the onset of DCM.