Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C (cMyBP-C)
Department of Cell Biology
Actins; Animals; Carrier Proteins; Chickens; Escherichia coli; Mice; Myocardium; Myosins; Peptide Fragments; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Tropomyosin
Myosin-binding protein C (MyBP-C) is an approximately 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 beta-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.
DOI of Published Version
J Mol Biol. 2011 Jul 8;410(2):214-25. Epub 2011 May 13. Link to article on publisher's site
Journal of molecular biology
Mun J, Gulick J, Robbins J, Woodhead JL, Lehman W, Craig RW. (2011). Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C (cMyBP-C). Craig Lab Publications. https://doi.org/10.1016/j.jmb.2011.05.010. Retrieved from https://escholarship.umassmed.edu/craig/23