Functional and spectroscopic studies of a familial hypertrophic cardiomyopathy mutation in Motif X of cardiac myosin binding protein-C

• Published in: European biophysics journal 2002-09, Vol.31 (5), p.400-408
• Main Authors: Brown, Louise J, Singh, Leena, Sale, Kenneth L, Yu, Bing, Trent, Ronald, Fajer, Peter G, Hambly, Brett D
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino acids; Animals; Biophysics; Cardiomyopathy, Hypertrophic, Familial - genetics; Cardiomyopathy, Hypertrophic, Familial - metabolism; Cardiovascular disease; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cattle; Cells, Cultured; Circular Dichroism - methods; Humans; Immunoglobulins; Models, Molecular; Mutagenesis, Site-Directed; Mutants; Mutation; Myocytes, Cardiac - metabolism; Myosins - metabolism; Protein Binding; Protein Denaturation; Proteins; Sensitivity and Specificity; Spectrum analysis; Spectrum Analysis - methods; Structure-Activity Relationship; Temperature
• ISSN: 0175-7571; 1432-1017
• DOI: 10.1007/s00249-002-0236-0

Familial hypertrophic cardiomyopathy is an autosomal dominant genetic disorder caused by mutations in cardiac sarcomeric proteins. One such mutation is a six amino acid duplication of residues 1248-1253 in the C-terminal immunoglobulin domain of cardiac myosin binding protein-C, referred to as Motif X. Motif X binds the myosin rod and titin. Here we investigate the structural and functional alteration in the mutant Motif X protein to understand how sarcomeric dysfunction may occur. The cDNA encoding Motif X was cloned, mutated and expressed as wild-type and mutant proteins in a bacterial expression system. Circular dichroism spectroscopy confirmed that the normal and mutant Motif X exhibited a high beta-content, as predicted for immunoglobulin domains. Thermal denaturation curves showed that Motif X unfolded with at least two structural transitions, with the first transition occurring at 63 degrees C in the wild-type but at 40 degrees C in the mutant, consistent with the mutant being structurally less stable. Sedimentation binding studies with synthetic myosin filaments revealed no significant difference in binding to myosin between the wild-type and the mutant Motif X. Molecular modeling of this duplication mutation onto an homologous IgI structure (telokin) revealed that the duplicated residues lie within the F strand of the immunoglobulin fold, on a surface of Motif X distant from residues previously implicated in myosin binding. Taken together, these data suggest that the Motif X mutation may interfere with other, as yet unidentified, functional interactions.