Properties of Titin Immunoglobulin and Fibronectin-3 Domains

Immunoglobulin (Ig) 1The abbreviations used are: Ig, immunoglobulin; Fn3, fibronectin-3; pN, piconewtons.and fibronectin-3 (Fn3) domains are common building blocks of many extracellular proteins involved in ligand recognition and cell adhesion. Ig and Fn3 domains are also the main components of a group of intracellular proteins associated with the contractile apparatus of muscles. The largest of the intracellular group is titin (∼3 MDa), which has key roles in the assembly and elasticity of vertebrate striated muscles. The titin molecule spans one-half of the sarcomere, the repeating contractile unit that gives striated muscle its characteristic striped appearance. Titin interacts with a majority of sarcomere proteins and is also likely to bind a variety of cytoplasmic signaling molecules. The Ig and Fn3 domains are important in titin interactions, whereas the levels of interdomain mobility and structural stability relate directly to mechanical functions. Titin and the other muscle Ig/Fn3 proteins are likely to have a common ancestry. It is plausible that the emergence and evolution of the muscle Ig/Fn3 proteins were closely related to the origin and evolution of multicellularity and cell differentiation. This review summarizes current knowledge regarding the properties of the Ig and Fn3 domains of titin. General properties of titins are presented only briefly and are described in detail in recent reviews (1Tskhovrebova L. Trinick J. Nat. Rev. Mol. Cell. Biol. 2003; 4: 679-689Crossref PubMed Scopus (284) Google Scholar, 2Granzier H.L. Labeit S. Circ. Res. 2004; 94: 284-295Crossref PubMed Scopus (469) Google Scholar, 3Miller M.K. Granzier H. Ehler E. Gregorio C.C. Trends Cell Biol. 2004; 14: 119-126Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Titins are formed from the largest polypeptides so far found in nature. The 363 exons estimated to comprise the human titin gene contain 114,114 bp, corresponding to a polypeptide of up to 38,138 amino acid residues long (4Labeit S. Kolmerer B. Science. 1995; 270: 293-296Crossref PubMed Scopus (1004) Google Scholar, 5Bang M.L. Centner T. Fornoff F. Geach A.J. Gotthardt M. McNabb M. Witt C.C. Labeit D. Gregorio C.C. Granzier H. Labeit S. Circ. Res. 2001; 89: 1065-1072Crossref PubMed Scopus (518) Google Scholar) (Swiss-Prot/TrEMBL accesion numbers Q10465, Q10466, and Q8WZ42). Isoforms sequenced from cardiac and skeletal muscle cDNAs contain between 240 and 300 of the Ig- and Fn-like domains. Assuming a linear arrangement of domains each ∼4 nm long, this indicates a molecule more than 1 μm long, which compares well with electron microscope measurements of the purified protein. The domain arrangement is also visible directly in a “string-of-beads” appearance in micrographs (6Trinick J. Knight P. Whiting A. J. Mol. Biol. 1984; 180: 331-356Crossref PubMed Scopus (142) Google Scholar). In situ the titin molecule spans one-half of the sarcomere, which is the repeating contractile unit of muscle, composed mainly of ordered arrays of thin (actin) and thick (myosin) filaments (Fig. 1). The N terminus is at the boundary of sarcomere, the Z-line, and the C terminus is in the M-line at the center of the sarcomere. More than one-half of the molecule (the A-band section) is an integral part of the myosin filament. This region is formed mainly by Ig and Fn3 domains. The I-band part of titin forms an elastic connection between the end of the thick filament and the Z-line, which changes its end-to-end distance when muscle contracts or extends. This part of titin does not contain Fn3 domains and has only Ig and unique sequences. Most of the Ig domains are arranged in tandem in two segments, “proximal” and “distal” to the Z-line, which flank the N2-PEVK region containing mainly unique sequences. On average, the titin Ig and Fn3 sequence motifs are less than 50% conserved within each family. However, each family has distinct subfamilies with sequence identity up to 90%. In the I-band part, the Ig domains constitutively expressed and those differentially expressed in isoforms fall into different subfamilies (7Witt C.C. Olivieri N. Centner T. Kolmerer B. Millevoi S. Morell J. Labeit D. Labeit S. Jockusch H. Pastore A. J. Struct. Biol. 1998; 122: 206-215Crossref PubMed Scopus (45) Google Scholar). The differentially expressed segment contains Ig domains from several different subfamilies that are arranged in long range patterns or “super-repeats” consisting of 6 and 10 domains (8Gautel M. Adv. Biophys. 