Effects of Mavacamten and Blebbistatin on the Small-Angle X-ray Scattering Structure of Human β-cardiac Myosin

Abstract

Cardiac myosin is a motor protein that converts chemical energy from ATP hydrolysis to mechanical work that keeps the heart beating. Full-length myosin and even the smaller sub-fragment, heavy meromyosin (HMM) are large and flexible molecules and have not been possible to crystallize and therefore has been unavailable for structural studies. These properties make myosin a good candidate for small-angle X-ray scattering (SAXS) studies. Here we use size exclusion chromatography coupled with SAXS to study structural changes of human β-cardiac 2-hep and 25-hep HMM constructs in relaxing conditions treated with two different myosin inhibitors: blebbistatin and mavacamten. Under resting conditions, the 25-hep HMM had a radius of gyration (Rg) of ∼ 118 Å (Guinier fit) to ∼ 137 Å (GNOM fit) and the maximum dimension of the molecule (Dmax) of 526 Å. The Rg of 25-hep HMM decreased by about 5-10% with either blebbistatin or mavacamten treatment. The Dmax of 25-hep HMM decreased ∼ 20 Å by blebbistatin treatment while it decreased ∼ 80 Å by mavacamten treatment. There were only moderate structural changes (< 2%) for the 2-hep HMM with either blebbistatin or mavacamten treatment. Our results indicate that most of the HMM molecules in relaxing conditions maintain an open configuration and that the myosin S2 region is essential for HMM to adopt the folded back closed configuration in presence of mavacamten and blebbistatin. Supported, in part, by NIH P41 GM103622.