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A New Member of the I Set Mark Pfuhl and Annalisa Pastore

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A New Member of the I Set Mark Pfuhl and Annalisa Pastore View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Tertiary structure of an immunoglobulin-like domain from the giant muscle protein titin: a new member of the I set Mark Pfuhl and Annalisa Pastore EMBL, Heidelberg, Meyerhofstrasse 1, 691 17 Heidelberg, Federal Republic of Germany Background: Titin is a gigantic protein located in the two P-sheets packed against each other. Each sheet thick filament of vertebrate muscles. The putative contains four strands. The structure of M5 belonau to the functions of titin range from interactions with myosin I (intermediate) set of the immunoglobulin superfamily and other muscle proteins to a role in muscle recoil. and is very similar to telokin, which is also found in mus- Analysis of its complete sequence has shown that titin cles. Although M5 and telokin have relatively little is a multi-domain protein containing several copies of sequence similarity, the two proteins clearly share the modules of 100 amino acids each. These are thought same hydrophobic core. The major difference between to belong to the fibronectin type-111 and immuno- telokin and the titin M5 module is the absence of the C' globulin superfamilies. So far, a complete structural strand in the latter. determination has not been carried out on any of the Conclusions: The titin domains and several of the titin modules. immunoglobulin-like domains from other modular Results: The three-dimensional structure of an immuno- muscle proteins are highly conserved at the positions globulin module, located in the M-line of the sarcomere corresponding to the hydrophobic core of M5. Our close to the titin C terminus and called 'MS', was deter- results indicate that it may be possible to use the structure mined by multi-dimensional NMR spectroscopy. The of M5 as a molecular temdate to model most of the other structure has the predicted immunoglobulin fold with immunoglobulin-like domains in muscle titin. Structure 15 April 1995, 3:391-401 Key words: immunoglobulin-like modules, muscles, NMR, titin Introduction containing these domains are found in a broad range of The increasing amount of information on both the ter- organisms (from molluscs to vertebrates) and show a wide tiary structures and amino acid sequences of proteins has variety of complexity [2]. They range from the rather revealed the existence of families that share a very con- simple myosin light-chain kinase (e.g. telokin [4]) and served folding pattern but have extremely divergent proteins such as M-protein [5], H-protein [6] and C-pro- sequences [I,2]. Among these families, the immunoglob- tein [7-91 with only a few immunoglobulin-like ulin superfamily is one of the most divergent, both in domains, through kettin [lo] with at most 30 units, up to terms of sequence and function. All of the members of the giant protein titin. the superfamily share, with only minor variations, the same fold (-100 amino acids long) assembled as two Titin has a molecular weight of -3 million Daltons, P-sheets that pack tightly to form a P-sandwich [3]. which makes it the largest single-chain protein known to date (for review, see [11,12]). It is thought to form a This fold places the two termini of the polypeptide chain fibrous intracellular system in vertebrate striated muscle at far ends of the structure, in contrast to most other and to play an important role in sarcomere alignment single-domain globular proteins. In this way, it allows the during muscle contraction [13-151. It has also been sug- single domains to be arranged in linear arrays, a useful gested that titin may act as a molecular ruler regulating feature for building long proteins used in intracellular the assembly and the precise length of the thick filament or extracellular scaffolds. Furthermore, the common in muscles [16]. The titin found in cardiac muscles is 'theme' for immunoglobulin-like modules is the possibil- one of the smallest isoforms, and contains about 240 ity of forming highly specific interactions with other mol- modules almost equally divided between immunoglobu- ecules [2]. The conserved P-sandwich fold is regarded as lin-like and fibronectin type-111-like domains with only providing a stable scaffold to 'host' highly specific binding a few intervening sequence elements ([17,18]; S Labeit, sites on its surface. Small variations of the fold may add personal communication). The most notable of these additional flexibility to fulfil particular functions. intervening sequences are the repeats of a KSP (Lys-Ser- Pro) phosphorylation motif placed as a linker between Among the few intracellular proteins containing two immunoglobulin modules located in the C terminus immunoglobulin-like domains reported so far, the of