Abstract
The relay domain of the myosin molecular motor communicates with the nucleotide binding site of the molecule, the actin-binding region, and the converter domain. Interactions between the relay and converter domains are critical for the converter to elicit movement of the lever arm, which yields the molecule's power stroke. An invariant tryptophan relay residue at the converter interface is flanked by two poorly conserved amino acids. Here we utilize mutant versions of the Drosophila melanogaster muscle myosin heavy chain to probe the function of these variable relay residues, residues 509 and 511. We replaced either or both poorly-conserved residues within an embryonic myosin isoform with those of the indirect flight muscle isoform. All such replacements eliminate in vitro actin motility, suggesting abnormal communication between the relay and converter domains that disables the power stroke. Interestingly, we found that the variations at residues 509 and 511 differentially disrupt both myosin ATPase activity and indirect flight muscle ultrastructure, with the double mutant showing the most severe effect upon ATPase activity and the least severe effect on emergent adult ultrastructure. Isoform-specific interactions may be implicated in these differential effects, as our molecular modeling at two stages of the mechanochemical cycle defined differences in contacts between the embryonic, flight muscle and chimeric 509 and 511 relay residues with a key residue in the converter domain. Overall, our studies show that two poorly conserved amino acid residues at the relay-converter interface are important to isoform-specific function of myosin and to the structural integrity of muscle.
