© 2006 Nature Publishing Group Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk Peijian Zou 1 *, Nikos Pinotsis 1,2 *, Stephan Lange 3,4 , Young-Hwa Song 1 , Alexander Popov 1 , Irene Mavridis 2 , Olga M. Mayans 1,5 , Mathias Gautel 3 & Matthias Wilmanns 1 The Z-disk of striated and cardiac muscle sarcomeres is one of the most densely packed cellular structures in eukaryotic cells 1 . It provides the architectural framework for assembling and anchoring the largest known muscle filament systems by an extensive network of protein–protein interactions, requiring an extraordinary level of mechanical stability. Here we show, using X-ray crystallography, how the amino terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the Z-disk ligand telethonin. The pseudosymmetric structure of telethonin medi- ates a unique palindromic arrangement of two titin filaments, a type of molecular assembly previously found only in protein– DNAcomplexes. We have confirmed its unique architecture in vivo by protein complementation assays, and in vitro by experiments using fluorescence resonance energy transfer. The model proposed may provide a molecular paradigm of how major sarcomeric filaments are crosslinked, anchored and aligned within complex cytoskeletal networks. The Z-disk of the sarcomere defines the lateral boundary of sarcomeric units within the myocyte cytoskeleton. In higher verte- brates it anchors and aligns at least three major sarcomeric filament systems, including actin, titin and nebulin 1,2 . It also harbours many smaller protein components, some of which, including a-actinin and telethonin, have been mapped to distinct binding sites at the N terminus of titin 3–7 . Their presence, proper sorting and localization within the Z-disk region are critical for myofibril assembly and for the maintenance of an intact Z-disk structure 8 . Components of the Z-disk are also involved in signalling processes that may regulate muscle development and degradation, as well as in linking contractile functions of muscle sarcomeres to membrane systems such as the sarcoplasmic reticulum or the T-tubules associated with excitation– contraction coupling 2,8 . The very N-terminal region of titin comprises a domain topology that has been predicted to consist of two immunoglobulin-like domains, referred to as Z1 and Z2 (ref. 9). Co-localization studies, two-hybrid interaction screens and pull-down assays have demon- strated that they interact with the N-terminal region of telethonin at the Z-disk periphery 3,4 . However, although the interaction with telethonin has been considered as a ‘cap’ (hence the alternative name ‘T-cap’), or a ‘bolt’ 3 , it is not known how titin–telethonin binding affects the overall architecture of myofibrils and their associated functions. The physiological importance of this inter- action has been supported by evidence linking mutations in the N-terminal regions of titin, telethonin and the telethonin-binding site of muscle LIM protein (MLP) to different familial forms of limb-girdle muscular dystrophy, as well as hypertrophic and dilated cardiomyopathy 10–12 . These findings, together with complementary data from animal models, indicate the possible existence of a link between the titin–telethonin–MLP interaction and mechanical stress sensor pathways 10 . Depending on the specific isoform of titin, 200–700 N-terminal residues of multimeric titin filaments are located within and cross over most of the Z-disk of striated muscle sarcomeres 2,3,6 . However, no data are yet available on the molecular nature of the titin–titin association. Here we have determined the crystal structure of its N-terminal region in complex with the titin-binding domain of telethonin (Fig. 1, Table 1 and Supplementary Fig. S1). The latter domain is sufficient to localize telethonin to the Z-disk of cardiac myofibrils (Supplementary Fig. S2). Contrary to previous expec- tations 3 and previous structural findings on other immunoglobulin- like domain-containing proteins (see Supplementary Information), our data reveal an antiparallel assembly of two titin molecules mediated by telethonin, indicating that telethonin might have a key role in titin assembly and Z-disk anchoring. In the complex, the two N-terminal immunoglobulin-like domains of titin, Z1 and Z2 are in an extended conformation and are connected by a short three-residue linker. The two domains have similar structures (root-mean-square deviation (r.m.s.d.) ¼ 0.66 A ˚ , for all common main-chain atoms) and sequences (40 of 98 residues are identical). In each of the two titin molecules, domains Z1 and Z2 are almost equally translated by 48 A ˚ and rotated by 538 and 618 with respect to each other, generating a superhelical coil arrangement of each titin N terminus. The second component of the complex, telethonin, forms a unique elongated structure with a central five-stranded antiparallel b-sheet that is extended by two exposed LETTERS Table 1 | Refinement statistics Resolution (A ˚ ) 15.0–2.45 R work /R free (%) 23.2/26.5 Number of atoms Protein 3,726 Ion 25 Water 179 B factors (A ˚ 2 ) Protein 49.7 Ion 51.2 Water 48.6 R.m.s.d. Bond lengths (A ˚ ) 0.011 Bond angles (8) 1.370 1 EMBL-Hamburg c/o DESY, Notkeststrasse 85, D-22603 Hamburg, Germany. 2 Institute of Physical Chemistry, National Center for Scientific Research ‘Demokritos’, Aghia Paraskevi 15310, Athens, Greece. 3 The Randall Division of Cell and Molecular Biophysics and Cardiovascular Division, King’s College London, Guy’s Campus, London SE1 1UL, UK. 4 Institute of Cell Biology, ETH Zurich Hoenggerberg, CH-8093 Zurich, Switzerland. 5 Biozentrum, University of Basel, Division of Structural Biology, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. *These authors contributed equally to this work. Vol 439|12 January 2006|doi:10.1038/nature04343 229