title: The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position.
abstract: Mutations in cardiac myosin binding protein C (cMyBP-C), a thick filament protein that modulates contraction of the heart, are a leading cause of hypertrophic cardiomyopathy (HCM). Electron microscopy and 3D reconstruction of thin filaments decorated with cMyBP-C N-terminal fragments suggest that one mechanism of this modulation involves the interaction of cMyBP-C's N-terminal domains with thin filaments to enhance their Ca(2+)-sensitivity by displacement of tropomyosin from its blocked (low Ca(2+)) to its closed (high Ca(2+)) position. The extent of this tropomyosin shift is reduced when cMyBP-C N-terminal domains are phosphorylated. In the current study, we have examined L348P, a sequence variant of cMyBP-C first identified in a screen of patients with HCM. In L348P, leucine 348 is replaced by proline in cMyBP-C's regulatory M-domain, resulting in an increase in cMyBP-C's ability to enhance thin filament Ca(2+)-sensitization. Our goal here was to determine the structural basis for this enhancement by carrying out 3D reconstruction of thin filaments decorated with L348P-mutant cMyBP-C. When thin filaments were decorated with wild type N-terminal domains at low Ca(2+), tropomyosin moved from the blocked to the closed position, as found previously. In contrast, the L348P mutant caused a significantly larger tropomyosin shift, to approximately the open position, consistent with its enhancement of Ca(2+)-sensitization. Phosphorylated wild type fragments showed a smaller shift than unphosphorylated fragments, whereas the shift induced by the L348P mutant was not affected by phosphorylation. We conclude that the L348P mutation causes a gain of function by enhancing tropomyosin displacement on the thin filament in a phosphorylation-independent way.
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Mun
authors.affiliation: Department of Anatomy and Neurobiology, University of Puerto Rico School of Medicine, San Juan, PR 00936, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Kensler
authors.affiliation: Department of Cellular and Molecular Medicine, University of Arizona College of Medicine, Tucson, AZ 85724, USA.
authors.forename: Samantha P
authors.initials: SP
authors.lastname: Harris
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address: roger.craig@umassmed.edu.
authors.forename: Roger
authors.initials: R
authors.lastname: Craig
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D003599:Cytoskeleton / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D015398:Signal Transduction; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099163:Myosin Binding Protein C
chemical_list: D000199:Actins; D002118:Calcium; D002352:Carrier Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; D000099163:Myosin Binding Protein C
keywords: Cardiac muscle; Electron microscopy; Hypertrophic cardiomyopathy; Myosin binding protein C; Thin filament; cMyBP-C