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Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T.

ABSTRACT: Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 A resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 A resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix.

SUBMITTER: Murakami K

PROVIDER: S-EPMC2438227 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T.

Murakami Kenji K Stewart Murray M Nozawa Kayo K Tomii Kumiko K Kudou Norio N Igarashi Noriyuki N Shirakihara Yasuo Y Wakatsuki Soichi S Yasunaga Takuo T Wakabayashi Takeyuki T

Proceedings of the National Academy of Sciences of the United States of America 20080515 20

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 A resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 A resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing ...[more]

PMID: 18483193

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Project description:Complete description of thin filament conformational transitions accompanying muscle regulation requires ready access to atomic structures of actin-bound tropomyosin-troponin. To date, several molecular-docking protocols have been employed to identify troponin interactions on actin-tropomyosin because high-resolution experimentally determined structures of filament-associated troponin are not available. However, previously published all-atom models of the thin filament show chain separation and corruption of components during our molecular dynamics simulations of the models, implying artifactual subunit organization, possibly due to incorporation of unorthodox tropomyosin-TnT crystal structures and complex FRET measurements during model construction. For example, the recent Williams et al. (2016) atomistic model of the thin filament displays a paucity of salt bridges and hydrophobic complementarity between the TnT tail (TnT1) and tropomyosin, which is difficult to reconcile with the high, 20 nM Kd binding of TnT onto tropomyosin. Indeed, our molecular dynamics simulations show the TnT1 component in their model partially dissociates from tropomyosin in under 100 ns, whereas actin-tropomyosin and TnT1 models themselves remain intact. We therefore revisited computational work aiming to improve TnT1-thin filament models by employing unbiased docking methodologies, which test billions of trial rotations and translations of TnT1 over three-dimensional grids covering end-to-end bonded tropomyosin alone or tropomyosin on F-actin. We limited conformational searches to the association of well-characterized TnT1 helical domains and either isolated tropomyosin or actin-tropomyosin yet avoided docking TnT domains that lack known or predicted structure. The docking programs PIPER and ClusPro were used, followed by interaction energy optimization and extensive molecular dynamics. TnT1 docked to either side of isolated tropomyosin but uniquely onto one location of actin-bound tropomyosin. The antiparallel interaction with tropomyosin contained abundant salt bridges and intimately integrated hydrophobic networks joining TnT1 and the tropomyosin N-/C-terminal overlapping domain. The TnT1-tropomyosin linkage yields well-defined molecular crevices. Interaction energy measurements strongly favor this TnT1-tropomyosin design over previously proposed models.

Project description:Coiled-coil tropomyosin, localized on actin filaments in virtually all eukaryotic cells, serves as a gatekeeper regulating access of the motor protein myosin and other actin-binding proteins onto the thin filament surface. Tropomyosin's modular pseudo-repeating pattern of approximately 39 amino acid residues is designed to allow binding of the coiled-coil to successive actin subunits along thin filaments. Even though different tropomyosin isoforms contain varying numbers of repeat modules, the pseudo-repeat length, in all cases, matches that of a single actin subunit. Thus, the seven pseudo-repeats of 42nm long muscle tropomyosin bind to seven successive actin subunits along thin filaments, while simultaneously bending into a super-helical conformation that is preshaped to the actin filament helix. In order to form a continuous cable on thin filaments that is free of gaps, adjacent tropomyosin molecules polymerize head-to-tail by means of a short (∼9 residue) overlap. Several laboratories have engineered peptides to mimic the N- and C-terminal tropomyosin association and to characterize the overlap structure. All overlapping domains examined show a compact N-terminal coiled-coil inserting into a partially opened C-terminal partner, where the opposing coiled-coils at the overlap junction face each other at up to ∼90° twist angles. Here, Molecular Dynamics (MD) simulations were carried out to determine constraints on the formation of the tropomyosin overlap complex and to assess the amount of twisting exhibited by full-length tropomyosin when bound to actin. With the exception of the last 20-40 C- and N-terminal residues, we find that the average tropomyosin structure closely resembles a "canonical" model proposed in the classic work of McLachlan and Stewart, displaying perfectly symmetrical supercoil geometry matching the F-actin helix with an integral number of coiled-coil turns, a coiled-coil helical pitch of 137Å, a superhelical pitch of 770Å, and no localized pseudo-rotation. Over the middle 70% of tropomyosin, the average twisting of the coiled-coil deviates only by 10° from the canonical model and the torsional freedom is very small (std. dev. of 7°). This small degree of twisting cannot yield the orthogonal N- and C-termini configuration observed experimentally. In marked contrast, considerable coiled-coil unfolding, splaying and twisting at N- and C-terminal ends is observed, providing the conformational plasticity needed for head-to-tail nexus formation.

Project description:The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding of nonmuscle cell motility. Indeed, experimental approaches such as fiber diffraction, cryoelectron microscopy, and three-dimensional reconstruction have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryogenic electron microscopy reconstruction of myosin-S1-decorated F-actin-tropomyosin together with atomic scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle β-myosin complexes; masseter myosin, which shares sequence identity with β-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39-42 residue-long tropomyosin pseudorepeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation.

Dataset Information

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T.

Publications

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T.

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