Interaction Between the Relay Loop and the SH1-SH2 Helix Region in Drosophila Muscle Myosin is Essential for Normal Motor Function, Myofibril Stability and Muscle Contraction

Abstract

Molecular modeling of Drosophila muscle myosin reveals that relay domain residue E499 interacts with SH1-SH2 residue R714 in a charge-dependent manner. To explore the significance of this interaction, we generated transgenic lines expressing myosin with a mutation in the relay loop domain (E499R) or the SH1-SH2 helix (R714E). Both mutations yield ∼75% reductions in CaATPase as well as basal or actin-activated MgATPase activity. Actin-sliding velocity of E499R is reduced by 65% and R714E shows no motility. Indirect flight muscles in late pupae of each mutant display disrupted myofibril assembly. Two-hour- and two-day-old adults have severely abnormal myofibril morphology and poor sarcomere organization, with no flight ability. Therefore, the putative interaction of the relay with SH1-SH2 is indispensable for normal motor function, myofibril stability and locomotion. We next constructed a putative compensatory mutant designed to restore function of the E499R or R714E mutant by generating a transgenic line that expresses both E499R and R714E. Interestingly, calcium, basal, and actin-stimulated ATPase values are restored to 65–70% of wild type and actin-sliding velocity is at 40%. The compensatory mutation suppresses the assembly defects in pupal muscle that are seen in both E499R and R714E and reduces disrupted myofibril morphology in two-hour old indirect flight muscles. However, the E499R-R714E mutant myosin was unable to maintain sarcomere organization in two-day-old flies or to restore flight ability. Overall, our results reveal that interaction of residues at the relay/SH1-SH2 helix interface is important for myosin and muscle function.