Metabolism sustains life through enzyme-catalyzed chemical reactions within the cells of all organisms. The coupling of catalytic function to the structural organization of enzymes contributes to the kinetic optimization important to tissue-specific and whole-body function. This coupling is of paramount importance in the role that muscle plays in the success of Animalia. The structure and function of glycolytic enzyme complexes in anaerobic metabolism have long been regarded as a major regulatory element necessary for muscle activity and whole-body homeostasis. While the details of this complex remain to be elucidated through in vivo studies, this review will touch on recent studies that suggest the existence of such a complex and its structure. A potential model for glycolytic complexes and related subcomplexes is introduced.

Cardiac myosin binding protein-c (cMyBP-C) of mammalian cardiac muscle and flightin (FLN) of invertebrate indirect flight muscle (IFM) have been shown to contribute to thick filament stiffness, as determined by calculations of persistence length (PL), an index of flexural rigidity [1, 2] in their corresponding muscle systems. FLN and cMyBP-C in vitro bind to a common site in the coiled-coil region of myosin II, and both proteins are known to be regulated by phosphorylation [3, 4]. To test the hypothesis that FLN and cMyBP-C are functionally homologous, we have determined the extent to which cMyBP-C can rescue the phenotypes manifested in the Drosophila FLN knockout strain fln 0 . Structural characteristics of flight muscle sarcomeres were analyzed by transmission electron microscopy (TEM) and the contour and endto-end length of isolated, hydrated native thick filaments was measured by atomic force microscopy (AFM). ). In preparation for TEM, thoraces from newly eclosed (<1 hour) D. melanogaster were bisected, fixed, dehydrated, infiltrated, embedded, sectioned and imaged by TEM [5]. The length of sarcomeres from 4-5 flies for each Drosophila strain was measured using ImageJ. AFM data of isolated thick filaments were evaluated using the parameters and programs described by [6]. Statistical analysis was done using JMP 9 software.The TEM results confirmed both the sarcomere length measurements and level of structural order previously seen for fln 0 and fln 0 ;fln + , while revealing shorter sarcomeres in the transgenic lines involving cMyBP-C alone (Fig. 1, Table. 1). When cMyBP-C is expressed alongside FLN, sarcomere length is slightly but significantly longer than sarcomere length in the control fln 0 ;fln + . These results support the idea of cMyBP-C binding to myosin in thick filaments of D. melanogaster and influencing the length of the filaments. However, the length regulation exerted by cMyBP-C is surpassed by FLN when FLN is present, either by direct binding competition to a common myosin binding site or another regulatory mechanism.The PL for fln + ;cMyBPC + obtained by AFM was significantly higher than PL for fln 0 ;fln + , suggesting that cMyBP-C contributes to filament stiffness when expressed ectopically in IFM. However, the cMyBP-C effect is seen only in the presence of FLN as PL of fln 0 ;cMyBPC + was not different that PL of fln 0 . Our observations suggest that the presence of FLN influences the effects that cMyBP-C has on the mechanical properties of the thick filaments. This may possibly be due to FLN stabilizing the thick filaments to permit a more ideal environment for cMyBP-C binding. From these studies we conclude that 80

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.

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.