1996; 33: 27-37Crossref PubMed Scopus (21) Google Scholar). In the A-band part, the Ig and Fn3 domains are arranged in 7- and 11-domain super-repeats, which are repeated 6 and 11 times, respectively. The domains at similar positions within different super-repeats have higher sequence homology than the domains from the same super-repeat, indicating that they belong to different subfamilies (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar, 10Amodeo P. Fraternali F. Lesk A.M. Pastore A. J. Mol. Biol. 2001; 311: 283-296Crossref PubMed Scopus (28) Google Scholar). Structural modeling predicted that the subfamily-specific consensus sequences and residues would define the general surface and interface differences between domain subfamilies. Thus, in the I-band the constitutively and differentially expressed Ig domains are likely to differ systematically in exposed polar and charged residues and in the stiffness of domain interfaces (11Fraternali F. Pastore A. J. Mol. Biol. 1999; 290: 581-593Crossref PubMed Scopus (28) Google Scholar). Likewise, in the A-band the Fn3 domains are predicted to have subfamily-specific clusters of conserved residues, mostly charged, on their surfaces and interfaces (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar, 10Amodeo P. Fraternali F. Lesk A.M. Pastore A. J. Mol. Biol. 2001; 311: 283-296Crossref PubMed Scopus (28) Google Scholar, 12Muhle-Goll C. Habeck M. Cazorla O. Nilges M. Labeit S. Granzier H. J. Mol. Biol. 2001; 313: 431-447Crossref PubMed Scopus (85) Google Scholar). The structures of titin domains inferred from sequence were confirmed by NMR and x-ray crystallography. The expressed domains that have been solved are representative of the M-line region, Ig-M5 (13Pfuhl M. Pastore A. Structure. 1995; 3: 391-401Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), the constitutive distal segment, Ig-I27 (14Improta S. Politou A.S. Pastore A. Structure. 1996; 4: 323-337Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), the A-band, Fn3-A71 (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar), and the constitutive proximal Ig segment, Ig-I1 (16Mayans O. Wuerges J. Canela S. Gautel M. Wilmanns M. Structure. 2001; 9: 331-340Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In addition, atomic structures have been obtained of polydomain constructs (17Improta S. Krueger J.K. Gautel M. Atkinson R.A. Lefevre J.F. Moulton S. Trewhella J. Pastore A. J. Mol. Biol. 1998; 284: 761-777Crossref PubMed Scopus (74) Google Scholar), the kinase domain (18Mayans O. van der Ven P.F.M. Wilm M. Mues A. Young P. Furst D.O. Wilmanns M. Gautel M. Nature. 1998; 395: 863-869Crossref PubMed Scopus (315) Google Scholar), and a motif repeat of the unique PEVK region (19Ma K. Kan L.S. Wang K. Biochemistry. 2001; 40: 3427-3438Crossref PubMed Scopus (120) Google Scholar). The titin Ig structures have the β-sandwich fold characteristic of the immunoglobulin superfamily (Fig. 2). The fold contains 8–9 β-strands arranged in two sheets, ABED and A′GFC(C′), packed face-to-face. A disulfide bridge connecting the two sheets was observed only in one case, the I1 domain. This bridge, however, differs from the classical Ig disulfide, which links strands B and F; instead it connected strands C and E. Close inspection of the structures and sequences placed the domains in the intermediate I-set of the Ig superfamily (13Pfuhl M. Pastore A. Structure. 