Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T Kenji Murakami*, Murray Stewart†, Kayo Nozawa*, Kumiko Tomii*, Norio Kudou‡, Noriyuki Igarashi‡, Yasuo Shirakihara§, Soichi Wakatsuki‡, Takuo Yasunaga¶, and Takeyuki Wakabayashi*ʈ *Department of Biosciences, School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551, Japan; †Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 0QH, United Kingdom; ‡Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization, Oho 1-1, Tsukuba 305-0801, Japan; §Structural Biology Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan; and ¶Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Ooaza-kawazu 680-4, Iizuka, Fukuoka 820-850, Japan Communicated by Chikashi Toyoshima, University of Tokyo, Tokyo, Japan, February 27, 2008 (received for review September 13, 2007) Head-to-tail polymerization of tropomyosin is crucial for its actin one helix into the holes between residues on the other helix binding, function in actin filament assembly, and the regulation of [‘‘knobs-in-holes’’ packing (9)]. Some tropomyosin heptad repeats actin-myosin contraction. Here, we describe the 2.1 Å resolution deviate from the canonical pattern in having either bulky or small structure of crystals containing overlapping tropomyosin N and C hydrophobic residues in positions a and d (10). It has been proposed termini (TM-N and TM-C) and the 2.9 Å resolution structure of crystals (11) that the small side chains might be able to move within the large containing TM-N and TM-C together with a fragment of troponin-T holes facilitating the flexibility required for tropomyosin’s wide (TnT). At each junction, the N-terminal helices of TM-N were splayed, range of functions on actin filaments and this has been verified with only one of them packing against TM-C. In the C-terminal region experimentally (12). Tropomyosin (284 residues in most isoforms) of TM-C, a crucial water in the coiled-coil core broke the local 2-fold also has a 7-fold (Ϸ40 residue) sequence repeat (13), and each motif symmetry and helps generate a kink on one helix. In the presence of is thought to interact similarly with one actin monomer (8, 11). a TnT fragment, the asymmetry in TM-C facilitates formation of a To accommodate its wide range of functions, tropomyosin 4-helix bundle containing two TM-C chains and one chain each of exists in Ͼ40 isoforms, which are expressed from four genes TM-N and TnT. Mutating the residues that generate the asymmetry in (1). Each tropomyosin gene is composed of nine exons. Exons TM-C caused a marked decrease in the affinity of troponin for 1, 2, 6, and 9 are alternatively spliced, and splicing patterns are actin-tropomyosin filaments. The highly conserved region of TnT, in correlated with different functions (14). In striated muscle, which most cardiomyopathy mutations reside, is crucial for interact- anchoring the tropomyosin-binding subunit of troponin, tro- ing with tropomyosin. The structure of the ternary complex also ponin-T (TnT, 15), requires the C-terminal region encoded by explains why the skeletal- and cardiac-muscle specific C-terminal striated muscle-specific exon 9a (16). region is required to bind TnT and why tropomyosin homodimers Here, we describe crystal structures of overlapping fragments bind only a single TnT. On actin filaments, the head-to-tail junction from the N- and C-terminal regions of tropomyosin in the can function as a molecular swivel to accommodate irregularities in presence and absence of a fragment of TnT. These structures the coiled-coil path between successive tropomyosins enabling each show how TnT generates a molecular swivel that enables suc- to interact equivalently with the actin helix. cessive tropomyosins to interact equivalently with the actin helix. calcium ͉ cardiomyopathy ͉ troponin Results Tropomyosin Head–Tail Interaction. The C-terminal (TM-C) (resi- ropomyosin, the archetypal coiled-coil protein, is distributed dues 254–284, preceded by a 20 residue fragment of the GCN4 Twidely in eukaryotic cells where it is a key component of the leucine zipper to stabilize dimerization) and N-terminal (TM-N) actin cytoskeleton (1). Tropomyosin molecules lie in the groove of [residues 1–24, followed by a 12 residue fragment of the GCN4 the actin helix and successive molecules overlap head-to-tail so that leucine zipper, and preceded by N-terminal GlyAlaAlaSer- the C terminus of one binds the N terminus of the next. This overlap extension necessary to mimic N-acetylation; see refs. 17 and 18 