Functional and spectroscopic studies of a familial hypertrophic cardiomyopathy mutation in Motif X of cardiac myosin binding protein-C.
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.
Department of Pathology, University of Sydney, NSW 2006, Australia.
Louise J
LJ
Brown
Leena
L
Singh
Kenneth L
KL
Sale
Bing
B
Yu
Ronald
R
Trent
Peter G
PG
Fajer
Brett D
BD
Hambly
D020816:Amino Acid Motifs; D000818:Animals; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002942:Circular Dichroism / Q000379:methods; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods; D013329:Structure-Activity Relationship; D013696:Temperature; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D009218:Myosins; D000099163:Myosin Binding Protein C