1995; 3: 391-401Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This implied that, unlike domains of the constant C2-set to which they were originally assigned, titin Ig domains are more similar to the V-set (variable) domains. The conserved pattern of residues specific to the I-set, the V-frame (20Harpaz Y. Chothia C. J. Mol. Biol. 1994; 238: 528-539Crossref PubMed Scopus (377) Google Scholar), had earlier been detected in other muscle Ig proteins and was also found in all the titin Ig domains. Thus I27, M5, and I1 all are similar to the typical I-set domain, telokin (20Harpaz Y. Chothia C. J. Mol. Biol. 1994; 238: 528-539Crossref PubMed Scopus (377) Google Scholar). The deviations from this pattern include inconsistency in structuring of the A, A′ and C′ strands and absence of some key interactions involving loops A′B and EF and strand D. There are also some variations between the domains in the length of the β-strands, in the size and conformation of loops, and in the overall shape. These differences seem to reflect general structural differences between the domain subfamilies (7Witt C.C. Olivieri N. Centner T. Kolmerer B. Millevoi S. Morell J. Labeit D. Labeit S. Jockusch H. Pastore A. J. Struct. Biol. 1998; 122: 206-215Crossref PubMed Scopus (45) Google Scholar, 11Fraternali F. Pastore A. J. Mol. Biol. 1999; 290: 581-593Crossref PubMed Scopus (28) Google Scholar). Titin domain A71, the only structure of an intracellular Fn3 domain so far solved, was confirmed to have the characteristic Fn3 family fold (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar). The strongest resemblance is to the first Fn3 domain of Drosophila neuroglian and to Fn3 domains in fibronectin and tenascin. Sequence analysis indicated that all titin Fn3 domains contain key residues specific to the Fn3 family that form the hydrophobic core of the domain. In addition to these residues, titin domains were also found to contain titin-specific sets of conserved residues. Insights into the relative arrangement of domains and the overall conformation of the titin molecule have come from combined experimental and modeling studies. A model of the constitutively expressed I-band part, calculated from NMR data, supported the expected beads-on-a-string arrangement (17Improta S. Krueger J.K. Gautel M. Atkinson R.A. Lefevre J.F. Moulton S. Trewhella J. Pastore A. J. Mol. Biol. 1998; 284: 761-777Crossref PubMed Scopus (74) Google Scholar). This also suggested bending (∼155°) and twisting (∼90°) between successive domains, resulting in 10–17% shortening of the end-to-end distance from a fully extended conformation. The interdomain mobility appeared to be low, indicating a relatively well defined overall conformation of the constitutively expressed segment. The differentially expressed Ig segment was predicted to have an even more regular and stiff conformation, because of the presence of super-repeats and higher stiffness of interdomain interfaces suggested by sequence analysis. A-band titin is also likely to have an extended and relatively stiff conformation, based on modeling of the arrangement of the Fn3 domains, which are predominant in this part of the molecule (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar, 10Amodeo P. Fraternali F. Lesk A.M. Pastore A. J. Mol. Biol. 2001; 311: 283-296Crossref PubMed Scopus (28) Google Scholar, 12Muhle-Goll C. Habeck M. Cazorla O. Nilges M. Labeit S. Granzier H. J. Mol. Biol. 2001; 313: 431-447Crossref PubMed Scopus (85) Google Scholar). The super-repeat pattern of A-band titin and the predicted high stiffness of the interdomain interfaces again suggest a regular conformation. Mobility between domains is likely to be the major source of the flexibility in titin. Flexibility is especially important in the I-band region that connects thick filaments to the Z-line. As described above, this region has important mechanical roles and is the origin of elasticity in relaxed sarcomeres. It also maintains the central position of the thick filament in the sarcomere, which ensures balance of forces between both its halves during the contractile cycle. As the sarcomere changes length during muscle contraction or passive extension, the I-band titin changes its conformation, coiling up or elongating. Although mobility between domains is predicted to be