titin. In contrast to other muscle proteins containing muscle-related family is the most numerous. Proteins immunoglobulin-like domains, titin extends throughout O Current Biology Ltd ISSN 0969-2126 392 Structure 1995, Vol 3 No 4 the whole length of each half sarcomere [15,19,20] the linker sequence that contains the KSP phosphoryla- (Fig. 1). Immunoglobulin-like domains are common tion repeats [18]. High levels of KSP phosphorylating throughout the sarcomere, in contrast to the fibronectin kinase were detected (in vitro) in developing but not in type-Ill domains, which are present only near to the differentiated muscle. This suggests that phosphorylation centre of the molecule (in the A-band). Immunoglobu- of the titin C terminus is necessary during muscle differ- lin-like domains located in different regions of the sarco- entiation and it is possible that this phosphorylation may mere must have adapted to satisfy different functions of control the assembly of M-line proteins into regular titin, from being a major organizer of the thick filament structures in myogenesis. in the A-band [21], interacting with myosin and C-protein, to anchoring to the Z-line and the M-line, and to providing elasticity in the I-band [15,22]. The Results and discussion presumed involvement of titin in elasticity and the pas- Structural statistics sive generation of force adds a previously unobserved A detailed account of the complete assignment procedure function to the already numerous ones known for the of the NMR spectrum of the M5 domain has already immunoglobulin fold. It is therefore of great interest to been presented elsewhere [25]. A total of 872 nuclear solve the structures of representative immunoglobulin Overhauser effect enhanced (NOE) cross-peaks distance domains from different regions of the titin molecule. By restraints were obtained from NOE spectroscopy correlating the structures to the sequences on the one (NOESY) experiments (Table 1). Eighteen pairs of hand and to the specific functions on the other, a much ,B-methylene protons were assigned stereospecifically. better understanding of the immunoglobulin fold may The residues of a histidine tag (MHHHHHHSS), intro- be achieved. duced to simplify purification of the M5 module [26] and possibly to help increase the solubility of this domain, With this aim in mind, we have begun the characteriza- were not included in the structure calculation because no tion of several immunoglobulin-like modules selected NOEs were observable for this part of the molecule. The from different regions of titin. By preliminary circular histidine tag region is presumed to be highly mobile, as dichroism, fluorescence and NMR spectroscopy we indicated by the random-coil chemical shifts [27] of the found that titin's immunoglobulin-like modules adopt a histidine tag a-protons. Therefore, it should have little stable fold in solution and exhibit structural features con- influence on the structure of the domain. sistent with predictions [23,24]. Among them, domains from the M-line are the best-behaved in terms of high- Table 1. Statistics of structure calculation. resolution structural determination. Based on the com- plete assignment of the NMR spectrum of this module, NOE and H-bond derived distances: we describe here the structure of M5, which corresponds No. of NOEs 872 to the fifth immunoglobulin module of the 10 located in No. of intra-residual NOEs 180 the titin C terminus (Fig. lb). It is followed directly by No. of sequential NOEs 224 No. of NOEs (i-j, i+1 <j<i+5) 126 No. of NOEs (i-j, j>i+5) 342 No. of hydrogen bond constraints 23 Calculation results: No. of structures at start 500 No. accepted after first redac run 273 Min/max target function after second redac run 27/400 No. accepted after second redac run 129 Min/max target function after second redac run 14/98 No. accepted after third redac run 79 Min/max target function after third redac run 10/28 No. of structures used for MD 40 Parameters for 16 structures after MD: No. of NOE violations>0.4A 0 No. of NOE violations 0.3-0.4A 1 No. of NOE violations 0.2-0.3 A 16 No. of NOE violations 0.1-0.2A 70 Total no. of NOE violations 210 Average NOE violation (A) 0.053 Average bond length deviation (A) 0.006 Fig. 1. (a) Schematic representation of the sarcomere. The thick Maximal bond length deviation (A) 0.03 and the thin filaments are coloured red and blue, respectively. Average bond angle deviation (°) 1.1 Titin is shown in green and spans the whole length between the Maximal bond angle deviation (°) 4.3 Z-disk and the M-line. (b) The C-terminal region of the titin mol- ecule (from 118]). The titin kinase (in black) is on the left fol- Abbreviations: NOE, nuclear Overhauser enhancement; MD, molecular lowed by 10 immunoglobulin domains (grey) and insertion dynamics. sequences (white). The module M5 is highlighted in orange.
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