Converter Domain Residue R759 Interaction with Relay Loop Residue N509 in Drosophila Muscle Myosin is Critical for Motor Function, Myofibril Stability and Flight Ability

Abstract

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of Drosophila melanogaster skeletal muscle myosin. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at the relay loop interaction site. The mutation depresses calcium, basal or actin-activated MgATPase values (Vmax) by ∼60% and actin sliding velocity ∼35% compared to wild-type myosin. Ultrastructure of two-day-old adult fibers shows cracking and frayed myofibrils with some disruption of the myofilament lattice which becomes more severe in one-week-old adults. Flight ability is reduced in two-day-old flies compared to controls and is absent in 1-week-old adults. Thus appropriate interaction between the relay loop and converter domain is essential for normal motor function, myofibril stability and locomotion. To examine the specificity of this interaction, we used a compensatory mutational approach to attempt to restore the function of the R759E mutant myosin. Our modeling indicates that relay loop residues N509 and D511 interact with converter domain residue R759. To verify our model, we generated two transgenic lines that express R759E and either the N509K or D511K mutations. Interestingly, calcium, basal, and actin stimulated ATPase values are restored to 70% and actin sliding velocity is restored to 90% in N509K/R759E but not in D511K/R759E. Structurally fibers from 2-day or one-week -old adults appear morphologically normal in N509K/R759E and their flight ability is like wild type. However, D511K/R759E myofibrils do not show any improvement compared to R759E and flight ability is worse than R759E. Overall, our results reveal the critical interaction between the converter domain with relay loop residues and their role in myosin motor function and myofibril assembly/stability.