Contiguity and Structural Impacts of a Non-Myosin Protein within the Thick Filament Myosin Layers

Abstract

Myosin dimers arranged in layers and interspersed with non-myosin densities have been described by cryo-EM 3D reconstruction of the thick filament in Lethocerus at 5.5 Å resolution. One of the non-myosin densities, denoted the ‘red density’, is hypothesized to be flightin, an LMM-binding protein essential to the structure and function of Drosophila indirect flight muscle (IFM). Here, we build upon the 3D reconstruction results specific to the red density and its engagement with the myosin coiled-coil rods that form the backbone of the thick filament. Each independent red density winds its way through the myosin dimers, such that it links four dimers in a layer and one dimer in a neighboring layer. This area in which three distinct interfaces within the myosin rod are contacted at once and the red density extends to the thick filament core is designated the “multiface”. Present within the multiface is a contact area inclusive of E1563 and R1568. Mutations in the corresponding Drosophila residues (E1554K and R1559H) are known to interfere with flightin accumulation and phosphorylation in Drosophila. We further examine the LMM area in direct apposition to the red density and identified potential binding residues spanning up to ten helical turns. We find that the red density is associated within an expanse of the myosin coiled-coil that is unwound by the third skip residue and the coiled-coil is re-oriented while in contact with the red density. These findings suggest a mechanism by which flightin induces ordered assembly of myosin dimers through its contacts with multiple myosin dimers and brings about reinforcement on the level of a single myosin dimer by stabilization of the myosin coiled-coil.

Molecular Characterization and Phylogenetic Analysis of Flightin Gene in Vespa basalis (Hymenoptera, Vespidae)

“…These findings are consistent with the secondary structure of flightin in Drosophila , which exhibits two helical regions [ 13 ]. Cryo-electron microscopy studies by Menard et al on the thick myofilament of Lethocerus identified a specific red density in the contact area of the thick myofilament skeleton, suggesting the presence of flightin [ 13 , 29 , 30 ]. Our findings suggest that flightin facilitates the contact between multiple myosin dimers, promoting their orderly assembly and stabilizing myosin coils.…”

Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution

“…In Lethocerus, stretch activation has been shown to occur at submaximal calcium concentrations [80]. Many features of the flight muscle lattice have been shown to affect stretch activation including troponin-C isoforms [81], flightin [82,83], the N-terminal extension on the RLC [84], extensions on tropomyosin and troponin [85], and troponin bridges formed from myosin heads [38]. Another stretch activation model proposes that stretch produces enhanced alignment of myosin head origins with actin targets [59].…”