Missense mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-β aggregates

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations-L54R, A168D, A171P, and Y231N-on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded a-helical protein in solution, as is evident from its a-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR N-15-H-1 HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal (T-1) and transverse (T-2) relaxation experiments. Compared to WT, three mutants-L54R, A168D, and A171P-show a decreased alpha-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a molten globule state. In contrast, Y231N has an a-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-beta structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.