Obscurin Interacts with a Novel Isoform of MyBP-C Slow at the Periphery of the Sarcomeric M-Band and Regulates Thick Filament Assembly

Ackermann, Maegen A.; Hu, Li-Yen R.; Bowman, Amber L.; Bloch, Robert J.; Kontrogianni‐Konstantopoulos, Aikaterini

doi:10.1091/mbc.e08-12-1251

Cited by 54 publications

(81 citation statements)

References 57 publications

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“…Given that the C-terminal region is responsible for anchoring MyBP-C to the thick filament by binding to LMM and titin 20; 21; 22 , and that the other antibodies labeled the A-band, a simple interpretation is that the C10 domain is masked by binding to these other components, and that unbound antibodies bind non-specifically to the Z-line (like the pre-immune serum). We tested this idea by carrying out antibody labeling of myofibrils that had first been fixed with glutaraldehyde, which, we reasoned, could potentially alter epitope availability or reactivity 54 . C10 labeling was now found in the A-band and was absent from the Z-line (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Lee

Harris

Sadayappan

et al. 2015

Journal of Molecular Biology

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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.

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Section: Resultsmentioning

confidence: 99%

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

Lee

Harris

Sadayappan

et al. 2015

Journal of Molecular Biology

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“…M-band assembly involves multiple interactions: those of myomesin with myosin, of titin with myomesin and obscurin/obsl1, of obscurin/obsl1 with myomesin (3,5,6) and possibly a novel splice variant of MyBP-C (27). The genetic diseases disrupting the titinobsl1/obscurin interaction are late-onset diseases, and although the Finnish and French titin mutations lead to a total loss of obscurin and obsl1 binding, they are not embryonically lethal, possibly because the myomesin interactions will be retained.…”

Section: Discussionmentioning

confidence: 99%

Structural insight into M-band assembly and mechanics from the titin-obscurin-like-1 complex

Pernigo

Fukuzawa

Bertz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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In the sarcomeric M-band, the giant ruler proteins titin and obscurin, its small homologue obscurin-like-1 (obsl1), and the myosin cross-linking protein myomesin form a ternary complex that is crucial for the function of the M-band as a mechanical link. Mutations in the last titin immunoglobulin (Ig) domain M10, which interacts with the N-terminal Ig-domains of obscurin and obsl1, lead to hereditary muscle diseases. The M10 domain is unusual not only in that it is a frequent target of disease-linked mutations, but also in that it is the only currently known muscle Ig-domain that interacts with two ligands-obscurin and obsl1-in different sarcomeric subregions. Using x-ray crystallography, we show the structural basis for titin M10 interaction with obsl1 in a novel antiparallel Ig-Ig architecture and unravel the molecular basis of titin-M10 linked myopathies. The severity of these pathologies correlates with the disruption of the titin-obsl1/obscurin complex. Conserved signature residues at the interface account for differences in affinity that direct the cellular sorting in cardiomyocytes. By engineering the interface signature residues of obsl1 to obscurin, and vice versa, their affinity for titin can be modulated similar to the native proteins. In single-molecule force-spectroscopy experiments, both complexes yield at forces of around 30 pN, much lower than those observed for the mechanically stable Z-disk complex of titin and telethonin, suggesting why even moderate weakening of the obsl1/obscurintitin links has severe consequences for normal muscle functions.immunoglobulin domain | protein complex | x-ray crystallography | mechanosensor | myopathy S arcomeres are the smallest contractile units of striated muscles. They are highly ordered assemblies of precisely tailored actin and myosin filaments that are crosslinked at Z-disks and Mbands, respectively. The assembly of hundreds of protein subunits into ordered sarcomeres forms the structural basis for striated muscle contraction. The global layout of sarcomere assembly requires a giant ruler protein, titin, a 3000 kDa modular protein with a length of over 1.2 μm, displaying binding sites for proteins along the entire distance from Z-disk to M-band (1-3). Titin is composed of hundreds of immunoglobulin-and fibronectin-3-like (Ig and Fn3) domains that are arranged in specific patterns in the subcompartments of the sarcomere (3). The Ig-domains in sarcomeric proteins like titin, myomesin, or obscurin present a functionally versatile surface around a highly stable structural scaffold (4).At the M-band, the centers of myosin filaments are crosslinked into a mechanically stable network that involves titin, the cytoskeletal protein myomesin (5), and the giant obscurin (6), the latter acting as a linker to the sarcoplasmic reticulum (SR) (7). Obscurin, with a mass of around 800 kDa, was initially discovered as a ligand of Z-disk titin (3). Like titin, it is a modular protein composed of Ig and Fn3 domains. Intriguingly, obscurin localizes predominantly to the M-band ...

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“…Obscurin proteins serve multiple roles during myofibrillogenesis, including providing overall structural integrity and myofibril stabilization and, more specifically, providing proper spatial positioning of other contractile proteins, such as myosin (Borisov et al 2006;KontrogianniKonstantopoulos et al 2009). The specific role played by the obscurin proteins in myofibrillogenesis depends on their spatial/temporal distribution and interactions with key ABD Ankyrin-binding domain; ID, intercalated disc; Ob, obscurin; RhoGEF, rho-guanine nucleotide exchange factor; SK2, one of two active Ser/Thr kinase motifs in the COOH-terminus of obscurin-B a Epitope number corresponds to the epitope map found in Fig.…”

Section: Obscurins In Skeletal Musclementioning

confidence: 99%

Obscure functions: the location–function relationship of obscurins

2017

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The obscurin family of polypeptides is essential for normal striated muscle function and contributes to the pathogenesis of fatal diseases, including cardiomyopathies and cancers. The single mammalian obscurin gene, OBSCN, gives rise to giant (∼800 kDa) and smaller (∼40-500 kDa) proteins that are composed of tandem adhesion and signaling motifs. Mammalian obscurin proteins are expressed in a variety of cell types, including striated muscles, and localize to distinct subcellular compartments where they contribute to diverse cellular processes. Obscurin homologs in Caenorhabditis elegans and Drosophila possess a similar domain architecture and are also expressed in striated muscles. The long sought after question, "what does obscurin do?" is complex and cannot be addressed without taking into consideration the subcellular distribution of these proteins and local isoform concentration. Herein, we present an overview of the functions of obscurins and begin to define the intricate relationship between their subcellular distributions and functions in striated muscles.

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Obscurin Interacts with a Novel Isoform of MyBP-C Slow at the Periphery of the Sarcomeric M-Band and Regulates Thick Filament Assembly

Abstract: Obscurin is a multidomain protein composed of adhesion and signaling domains that plays key roles in the organization of contractile and membrane structures in striated muscles. Overexpression of the second immunoglobulin domain of obscurin (Ig2)

Cited by 54 publications

References 57 publications

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

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

Structural insight into M-band assembly and mechanics from the titin-obscurin-like-1 complex

Obscure functions: the location–function relationship of obscurins

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