and is crucial for function, because only polymerizable tropomyosin the top line of supporting information (SI) Fig. S1] regions of binds to the actin cytoskeleton, stabilizes its structure, and influ- skeletal-muscle ␣-tropomyosin, which is identical to cardiac tropo- ences the function of actin-binding proteins, such as ADF/cofilin, myosin, were expressed in bacteria (Fig. 1A). The structure of P21 Arp2/3, and gelsolin (2, 3). Tropomyosin plays a pivotal role in crystals containing both TM-N and TM-C was determined to 2.1 Å regulating the actin-myosin interaction in skeletal and cardiac resolution by molecular replacement using the C-terminal region of muscle contraction, where its azimuthal position on actin filaments tropomyosin (19) as template. The resultant composite omit map is critical. Cardiac tropomyosin is identical to skeletal ␣-tropomy- showed that residues 254–281 formed a coiled coil, even though the osin. At a high Ca2ϩ concentration, tropomyosin equilibrates between two azimuthal positions (‘‘closed’’ and ‘‘open’’ states). Binding myosin shifts tropomyosin from the closed state (where Author contributions: K.M. and T.W. designed research; K.M., K.N., K.T., N.K., N.I., Y.S., S.W., T.Y., and T.W. performed research; K.M., M.S., and T.W. analyzed data; and K.M. and myosin binds actin only weakly) to the open state (where myosin T.W. wrote the paper. 2ϩ binds actin strongly) (4, 5). At low Ca concentration, the azi- The authors declare no conflict of interest. muthal shift toward the outer domain of actin is induced by Data deposition: Coordinates and diffraction data have been deposited in the Protein Data troponin (6, 7) (the ‘‘blocked’’ state, where myosin cannot bind Bank, www.pdb.org [PDB ID codes 2Z5I (binary complex) and 2Z5H (ternary complex)]. actin). ʈTo whom correspondence should be addressed. E-mail: [email protected]. Sequences of coiled-coil proteins such as tropomyosin contain ac.jp. seven-residue heptad repeats [designated a, b, c, d, e, f, g (8)]. At the This article contains supporting information online at www.pnas.org/cgi/content/full/ interface between the two ␣-helices, side chains of residues in 0801950105/DCSupplemental. positions a and d (which are generally hydrophobic) protrude from © 2008 by The National Academy of Sciences of the USA 7200–7205 ͉ PNAS ͉ May 20, 2008 ͉ vol. 105 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801950105 Downloaded by guest on September 29, 2021 four TM-C molecules (eight chains: AB, CD, EF, and GH). A Although the four TM-C molecules were similar (C␣ RMSD ϭ 0.75 Å), the two chains within each molecule were systematically dif- pointed barbed end end ferent (C␣ RMSD ϭ 0.98 Å). The asymmetric unit contained a GAAS 25 GCN4 36 233 GCN4 253 1 284 15 COOH single TM-N molecule (two chains: IJ). The TM-N helices were COOH TM-C GAAS TM-N splayed apart from Met-1 to Lys-15 (Fig. 1B), of which residues 1–11 112 TnT 58 interacted with one TM-C. Chains ABCD of TM-C were related to chains EFGH by a noncrystallographic 2-fold axis, which also G233 G233 TM-C S-arm related chains I and J of TM-N (Fig. 1B). Thus, one TM-N molecule B )Aniahc( L-arm )Bn (chain B) (chai )An interacted with two TM-C molecules and the interaction between i chain I and molecule AB was essentially same as that between chain (cha J and molecule EF. The two other TM-C molecules (chains CD and ra GH) did not interact with a TM-N. - Sm L-arm At the TM-N:TM-C junction, hydrophobic residues of TM-N (Met-1 and Met-8 in position a; Ile-4 and Leu-11 in position d) packed against TM-C, but not against the other TM-N chain (Fig. 1B). The TM-N:TM-C interaction showed ‘‘knob-in-hole’’ packing: -C MT Leu-278 (position e) of the TM-C B chain was inserted as a single ) knob into a single hydrophobic hole formed by Ile-4, Lys-7, and M1 5(b 27 ) D L278(e) I4(d Met-8 of TM-N (Fig. 1 B and C). The importance of Leu-278 is highlighted by its conservation (Leu or Phe) between isoforms (Fig. ) K7(g S1) and suggests that in all isoforms except Tmbr2, tropomyosin K15 M8 (a) molecules overlap in a similar way. ) d -N L11( A Water Molecule in the Coiled-Coil Core Breaks the Symmetry in the in J) TM )Ini ha ain J) C-Terminal Region of Tropomyosin. Although each TM-C molecule (c a (ch h TM-N (c formed a coiled coil over its entire length, the local 2-fold symmetry CELL BIOLOGY V36 (chain I) was broken. One arm [designated L (long)] was more bent and V36 followed a longer path than the other [designated S (short)] (Fig. 2C;alsoseeFig. S3).