Orientation of Myosin Binding Protein C in the Cardiac Muscle Sarcomere Determined by Domain-Specific Immuno-EM

Abstract

Myosin binding protein-C is a thick filament protein of vertebrate striated muscle. The cardiac isoform (cMyBP-C) is essential for normal cardiac function, and mutations in cMyBP-C cause cardiac muscle disease. The rod-shaped molecule is composed primarily of 11 immunoglobulin- or fibronectin-like domains, and is located at 9 sites, 43 nm apart, in each half of the A-band. To understand how cMyBP-C functions, it is important to know its structural organization in the sarcomere, as this will affect its ability to interact with other sarcomeric proteins. Several models have been proposed, in which cMyBP-C wraps around, extends radially from, or runs axially along the thick filament. Our goal was to define cMyBP-C orientation by determining the relative axial positions of different cMyBP-C domains. Immuno-electron microscopy was performed using mouse cardiac myofibrils labeled with antibodies specific to the N- and C-terminal domains and to the middle of cMyBP-C. Antibodies to all regions of the molecule, except the C-terminus, labeled at the same nine axial positions in each half A-band, consistent with a circumferential and/or radial rather than an axial orientation of the bulk of the molecule. The C-terminal antibody stripes were slightly displaced axially, demonstrating an axial orientation of the C-terminal 3 domains, with the C-terminus closer to the M-line. These results, combined with previous studies, suggest that the C-terminal domains of cMyBP-C run along the thick filament surface, while the N-terminus extends towards neighboring thin filaments. This organization provides a structural framework for understanding cMyBP-C’s modulation of cardiac muscle contraction.

Phosphorylation and calcium antagonistically tune myosin-binding protein C’s structure and function

“…1A) (4,5). The C-terminal domain (C10) is tightly bound to the thick filament backbone, and the N-terminal domains extend radially from the thick filament (6,7). Thus cMyBP-C's N-terminal domains are positioned to bind to neighboring actin filaments and/or the myosin S2 domain to modulate actomyosin activity.…”

The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency

“…To determine if the A31P mutation altered antibody recognition of epitopes in C0 we made use of a polyclonal antibody raised against full-length cMyBP-C that preferentially recognizes epitopes within the C0 domain (C-pro [23, 24]). Figure 2 shows western blots (WB) and Coomassie (C) stained gels loaded with increasing amounts of C0C2-WT and C0C2-A31P.…”

“…The sensitivity of the C-pro antibody to a single epitope is surprising because this polyclonal antibody was raised against full-length native cMyBP-C purified from rat heart [23] and is thus expected to react with numerous epitopes throughout the entire length of cMyBP-C. However, recent epitope mapping experiments revealed that the C-pro polyclonal antibody preferentially reacts with epitopes in C0 and to a lesser extent with other N-terminal domains of cMyBP-C such as the regulatory M-domain [24]. The significance of the strong antigenic nature of C0 is currently unknown, but the N-terminus of cMyBP-C has been identified in the development of immunogenic myocarditis suggesting that it is especially provocative to immune cells [30, 31].…”

“…Proteins were separated on SDS-PAGE (10% polyacrylamide) and transferred to nitrocellulose membranes (Biorad) for detection of proteins by western blot. Two antibodies were used to quantify total amounts of cMyBP-C: a rabbit polyclonal antibody that preferentially recognizes epitopes near the N-terminus of cMyBP-C (C-pro, [23, 24] 1:10,000) and a commercially available goat polyclonal antibody raised against internal domains of cMyBP-C (K-16, 1:200, Santa Cruz Biotechnology). An additional rabbit polyclonal antibody was raised against a short peptide sequence containing the A31P substitution to specifically detect mutant A31P cMyBP-C (rabbit polyclonal, 1:1,000, Invitrogen).…”

Molecular mechanisms of cardiomyopathy phenotypes associated with myosin light chain mutations

“…These phenotypes were not observed for the RLC mutants. One common denominator for both light chains could be the interaction between the ELC- and RLC-containing lever arm domain and actin via the cardiac MYBP-C, which was recently shown to span the neck domain of myosin head and actin with the C-terminal domains of cMyBP-C positioned along the thick filament surface, and the N-terminus of cMyBP-C extending toward neighboring thin filaments (Lee et al 2015). Interestingly, even though the ELC does not bind calcium, A57G-ELC and M173V-ELC both left shifted the calcium sensitivity of force (Table 1), a hallmark of HCM disease, suggesting the possibility of the N-terminus ELC mediated cross talk between the thick and thin filaments and resultant alterations in pCa 50 .…”