Modulating Contraction by Binding of MyBP-C to Actin

Abstract

Myosin-binding protein C (MyBP-C) is a ∼130 kDa protein of the thick filaments of vertebrate skeletal and cardiac muscle. It consists of a linear array of ten or eleven globular, 10-kDa domains from the immunoglobulin (Ig) and fibronectin type III families, and an additional, MyBP-C-specific motif. The cardiac isoform, cMyBP-C, plays a key role in modulating cardiac function, and mutations in MyBP-C cause heart disease. Despite its discovery 40 years ago, the mechanism of MyBP-C function remains poorly understood. In vitro studies suggest that it could modulate contraction by binding to thin filaments, but there has been no evidence for this in situ. We used electron tomography of exceptionally well-preserved skeletal muscle to study the 3D organization of MyBP-C in the intact sarcomere. The tomogram shows that MyBP-C projects perpendicular to the thick filament surface and reaches neighboring thin filaments. This thick-thin filament bridge suggests a possible physical basis for modulating filament sliding and thus contraction. In vitro, binding to actin has been shown to occur via MyBP-C's N-terminal end. To understand the structural basis of this binding, we used negative stain electron microscopy and 3D reconstruction to study F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions. 3D reconstructions showed MyBP-C density starting over subdomain 1 of actin and extending tangentially towards 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 appeared to overlap the relaxed (low Ca2+) position of tropomyosin but not the activated position. This suggests that MyBP-C might help determine the state of thin filament activity by modulating tropomyosin position on actin.