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Tropomyosin Movement Is Described by a Quantitative High‑Resolution Model of X‑Ray Diffraction of Contracting Muscle

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Tropomyosin Movement Is Described by a Quantitative High‑Resolution Model of X‑Ray Diffraction of Contracting Muscle This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Tropomyosin movement is described by a quantitative high‑resolution model of X‑ray diffraction of contracting muscle Koubassova, Natalia A.; Bershitsky, Sergey Y.; Ferenczi, Michael Alan; Narayanan, Theyencheri; Tsaturyan, Andrey K. 2016 Koubassova, N. A., Bershitsky, S. Y., Ferenczi, M. A., Narayanan, T., & Tsaturyan, A. K. (2017). Tropomyosin movement is described by a quantitative high‑resolution model of X‑ray diffraction of contracting muscle. European Biophysics Journal, 46(4), 335‑342. https://hdl.handle.net/10356/82598 https://doi.org/10.1007/s00249‑016‑1174‑6 © 2016 European Biophysical Societies' Association. This is the author created version of a work that has been peer reviewed and accepted for publication in European Biophysics Journal, published by Springer Berlin Heidelberg on behalf of European Biophysical Societies' Association. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1007/s00249‑016‑1174‑6]. Downloaded on 26 Sep 2021 21:50:01 SGT Tropomyosin movement is described by a quantitative high resolution model of x- ray diffraction of contracting muscle Pre-publication : not for distribution Koubassova N.A.1, Bershitsky S.Y.2, Ferenczi M.A.3, Narayanan T.4, Tsaturyan A.K.1 1Institute of Mechanics, M.V. Lomonosov Moscow University, Moscow, Russia; 2Institute of Immunology and Physiology, Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russia; 3Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore 4European Synchrotron Radiation Facility, Grenoble, France Contact information: Dr. Natalia Koubassova Institute of Mechanics Moscow University 1 Michurinsky prosp. Moscow 119192 Russia E-mail: [email protected] 1 Abstract Contraction of skeletal and cardiac muscle is controlled by Ca2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca2+, Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the 3-state model (McKillop, Geeves 1993), upon Ca2+ binding to Tn, Tpm rotates about the filament axis to a ‘closed state’ where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an ‘open’ position where neighbouring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low angle x- ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of x-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much improved fit to the experimental data obtained from rabbit muscle fibres in relaxed and rigor states than previous lower resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 nm-1 to 0.3 nm-1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin. Keywords: actin, tropomyosin, myosin heads, x-ray diffraction, modelling INTRODUCTION Actin-myosin interaction that powers contraction of skeletal and cardiac muscles is controlled by calcium ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) (Gordon et al. 2000; Nevzorov and Levitsky 2011). Tpm is a ~40 nm-long coiled-coil dimer of a-helices. Tpm molecules bind each other in a tail-to-head manner forming two stiff continuous strands, which lie in two grooves of a pseudo-two-fold long helix on the surface of F-actin (Moore et al. 2016). Every Tpm molecule forms a complex with Tn which consists of Tn-C, Tn-I and Tn-T subunits. The steric blocking theory of muscle regulation was put forward more than 40 years ago (Haselgrove 1972, Huxley 1972, Spudich et al. 1972). It postulates that in the absence of Ca2+ regulatory proteins block myosin-binding sites on actin and keep muscle relaxed, while Ca2+ binding to Tn releases the block and allows muscle to contract. Modern versions of the theory, the 3-state model (McKillop, Geeves 1993) now supported by protein crystallography 2 (Vinogradova et al. 2005) suggests that in the absence of Ca2+, Tn holds Tpm in a blocked, or B- state, where it protects actin monomers from myosin binding. In the presence of Ca2+ and in the absence of myosin heads, Ca2+ binds Tn-C and causes conformational changes in other Tn subunits. This releases Tpm from the B-state and allows a transition to so called closed, or C- state. During the B- to C-state transition Tpm rotates azimuthally about the filament axis by ~25 (Vibert et al. 1997) and partially opens actin sites for myosin binding. When a myosin head binds actin in a strong, stereospecific manner, it causes the Tpm strand to move further by ~10 (Vibert