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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-08, Vol.107 (34), p.15069-15074
• Main Authors: Singh, Surinder M, Kongari, Narsimulu, Cabello-Villegas, Javier, Mallela, Krishna M.G
• Format: Article
• Language: English
• Subjects: Actins; Aggregation; Biological Sciences; Biophysical Phenomena; Cell aggregates; Duchenne muscular dystrophy; Dystrophin - chemistry; Dystrophin - deficiency; Dystrophin - genetics; Dystrophin - ultrastructure; Fluorescence; Genetic mutation; Humans; In Vitro Techniques; Microscopy, Electron, Transmission; Models, Molecular; Multiprotein Complexes - chemistry; Muscular dystrophies; Muscular Dystrophies - etiology; Muscular Dystrophies - genetics; Muscular Dystrophies - metabolism; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - ultrastructure; Mutation, Missense; Protein Folding; Protein refolding; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - ultrastructure; Thermodynamics; Wavelengths
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1008818107

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.