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

SIMPLE SUMMARY: WYR is a conserved protein domain characteristic of the muscle protein flightin, estimated to have originated in the ancestor to hexapods and crustaceans ~500 MYA. This study characterizes the secondary structure of WYR and shows that it binds the coiled-coil motif of muscle myosin and changes its structural properties. Characterizing WYR and its role in conjunction with myosin provides valuable insight into enduring evolutionary processes driving the success of Insecta. The relationship between WYR and myosin further reveals a means of ultrastructural regulation capable of informing the molecular structure–function relationships essential for the mechanical properties and structural stability of muscle. ABSTRACT: 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 β, and 48% ‘other’ content. We propose a structural model of WYR consisting of an antiparallel β hairpin between Q92-K114 centered on an ASX or β 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.