title: Determination of the critical residues responsible for cardiac myosin binding protein C's interactions.
abstract: Despite early demonstrations of myosin binding protein C's (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure-function relationships is needed to fully understand cMyBP-C's ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-C's N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-C's C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for the formation and/or maintenance of normal cMyBP-C sarcomeric distribution.
authors.affiliation: Md_Shenuarin.Bhuiyan@cchmc.org
authors.forename: Md Shenuarin
authors.initials: MS
authors.lastname: Bhuiyan
authors.forename: James
authors.initials: J
authors.lastname: Gulick
authors.forename: Hanna
authors.initials: H
authors.lastname: Osinska
authors.forename: Manish
authors.initials: M
authors.lastname: Gupta
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Robbins
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D051379:Mice; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism*; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D051381:Rats; D016415:Sequence Alignment; D000099163:Myosin Binding Protein C
chemical_list: D000199:Actins; D002352:Carrier Proteins; D015879:Myosin Subfragments; D000099163:Myosin Binding Protein C