Myosins Active Site Closure via Switch-1 Allosterically Regulates the Motor-Actin Interaction during the ATPase Cycle for Productive Force Generation

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. Drosophila melanogaster contains one gene encoding all striated muscle myosin II isoforms. The objective of this study is to gain insight into how alternative exon selection imparts myosin protein isoform biochemical and biophysical specificity. To this end, two His-tagged recombinant proteins encoding an indirect flight muscle myosin isoform (IFI) and an embryonic body wall myosin isoform (EMB) were expressed in and purified from the indirect flight muscles (IFM) of engineered fly lines lacking endogenous IFM myosin (Caldwell et al., Methods, 2012). The purified His-tagged myosins retain ATPase activity similar to their corresponding untagged isoforms. The myosin subfragment-1 (S1) contains the myosin heavy chain motor domain and the essential light chain. We determined the three-dimensional structure of IFI S1 at 2.5 Å resolution and EMB S1 at 2.2 Å resolution. They are the first insect myosin protein structures determined by X-ray crystallography (EMB is PDB 4QBD). Both structures are in the post rigor enzymatic state, as determined by comparisons with known myosin structures. For the EMB structure, two copies of the myosin molecule with slight conformational differences were resolved in the asymmetric unit. The electron density revealed a citrate molecule (contained in the crystallization solution) bound in the nucleotidebinding pocket. For the IFI structure, there is one myosin molecule in the asymmetric unit. The nucleotide-binding pocket contains an ADP molecule. Differences in conformation within regions of the proteins encoded by alternative exons suggest a source of the observed physiological differences in the embryonic and adult flight muscle fibers. (Funded by NIH R01GM32443 to SIB).