et al., 1997) to the open, or O-state, where neighbouring actin sites are fully exposed for myosin binding. The structural changes associated with Tpm movement are studied by electron microscopy (Vibert et al. 1997) or low-angle x-ray diffraction. The latter is the only method that allows the monitoring of Tpm movement in functioning contracting muscle, either intact (Kress et al. 1986) or chemically permeabilised (Poole et al. 2006). As F-actin with bound Tpm strands forms a near-helical structure with a pitch of ~36 nm, azimuthal Tpm movement can be monitored by measuring the intensity of the actin layer lines, especially the second actin layer line, A2, that has spacing of ~1/(18 nm) (Haselgrove 1972, Huxley 1972). More recently, using time-resolved x- ray diffraction on a synchrotron radiation source and 1D detector positioned at a radial spacing of ~0.23 nm-1, Kress et al. (1986) observed changes in the intensity of the A2 layer line which reported Tpm movement during the onset of contraction of frog skeletal muscle. An increase in the A2 intensity precedes the tension rise and changes in the equatorial x-ray reflections showing that Tpm rotation precedes not only force generation but also the radial movement of myosin heads associated with their binding to actin (Kress et al. 1986). The data demonstrated that low angle x-ray diffraction reports Tpm transition from B- to C- and O-states. Monitoring the Tpm transition from B- to O-state and vice versa in fully activated muscle is more difficult as the expected changes in the x-ray intensities upon mechanical perturbations are smaller, and are accompanied by a change in the number of myosin heads bound to actin, which also contribute to the intensities of the actin layer lines. As phases of the x-ray reflections cannot be found experimentally, direct mathematical modelling (Koubassova et al. 2002, 2008) is required for quantitative interpretation of the changes in the x-ray diffraction intensities in terms of molecular movement. Early models based on low resolution EM data (Poole et al. 2006, Koubassova 2008) provided some useful insight into the problem. However, the quality of those models was insufficient for making convincing estimates and predictions. For example, the ratio of the intensities of generic A6 and A7 actin layer lines at 1/(5.9 nm) and 1/(5.1 nm), respectively, predicted by the models was twice that observed experimentally (Koubassova 2008). 3 Here we report the results of modelling of the intensities of the actin layer lines using new high- resolution near-atomic models of the actin-Tpm complex obtained in the presence (O-state, Behrmann et al. 2012) and in the absence (von der Ecken et al. 2015) of myosin heads strongly bound to actin. The results of simulations were compared to experimental x-ray intensities obtained at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) from thin bundles of permeabilised fibres from fast rabbit skeletal muscle in relaxed and rigor states. Our aims were to determine how the high-resolution models of thin filament quantitatively derive the intensities of the actin layer lines experimentally determined by x-ray diffraction, and to identify the x-ray reflections whose intensities provide a quantitative estimate of the transition between C- and O-states independently of the number of myosin heads strongly bound to actin. METHODS Structural models of thin filament, intensity calculations. Two recently published atomic structures of the actin-tropomyosin complex were used for model calculations (Behrmann et al. 2012; von der Ecken et al. 2015). As actin monomers in these structures do not show a preserved helical periodicity, an ideal 13/6 helical model with axial shift of 2.75 nm and rotation angle of 12p/13 between neighbour monomers was built using an actin monomer and an adjacent 5.5 nm- long segment of Tpm dimer as a repeating unit of the actin helix. Additional calculations have shown that the choice of a particular actin monomer from a PDB file (PDB code 4AF7 or 3J8A) exerted a negligible effect on the calculated layer line intensities. The helical repeat contained 13 actin monomers and tropomyosin segments in 6 turns of the left-handed helix with a pitch of 35.75 nm. The rotation angle was assumed to be 25 for the B- to C-state transition (Vibert et al. 1997) and 10 or 16 for the transition between C- and O-states. The smaller figure was reported by Vibert et al. (1997), the higher figure is the difference in the position of the Tpm coiled-coil in the cryo-EM models obtained in the presence (Behrmann et al.
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