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

Abstract

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.

• Authors: Louise J Brown, Leena Singh, Kenneth L Sale, Bing Yu, Ronald Trent, Peter G Fajer, Brett D Hambly
• Journal: European biophysics journal : EBJ (Eur Biophys J) Vol. 31 Issue 5 Pg. 400-8 (Sep 2002) ISSN: 0175-7571 [Print] Germany
• PMID: 12202917 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Carrier Proteins
• myosin-binding protein C
• Myosins

Topics

• Amino Acid Motifs
• Animals
• Cardiomyopathy, Hypertrophic, Familial (genetics, metabolism)
• Carrier Proteins (chemistry, genetics, metabolism)
• Cattle
• Cells, Cultured
• Circular Dichroism (methods)
• Humans
• Models, Molecular
• Mutagenesis, Site-Directed
• Mutation
• Myocytes, Cardiac (metabolism)
• Myosins (metabolism)
• Protein Binding
• Protein Denaturation
• Sensitivity and Specificity
• Spectrum Analysis (methods)
• Structure-Activity Relationship
• Temperature