Mutating the converter-relay interface of Drosophila myosin perturbs ATPase activity, actin motility, myofibril stability and flight ability.
We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (V(max)) by approximately 60% compared to wild-type myosin, but there is no change in apparent actin affinity (K(m)). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by approximately 15% or approximately 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by approximately 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional "cracking" of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.
Kronert
William A
WA
Department of Biology, Molecular Biology Institute and Heart Institute, San Diego State University, San Diego, CA 92182-4614, USA.
Melkani
Girish C
GC
Melkani
Anju
A
Bernstein
Sanford I
SI
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D004331:Drosophila melanogaster / Q000235:genetics / Q000502:physiology*; D005426:Flight, Animal; D008124:Locomotion; D008958:Models, Molecular; D020125:Mutation, Missense; D009210:Myofibrils / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000199:Actins; D000251:Adenosine Triphosphatases; D009218:Myosins