Muscle giants: molecular scaffolds in sarcomerogenesis

Abstract

Myofibrillogenesis in striated muscles is a highly complex process that depends on the coordinated assembly and integration of a large number of contractile, cytoskeletal, and signaling proteins into regular arrays, the sarcomeres. It is also associated with the stereotypical assembly of the sarcoplasmic reticulum and the transverse tubules around each sarcomere. Three giant, muscle-specific proteins, titin (3-4 MDa), nebulin (600-800 kDa), and obscurin (approximately 720-900 kDa), have been proposed to play important roles in the assembly and stabilization of sarcomeres. There is a large amount of data showing that each of these molecules interacts with several to many different protein ligands, regulating their activity and localizing them to particular sites within or surrounding sarcomeres. Consistent with this, mutations in each of these proteins have been linked to skeletal and cardiac myopathies or to muscular dystrophies. The evidence that any of them plays a role as a "molecular template," "molecular blueprint," or "molecular ruler" is less definitive, however. Here we review the structure and function of titin, nebulin, and obscurin, with the literature supporting a role for them as scaffolding molecules and the contradictory evidence regarding their roles as molecular guides in sarcomerogenesis.

FIG. 1

Schematic representation of titin at the level of the Z-disk, illustrating its domain orientation and the structure of some of its domains, as well as identifying its binding partners. Exons 1–27 of the TTN gene code for the Z-disk portion of the titin. This region is composed of seven Ig domains and two Z insertions (Zis) that are unique to titin and flank the third Ig domain. The second Z insertion is comprised of 7 Z repeats (Zr) that can be alternatively spliced, and a Zq region (see key for complete domain list and color coding). Proteins that bind to titin in this region are indicated at their sites of interaction. Structures of the complexes formed by two of the protein ligands, T-cap/telethonin with the two NH₂-terminal Ig domains, and α-actinin with the Zq domain, are shown as ribbon diagrams, with tan representing the ligands and other colors representing their binding regions on titin.

FIG. 2

Schematic representation of titin spanning the I-band, illustrating its domain orientation and the structure of some of its domains, as well as identifying its binding partners. The I-band region of titin, encoded by exons 28–251 of the TTN gene, is the most highly alternatively spliced region of titin. Exons 45–48 are excluded from titin in striated muscle and are alternatively spliced to produce smaller isoforms, termed Novex-1, -2, and -3. A: the hypothetical protein comprising all the other exons, with binding partners and their sites of interaction indicated. Some of the domains along the I-band region of titin have been characterized structurally, either by NMR or X-ray crystallography, and are shown as ribbon diagrams. B: actual variants of titin that are expressed in different muscle tissues and have been characterized by RT-PCR (soleus, psoas, and two forms found in cardiac muscle, N2B and N2BA). The I-band region of titin is composed primarily of Ig domains. The Ig domains at the Z-I junction (Z8-Z10, encoded by exon 28) are flanked by sequences of unknown structure and are followed by the first 15 Ig domains found at the level of the I-band which are present in all striated muscles. Immediately downstream of this group of Ig domains is the N2B region, composed of nonrepetitive sequence and Ig domains, which is present only in some isoforms of titin. Following the N2B region is another stretch of Ig domains (I27-I79), the N2A region, which also contains nonrepetitive sequence and Ig domains, and the PEVK domain, which is largely responsible for titin's elastic properties. This middle stretch of Ig domains varies greatly among muscle-specific isoforms. Slow-twitch fibers of the soleus muscle, for example, contain all the Ig domains linked to this region and the longest PEVK region of any known titin isoform. The psoas, which is a fast-twitch muscle, contains 19 fewer Ig domains and has a much shorter PEVK domain. Cardiac N2B possesses only 2 Ig domains and a very short PEVK region. Many isoforms of cardiac N2BA, containing both the N2B and N2A regions, have been detected. The differences among them can be attributed to the number of Ig domains between the N2B and N2A regions, which can be as few as 13 or as many as 25 (as shown by the Ig domains outlined by a dotted line). COOH terminal to the PEVK domain is the last set of Ig domains in the I-band region of titin; all isoforms contain these same 22 Ig domains.

FIG. 3

Schematic representation of titin spanning the A- and M-bands, showing its domain orientation and the structure of some of its key domains, as well as identifying its binding partners. This portion of titin is encoded by exons 252–363 of the TTN gene. The A-band region of titin, including domain A1 through the kinase domain, is composed of multiple Ig and FN-III domains. They are arranged in two types of super repeats in which stretches of FN-III domains are bisected by single Ig domains. The M-band region, from the end of the kinase domain to the COOH terminus of the molecule, lacks FN-III domains and is composed solely of Ig domains and M-insertions (Mis; please see key for a complete list of the domains, with color-coding). Binding partners and interaction sites that have been mapped to this region of titin are indicated. Myosin binding protein-C (MyBP-C) binds titin repeatedly along the length of the A-band, specifically to the first Ig domain of each of the second type of super repeat. The precise location of the binding site on titin for myosin is unknown, but myosin does bind several of titin's FN-III domains throughout the A band, with the affinity increasing with increasing numbers of the FN-III domains with which it interacts. The domains in this region of titin that have been characterized structurally, by NMR or X-ray crystallography, are represented as ribbon diagrams.

FIG. 4

Schematic representation of nebulin, illustrating its domain orientation, structure, and binding partners. Nebulin is composed mainly of ~35-amino acid-long repeating motifs, i.e., M1-M185. The middle repeats, where alternative splicing occurs, are further organized into 22 super repeats, each containing 7 modules each. The super repeat region spans the majority of the I-band and harbors binding sites for the major contractile proteins, including actin and myosin. The NH₂- and COOH-terminal repeats (denoted in yellow), located at the pointed and barbed ends of actin, at the tips of the thin filaments of the I-band and the Z-disk, respectively, differ in sequence from the middle repeats and do not form super repeats. The NH₂-terminal sequence is rich in glutamic acid residues, whereas the COOH terminus carries a serine-rich region and an SH3 domain. Binding partners that interact directly with nebulin are depicted with a solid line pointing to their binding sites on nebulin.

FIG. 5

Schematic representation of the known isoforms of obscurin, illustrating their motifs, structures, and binding partners. Alternative splicing of the OBSCN gene results in at least four forms of obscurin: two giant isoforms, Obscurin A and Obscurin B/giant (g) MLCK, and two smaller kinase containing isoforms, Obscurin tandem (t) MLCK and Obscurin single (s) MLCK. All isoforms are composed of multiple structural motifs and signaling domains (see key for complete domain list and color coding). The giant isoforms diverge at the COOH terminus. Obscurin A is composed of a nonmodular COOH terminus, which houses the ankyrin-binding site (maroon box). The COOH terminus of Obscurin B/gMLCK possesses two kinase domains along with two additional Ig domains and a FN-III domain. Only 11 of obscurin's many domains have been characterized structurally, either by NMR or X-ray crystallography, which are shown as a cartoon model with flat arrows and coils representing β-sheets and α-helices, respectively. Binding partners and interaction sites that have been mapped to obscurin are also shown. Binding partners shown to interact directly with obscurin are depicted with a gray background and solid line pointing to their binding site on obscurin. The interaction between obscurin and myosin is not as well defined and may only be indirect and is thus shown by a dotted line. The phosphatase SCPL-1 and the LIM-domain protein LIM-9 were shown to interact directly with the kinases and preceding Ig and FN-III domains of Unc89, obscurin's invertebrate homolog, and are shown with a white background.