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

Abstract

Simple SummaryHexapods and crustaceans (Pancrustacea) represent nearly 80% of known living animals. Species within this clade exhibit exquisite muscle types propelling ingenious means of locomotion, likely contributing to their evolutionary success. Flightin, a myosin-binding protein, first identified in the flight muscle of Drosophila, is defined by WYR, a protein domain exclusive to Pancrustacea. In Drosophila, flightin imparts stiffness to the thick filament and is essential for their length determination and structural integrity. Here, we build on results from the three-dimensional reconstruction of the Lethocerus flight muscle thick filament to advance the hypothesis that flightin influences thick filament mechanics, and by extension muscle function, by acting as a cinch in the filament core.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.