low throughout the molecule, its level appears to be enough to provide the considerable overall flexibility. Studies of the purified protein indicate a value of persistence length in the range 13–19 nm, implying substantial bending over molecule segments containing 3–4 domains (21Higuchi H. Nakauchi Y. Maruyama K. Fujime S. Biophys. J. 1993; 65: 1906-1915Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 22Tskhovrebova L. Trinick J. J. Mol. Biol. 2001; 310: 755-771Crossref PubMed Scopus (47) Google Scholar, 23Kellermayer M.S.Z. Bustamante C. Granzier H.L. Biochim. Biophys. Acta. 2003; 1604: 103-114Google Scholar). Thermal Stability—Thermal unfolding of individual expressed titin Ig domains has been shown to be reversible and to occur in a simple two-state reaction. The relatively wide range of the transition temperatures, 35–70 °C (24Politou A.S. Thomas D.J. Pastore A. Biophys. J. 1995; 69: 2601-2610Abstract Full Text PDF PubMed Scopus (107) Google Scholar), is similar to the range observed for extracellular Ig domains. Studies of domain pairs indicate independent unfolding of each domain, as well as mutual stabilization (25Politou A.S. Gautel M. Improta S. Vangelista L. Pastore A. J. Mol. Biol. 1996; 255: 604-616Crossref PubMed Scopus (70) Google Scholar). Confinement within narrow pores to mimic cellular environment also increases stability (26Bolis D. Politou A.S. Kelly G. Pastore A. Temussi P.A. J. Mol. Biol. 2004; 336: 203-212Crossref PubMed Scopus (67) Google Scholar). With the exception of M-line domains, the average domain stability appears to be higher in the I-band than in the A-band part of the molecule. Chemical Stability—Chemical denaturation of titin domains correlates with the thermal denaturation data. Unfolding again follows essentially a two-state process, although in some cases there is evidence of low stability intermediates (24Politou A.S. Thomas D.J. Pastore A. Biophys. J. 1995; 69: 2601-2610Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 27Carrion-Vazquez M. Oberhauser A.F. Fowler S.B. Marszalek P.E. Broedel S.E. Clarke J. Fernandez J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3694-3699Crossref PubMed Scopus (893) Google Scholar, 28Scott K.A. Steward A. Fowler S.B. Clarke J. J. Mol. Biol. 2002; 315: 819-829Crossref PubMed Scopus (83) Google Scholar). 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However, muscle is reversible unfolding of domains titin and the sarcomere structure from with its size and of Ig and Fn3 domains, a of to a pattern (Fig. the first titin were the thick filament myosin and many other have been mostly by the suggest that titin not only for structural proteins in the sarcomere, also for many cytoplasmic of titin in the and M-line have two they to the structural of these of the sarcomere and they provide links with The links are likely to differ in cardiac and skeletal in with the structural and differences of these muscle the elastic I-band part of which spans between the and the of thick of the interactions to be in the N2-PEVK are mainly with proteins of signaling The A-band part of titin is integral with the thick filament. major myosin and are in a regular with the structure of the thick filament. have been to between titin domains and other of the muscle Ig family Labeit S. Gautel M. J. Mol. Scopus Google Scholar, P.A. 1999; PubMed Scopus Google Scholar). of this analysis are as the different of the A-band part and the constitutively and differentially expressed I-band from common domains. of these involved a of gene at the level of of domains, to the domain titin and the other vertebrate and of the muscle Ig family have a common and a protein have the of and The origin and of proteins are to be closely related to the emergence and of multicellularity and the L. 2003; PubMed Scopus Google Scholar). The domains and were some of the first to be to which were for and It plausible that, in of and cell of intracellular of striated muscle in would have proteins to and the of the contractile The Ig with its low for sequence and high to structural was well to these in