The p.P51L mutation in human HspB5: Structural and functional changes linked to cardiomyopathy and cataract pathogenesis.

Human small heat shock protein B5 (HspB5) or αB-crystallin is essential for maintaining cellular proteostasis through its ATP-independent chaperone activity. Mutations in HspB5 have been recognized to be implicated in myopathies, cardiomyopathies, and cataracts; however, the structural and functional reasons for their pathogenicity remains poorly understood. The p.P51L mutation, located within the N-terminal domain critical for oligomerization and client recognition, has been associated with cardiomyopathy and cataracts. In this study, we generated the p.P51L variant using site-directed mutagenesis and confirmed the mutation using mass spectrometry. We employed spectroscopic, microscopic, NMR, and molecular dynamics (MD) simulation techniques to study the structural and functional consequences of this mutation. Our findings demonstrate that the p.P51L mutation induces alterations in secondary structures and conformation, accompanied by an increase in oligomer size and amyloid fibril formation. MD simulations also revealed enhanced structural stability and compactness in the mutant dimer, and lower binding affinity to the key client proteins, such as HspB4 (αA-crystallin) and desmin, suggesting a feasible mechanism of its association with cardiomyopathy and cataracts. These observations demonstrate how a point mutation can disrupt the conformational and functional properties of HspB5, its chaperone activity and client interactions. Our study provides new mechanistic insights into the molecular basis of diseases related to HspB5 protein misfolding.