X-Ray Structure Determination of the First Insect Skeletal Muscle Myosin II

Abstract

Skeletal muscle myosin class II proteins are molecular machines that convert the chemical energy derived from the hydrolysis of ATP into mechanical work used to power muscle contraction. The three-dimensional structure of the truncated head from an embryonic body wall myosin isoform (EMB) of the common fruit fly, Drosophila melanogaster, is reported at 2.2 A resolution. The histidine-tagged recombinant protein was expressed in and purified from the indirect flight muscles of an engineered fly line (Caldwell et al., Methods, 2012). The purified histidine-tagged EMB myosin retains ATPase activity similar to that of the untagged EMB isoform. EMB myosin subfragment 1 (S1), contains the myosin heavy chain motor domain (MD) and the essential light chain (ELC). Crystals of the complex belong to space group P212121 with the unit cell parameters of a = 108.5 A, b = 148.5 A, and c = 148.7 A. Two copies of the molecule were resolved in the asymmetric unit and these have slight conformational differences. Interestingly, a number of the domains encoded by alternative exons are in contact between the two molecules, suggesting potential isoform-specific interactions that may be relevant in vivo. While the nucleotide-binding site had some ligand electron density, the ligand was not resolved. The enzymatic conformational state of the myosin cross-bridge cycle was determined as post-rigor by comparisons with known myosin structures. Amino acid residue orientations are currently being compared between EMB S1 and other myosins, particularly in the alternatively-encoded regions, to gain insights into structure-function relationships that define isoform-specificity.