Journal of Molecular Biology
The N-Terminal Domains of Myosin Binding Protein C Can Bind Polymorphically to F-Actin
Introduction
Biological macromolecules have evolved to perform an enormous range of functions—from catalysis of chemical reactions to generation of mechanical force. Over the course of evolution, the regulation of such activity has become more complex, and the study of biology can be seen, in some manner, largely as the study of layered regulatory systems. The primary regulatory system in vertebrate striated muscle involves the Ca2+-activated troponin–tropomyosin system that either allows or prevents myosin heads from binding to F-actin. The troponin–tropomyosin system involves the troponin complex that binds Ca2+, which causes it to shift the position of tropomyosin on F-actin from an 'off', or blocked state, to an ‘on’ state, allowing myosin S1 to bind. This original steric blocking model1, 2, 3, 4 is supported by abundant structural data, yet the molecular details of the interactions are still not known. More recently, the role of myosin binding protein C (MyBP-C) in modulating this primary regulatory signal has been the subject of intense investigation5, 6 as, within cardiac muscle, mutations in the MYBPC3 gene can cause serious heart disorders. Approximately 42%7 of familial hypertrophic cardiomyopathy8 cases involved mutations in cardiac myosin binding protein C (cMyBP-C), with a number of single amino acid substitutions (n = 24) occurring in the N-terminal ‘regulatory’ region comprising the first four domains of the protein that modulate myosin–actin interactions to fine-tune heart muscle contraction.9 A very recent tomographic reconstruction from intact muscle fibers showed that MyBP-C does actually span the space between thick filaments and thin filaments in striated muscle,10 establishing that the N-terminal region of MyBP-C does contact the thin filaments within intact muscles.
MyBP-C is a large modular protein (128–137 kDa) that belongs to the immunoglobulin superfamily containing repeating Ig-like and fibronectin-like type 3 modules (designated C1 through C10; Fig. 1a) plus a ∼ 100-residue motif of unknown structure between the C1 domain and the C2 domain. The cardiac isoform has an additional Ig domain (C0) at the N-terminus that is not present in the skeletal striated muscle isoform. A proline/alanine-rich (PA) linker connects C0 and C1, and it has been found that the percentage of proline + alanine content correlates with the heart rate requirement of different species11 and confers different effects on thin-filament activation. In combination with C1, this linker can also modulate thin-filament-activated S1 ATPase activity.12 Furthermore, a knock-in mouse model that carried N-terminal-shortened cMyBP-C (without PA-C1 but retaining the C0 domain) exhibited an increased Ca2+ sensitivity in force development, whereas maximal active force levels remained unchanged.13
It has been shown, using biochemical assays, electron microscopy (EM), and neutron scattering, that the N-terminal portion of cMyBP-C (containing either C0–C2 or C1–C2) can bind F-actin.11, 12, 14, 15, 16 When exogenous N-terminal fragments of cMyBP-C were added to skinned cardiac muscle fibers, these were also shown to bind actin in the intact myofibrillar lattice.17 A model for mouse C0–C2/actin interaction has been proposed based on neutron scattering data, with C0 and C1 binding subdomain 1 of one actin protomer and subdomain 2 of an adjacent actin protomer such that these cMyBP-C domains would clash with the S1 binding site on actin and with the ‘off’ position of tropomyosin. The model suggested that actin-bound C0–C2 could interfere with myosin S1 attachment and activate thin filaments independent of Ca2+/troponin by shifting tropomyosin toward an ‘on’ position. As the C0–C2 construct has also been shown to bundle F-actin,18 it has been proposed that multiple F-actin binding sites exist within the N-terminal regulatory region. The possibility that human C0–C2 bundles actin due to dimerization is excluded by small-angle X-ray scattering data (C.M.J.J. and J.T., unpublished data) showing that human C0–C2 is monomeric in solution, as was previously shown for mouse C0–C2.16
We have used EM and three-dimensional reconstruction to analyze complexes of human C0C1, and of both C0 and C1 alone, with F-actin. Our results are quite surprising and show that each of these two Ig domains can bind to the same two distinctly different positions on each F-actin protomer. Our results show how these two binding modes would differently impact the binding of myosin and tropomyosin to F-actin, and help to resolve a number of seemingly contradictory observations in the literature. This dual mode of binding also indicates that the ability of an actin binding protein to interact in multiple modes with actin is not limited to tropomyosin.
Section snippets
Results
Actin filaments can be seen by the eye to be decorated with the C0C1 fragment (Fig. 1c) in comparison with naked F-actin (Fig. 1b). Extensive bundling and aggregation of the actin filaments occur when C0C1 is present, similar to a previous report showing such cross-linking by the C0–C1–m-C2 fragment.18 This is a prima facie argument that multiple actin binding sites also exist within the C0C1 fragment.18 Some effort was therefore invested to find single filaments suitable for three-dimensional
Discussion
The ability of two different cMyBP-C domains, C0 and C1, to interact with similar sites on actin protomers is quite striking, as is the ability of each domain to bind to the same two distinctly different positions on F-actin. Our observations of specific binding sites for both C0 and C1 on F-actin conflict with a recent report suggesting that these N-terminal domains interact with actin in a nonspecific manner.25 Our results can be reconciled with a model derived from small-angle neutron
Protein preparation and EM
A synthetic gene fragment encoding the N-terminal domains (C0–C1–m-C2–C3–C4; C0C4) of human cMyBP-C (GenPept accession code CAA71216.1) was designed to incorporate near-optimal Escherichia coli codon usage and was synthesized by Genscript, Inc. (USA) in a pUC57 cloning vector. BamHI and EcoRI restriction endonucleases (unique sites engineered into the 5′ and 3′ ends of the synthetic gene, respectively) were used to isolate the C0C4 gene that was then subcloned into the expression plasmid pETM
Acknowledgements
This work was supported by National Institutes of Health grant GM081303 (to E.H.E.) and US Department of Energy Grant DE-FG02-05ER64026 (to J.T.).
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