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

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.

Secondary Structure of the Novel Myosin Binding Domain WYR and Implications within Myosin Structure.

Structural changes in the myosin II light meromyosin (LMM) that influence thick filament mechanical properties and muscle function are modulated by LMM-binding proteins. Flightin is an LMM-binding protein indispensable for the function of Drosophila indirect flight muscle (IFM). Flightin has a three-domain structure that includes WYR, a novel 52 aa domain conserved throughout Pancrustacea. In this study, we (i) test the hypothesis that WYR binds the LMM, (ii) characterize the secondary structure of WYR, and (iii) examine the structural impact WYR has on the LMM. Circular dichroism at 260-190 nm reveals a structural profile for WYR and supports an interaction between WYR and LMM. A WYR-LMM interaction is supported by co-sedimentation with a stoichiometry of ~2.4:1. The WYR-LMM interaction results in an overall increased coiled-coil content, while curtailing ɑ helical content. WYR is found to be composed of 15% turns, 31% antiparallel ß, and 48% 'other' content. We propose a structural model of WYR consisting of an antiparallel ß hairpin between Q92-K114 centered on an ASX or ß turn around N102, with a G1 bulge at G117. The Drosophila LMM segment used, V1346-I1941, encompassing conserved skip residues 2-4, is found to possess a traditional helical profile but is interpreted as having <30% helical content by multiple methods of deconvolution. This low helicity may be affiliated with the dynamic behavior of the structure in solution or the inclusion of a known non-helical region in the C-terminus. Our results support the hypothesis that WYR binds the LMM and that this interaction brings about structural changes in the coiled-coil. These studies implicate flightin, via the WYR domain, for distinct shifts in LMM secondary structure that could influence the structural properties and stabilization of the thick filament, scaling to modulation of whole muscle function.