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

SM Singh, N Kongari… - Proceedings of the …, 2010 - National Acad Sciences
SM Singh, N Kongari, J Cabello-Villegas, KMG Mallela
Proceedings of the National Academy of Sciences, 2010National Acad Sciences
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 α-helical
protein in solution, as is evident from its α-helical circular dichroism spectrum, blue shift of …
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 α-helical protein in solution, as is evident from its α-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR 15N-1H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal (T1) and transverse (T2) relaxation experiments. Compared to WT, three mutants—L54R, A168D, and A171P—show a decreased α-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 α-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-β 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.
National Acad Sciences