Superhelical Architecture of the Myosin Filament-Linking Protein Myomesin with Unusual Elastic Properties

et al. (2012) Superhelical Architecture of the Myosin Filament-Linking Protein Myomesin with Unusual Elastic Properties. PLoS Biol 10(2): e1001261. doi:10.1371/journal.pbio.1001261 Superhelical Architecture of the Myosin Filament-Linking Protein Myomesin with Unusual Elastic Properties Nikos Pinotsis 0 1 Spyros D. Chatziefthimiou 0 1 Felix Berkemeier 0 1 Fabienne Beuron 0 1 Irene M. 0 1 Mavridis 0 1 Petr V. Konarev 0 1 Dmitri I. Svergun 0 1 Edward Morris 0 1 Matthias Rief 0 1 Matthias Wilmanns 0 1 Gregory A. Petsko, Brandeis University, United States of America 0 Current address: Max F. Perutz Laboratories, University of Vienna , Campus Vienna Biocenter 5, A-1030 Vienna , Austria 1 1 European Molecular Biology Laboratory Hamburg , Hamburg, Germany , 2 Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories , London , United Kingdom , 3 Institute of Physical Chemistry, National Centre for Scientific Research Demokritos , Athens , Greece , 4 Institute for Biophysics and Munich Center for Integrated Protein Science, Physics Department, Technical University of Munich , Garching , Germany Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, to maintain an ordered state, they require molecular structures of extraordinary stability. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain arrays. Members of the myomesin protein family function as molecular bridges that connect major filament systems in the central M-band of muscle sarcomeres, which is a central locus of passive stress sensing. To unravel the mechanism of molecular elasticity in such filament-connecting proteins, we have determined the overall architecture of the complete C-terminal immunoglobulin domain array of myomesin by X-ray crystallography, electron microscopy, solution X-ray scattering, and atomic force microscopy. Our data reveal a dimeric tailto-tail filament structure of about 360 A in length, which is folded into an irregular superhelical coil arrangement of almost identical a-helix/domain modules. The myomesin filament can be stretched to about 2.5-fold its original length by reversible unfolding of these linkers, a mechanism that to our knowledge has not been observed previously. Our data explain how myomesin could act as a highly elastic ribbon to maintain the overall structural organization of the sarcomeric M-band. In general terms, our data demonstrate how repetitive domain modules such as those found in myomesin could generate highly elastic protein structures in highly organized cell systems such as muscle sarcomeres. - Funding: The work has been supported by the grant PITN-GA2009-238423 from the European Commission to M.W. and M.R., and by grants Wi1058/8-1 (FOR 1352) and RI 990/4-1(FOR1352) from the Deutsche Forschungsgemeinschaft to M.W. and M.R., respectively. F. Berkemeier has been supported by Elitenetzwerk Bayern. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: AFM, atomic force microscopy; EM, electron microscopy; EOM, Ensemble Optimization Method; Fn-III, fibronectin type III; Ig, immunoglobulin-like; MBP, maltose binding protein; My9My13, My9My10My11My12(My13)2My129My119My109My99; SAXS, small angle X-ray scattering; SeMet, seleno-L-methionine . These authors contributed equally to this work. Striated myofibrils are found in skeletal and cardiac muscle cells and represent a highly organized cellular system for studying how active force can be generated while the overall structural organization of the underlying sarcomeric units is maintained. The principal protein components of myofibrils are large longitudinal filaments that include actin (thin filament), myosin (thick filament), titin, and nebulin [1]. These filaments form a well-established striated pattern of distinct zones, with the M-band at the center [2]. On activation, both substantial axial and radial forces are generated within the overall sarcomere structure [3]. To maintain a constant sarcomere volume under defined physiological conditions, these forces can lead to changes in both radial and longitudinal contour dimensions of the sarcomere. Under typical tension conditions, Mband-associated thick filaments can substantially move away from the sarcomeric center by 0.1 mm or more, which can lead to Mband-induced instability of the sarcomere [4]. Because of the presence of a stiff Z-disk architecture at the sarcomeric periphery, the amount of movement decreases with the overall sarcomere length so that the resting tension stays constant. In cardiac muscles, elastic Mband motions are thought to correlate with heart beat rate [5], rendering investigations of the underlying molecular parameters highly relevant to heart and skeletal muscle research. To ensure the restoration of sarcomere integrity on activation, there are two principal structural compartments with elastic properties. The first section is defined by the I-band segment, which is situated between the stiff and highly interconnected Z-disk at the sarcomere periphery and the more dynamic central A-band and M-band [1,69]. The second site for molecular elasticity is within the M-band, in which so-called M-bridges transversely connect thick filaments with each other and with titin filaments [2,10,11]. At the molecular level, M-bridges are thought to be primarily composed of myomesin (MYOM1), which is universally expressed, and two related isoforms, MYOM2 and MYOM3, which show tissue-specific expression [12]. The three proteins share a common domain topology that is characterized by a unique N-terminal myosin-binding domain, followed by an array of fibronectin type III (Fn-III) domains and immunoglobulin-like (Ig) domains. In addition, they are capable of forming C-terminal tailThe contraction and relaxation cycles of active muscles generate substantial mechanical forces, both axially and radially, that place extraordinary stress on the molecular structures within the muscle fibers. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain structures. Myomesin is one such repetitive filament protein that is thought to form bridges between the main contractile filaments of the muscle, providing the muscle structure with resistance in the radial dimension. To investigate how the repetitive structure of myomesin contributes to muscle elasticity, we determined the overall architecture of its complete repetitive domain array using a combination of four complementary structural biology methods. Our study reveals a long, dimeric tail-to-tail filament structure folded into an irregular superhelical coil arrangement of almost identical domain modules separated by short linkers. When we applied tension t (...truncated)