Electron Microscopy and 3D Reconstruction of F-Actin Decorated with Cardiac Myosin-Binding Protein C (cMyBP-C)

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Abstract

Myosin-binding protein C (MyBP-C) is an ∼ 130-kDa rod-shaped protein of the thick (myosin containing) filaments of vertebrate striated muscle. It is composed of 10 or 11 globular 10-kDa domains from the immunoglobulin and fibronectin type III families and an additional MyBP-C-specific motif. The cardiac isoform cMyBP-C plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon β-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart diseases. In addition to binding to myosin, MyBP-C can also bind to actin via its N-terminal end, potentially modulating contraction in a novel way via this thick–thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and three-dimensional reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25 to 180 mM KCl concentration. Three-dimensional helical reconstructions, carried out at the 180-mM KCl level to minimize nonspecific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin.

Introduction

Myosin-binding protein C (MyBP-C or C-protein) is an ∼ 130-kDa, 40-nm-long rod-shaped accessory protein of the thick (myosin containing) filaments of vertebrate striated muscle1, 2, 3, 4 located in the C-zone of the A-band.5, 6, 7 The skeletal isoform comprises 10 tandemly arranged 10-kDa immunoglobulin (Ig) and fibronectin type III domains, numbered C1–C10 from the N-terminus, together with a MyBP-C-specific motif (also called the M domain) between C1 and C2 and a Pro–Ala-rich sequence at the N-terminus.3, 8 The cardiac isoform (cMyBP-C) has an additional N-terminal Ig domain (C0), four phosphorylation sites in the M domain, and a 28-residue insert in the C5 domain (Fig. 1a).3, 4, 9, 10, 11

MyBP-C is essential for the normal functioning of striated muscle. The cardiac isoform plays a key role in the enhancement of cardiac contraction that occurs in response to β-adrenergic stimulation, and mutations in cMyBP-C are a major cause of inherited hypertrophic cardiomyopathy.3, 4, 11, 12, 13, 14 In the case of skeletal MyBP-C, mutations can cause distal arthrogryposis, a disease characterized by joint contractures and abnormal muscle development.15 Apart from its role in modulating cardiac contraction, MyBP-C has also been reported to function in the regulation of myosin filament assembly in sarcomerogenesis,16 in stabilization of the thick filament through its phosphorylation,17 and in the modulation of myosin head organization.18, 19, 20

MyBP-C attaches to the thick filament surface via its C-terminal domains (C8–C10; Fig. 1a).3 In addition, its N-terminus has been shown to bind reversibly to myosin subfragment 2.21,22 Surprisingly, MyBP-C, first isolated as a myosin-binding protein, can also bind to the other major filament of the sarcomere, the thin filament. In vitro evidence for MyBP-C-actin binding was first reported on the basis of centrifugation measurements and electron microscopy (EM).23 Later studies demonstrated that binding also occurs to regulated thin filaments (containing troponin and tropomyosin) in the presence24, 25, 26 and also in the absence of Ca2+.25, 26 Experiments with expressed MyBP-C fragments show that binding occurs via the N-terminus, primarily the C1 and M domains,25, 27, 28 at a saturating 1:1 molar ratio with actin,25 although additional binding through the C-terminal half has also been reported.29 Electron tomography of muscle sections has directly demonstrated that MyBP-C links thick and thin filaments in intact muscle, demonstrating the physiological relevance of these in vitro studies.30 In the simple model suggested by this work, the thick filament binding domains C8–C10 run longitudinally along the surface of the filament, while the rest of the molecule extends out from the thick filament, binding to neighboring thin filaments by its N-terminus.20, 30, 31

These structural studies all suggest the possibility that MyBP-C might play a key role in muscle contraction by modulating actin–myosin filament sliding through this physical link between filaments. Support for this comes from in vitro motility observations demonstrating that actin filament sliding over myosin is slowed by MyBP-C binding to actin.27, 3235 Interestingly, this effect is weakened when the M domain is phosphorylated.25 Despite its potential importance in contraction, however, the structural mechanism by which MyBP-C binding to actin might modulate filament sliding is not understood. For example, it is not known whether MyBP-C might simply act as a tether or play a more active role, possibly by physically interfering with attachment of myosin heads or affecting tropomyosin positioning or movement upon Ca2+ activation. Structural studies are needed to answer this question. Based on neutron scattering of F-actin decorated with the N-terminal fragment C0C2† a structure has been proposed in which the C0 and C1 domains bind specifically to actin near the DNase I binding loop and subdomain (SD) 1, resulting in a highly regular structure.36 However, this work was carried out at relatively low ionic strength, and the proposed structure depends on model building of relatively low resolution data, leading to uncertainty in interpretation. Direct imaging by EM of negatively stained F-actin decorated with C0C2 has shown an increase in filament diameter and alterations in the Fourier transform of decorated filaments, together demonstrating regular binding of the fragment.25, 37 However, the location and mode of binding of the fragment on actin were not revealed. Here, we have used negative staining EM to observe F-actin decorated with N-terminal fragments and have computed three-dimensional (3D) reconstructions of the filaments that reveal the mode and location of fragment binding. Comparison of structures decorated with three different fragments has helped in interpreting the location and organization of the different MyBP-C domains. We find that the N-terminus of cMyBP-C directly interacts with F-actin in a position that would compete with the binding of myosin heads and possibly also with tropomyosin.

Section snippets

Characterization of N-terminal MyBP-C fragments

Four N-terminal fragments of mouse cMyBP-C were expressed in Escherichia coli (see Materials and Methods). These corresponded to domains C0C1, C0C1f, C0C2, and C0C3 (Fig. 1a). All fragments contain C0 and the Pro–Ala-rich region, and C0C2 and C0C3 also contain the M domain. C0C1f was the same as C0C1 but contained, in addition, the first 15 amino acids of the M domain,38 which have been shown to be important in binding to actin.34 These fragments ran with SDS-PAGE mobilities of 31, 32, 54, and

Discussion

The concept that MyBP-C might bridge between thick and thin filaments and thus modulate muscle contraction has existed ever since it was first shown that this myosin-binding protein could also bind to F-actin.23 This possibility has been strengthened by subsequent in vitro studies showing binding to regulated thin filaments,24, 26 and the location of the interaction site has been narrowed to the N-terminal region, primarily the C1 and M domains25, 27, 28 (see also Ref. 29). In vitro motility

Proteins

F-actin was purified from chicken pectoralis muscle according to Pardee and Spudich.48 cMyBP-C fragments were bacterially expressed from mouse cardiac cDNA using the pET expression system (Novagen, Madison, WI). Four expressed N-terminal domains, C0C1 (amino acids 1–254), C0C1f (1–269), C0C2 (1–448), and C0C3 (1–539), were studied (Fig. 1a). The cDNA fragments were generated by PCR, incorporating compatible restriction enzyme sites for cloning into the pET28a vector, with 5′ NcoI and 3′ XhoI

Acknowledgements

We thank Samantha Beck-Previs for making the F-actin. This work was supported by National Institutes of Health grants AR034711 to R.C. and HL036153 to W.L. and Program Project Grants P01 HL059408 to David Warshaw (R.C., principal investigator on Project 1 and J.R., on Project 3) and P01 HL069779 to J.R.

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