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

*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)

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 Å resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 Å 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.

Footnotes

^‖To whom correspondence should be addressed. E-mail: wakabayashi{at}nasu.bio.teikyo-u.ac.jp
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 T.W. wrote the paper.
The authors declare no conflict of interest.
Data deposition: Coordinates and diffraction data have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 2Z5I (binary complex) and 2Z5H (ternary complex)].
This article contains supporting information online at www.pnas.org/cgi/content/full/0801950105/DCSupplemental.