1H, 15N and 13C backbone chemical shift assignment of titin domains A59–A60 and A60 alone

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone ¹⁵N, ¹³C and ¹H chemical shift and ¹³Cβ assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.     

Shape and Flexibility in the Titin 11-Domain Super-Repeat

Titin is a giant protein of striated muscle with important roles in the assembly, intracellular signalling and passive mechanical properties of sarcomeres. The molecule consists principally of ∼ 300 immunoglobulin and fibronectin domains arranged in a chain more than 1 μm long. The isoform-dependent N-terminal part of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The larger, constitutively expressed C-terminal part is bound to the thick filament. Through most of the thick filament part, the immunoglobulin and fibronectin domains are arranged in a repeating pattern of 11 domains termed the ‘large super-repeat’. There are 11 contiguous copies of the large super-repeat making up a segment of the molecule nearly 0.5 μm long. We have studied a set of two-domain and three-domain recombinant fragments from the large super-repeat region by electron microscopy, synchrotron X-ray solution scattering and analytical ultracentrifugation, with the goal of reconstructing the overall structure of this part of titin. The data illustrate different average conformations in different domain pairs, which correlate with differences in interdomain linker lengths. They also illustrate interdomain bending and flexibility around average conformations. Overall, the data favour a helical conformation in the super-repeat. They also suggest that this region of titin is dimerised when bound to the thick filament.     

Towards a molecular understanding of titin.

Titin is at present the largest known protein (M(r) 3000 kDa) and its expression is restricted to vertebrate striated muscle. Single molecules span from M- to Z-lines and therefore over 1 micron. We have isolated cDNAs encoding five distant titin A-band epitopes, extended their sequences and determined 30 kb (1000 kDa) of the primary structure of titin. Sequences near the M-line encode a kinase domain and are closely related to the C-terminus of twitchin from Caenorhabditis elegans. This suggests that the function of this region in the titin/twitchin family is conserved throughout the animal kingdom. All other A-band sequences consist of 100 amino acid (aa) repeats predicting immunoglobulin-C2 and fibronectin type III globular domains. These domains are arranged into highly ordered 11 domain super-repeat patterns likely to match the myosin helix repeat in the thick filament. Expressed titin fragments bind to the LMM part of myosin and C-protein. Binding strength increases with the number of domains involved, indicating a cumulative effect of multiple binding sites for myosin along the titin molecule. We conclude that A-band titin is likely to be involved in the ordered assembly of the vertebrate thick filament.     

Stability and folding rates of domains spanning the large A-band super-repeat of titin

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.     

The Structure of the FnIII Tandem A77-A78 Points to a Periodically Conserved Architecture in the Myosin-Binding Region of Titin

Titin is a large intrasarcomeric protein that, among its many roles in muscle, is thought to modulate the in vivo assembly of the myosin motor filament. This is achieved through the molecular template properties of its A-band region, which is composed of fibronectin type III (FnIII) and immunoglobulin (Ig) domains organized into characteristic 7-domain (D-zone) and 11-domain (C-zone) superrepeats. Currently, there is little knowledge on the structural details of this region of titin. Here we report the conformational characterization of three FnIII tandems, A77-A78, A80-A82, and A84-A86, which are components of the representative fourth C-zone superrepeat. The structure of A77-A78 has been elucidated by X-ray crystallography to 1.65 Å resolution, while low-resolution models of A80-A82 and A84-A86 have been calculated using small-angle X-ray scattering. A77-A78 adopts an extended “up-down” domain arrangement, where domains are connected by a hydrophilic three-residue linker sequence. The linker is embedded in a rich network of polar contacts at the domain interface that results in a stiff molecular conformation. The models of A80-A82 and A84-A86, which contain hydrophobic six-residue-long interdomain linkers, equally showed elongated molecular shapes, but with slightly coiled or zigzagged conformations. Small-angle X-ray scattering data further suggested that the long linkers do not result in a noticeable increase in molecular flexibility but lead to semibent domain arrangements. Our findings indicate that the structural characteristics of FnIII tandems from A-band titin contrast markedly with those of poly-Ig tandems from the elastic I-band, which exhibit domain interfaces depleted of interactions and compliant conformations. Furthermore, the analysis of sequence conservation in FnIII domains from A-band titin points to the existence of conformationally defined interfaces at specific superrepeat positions, possibly leading to a periodic and locally ordered architecture supporting the molecular scaffold properties of this region of titin.     

Molecular evolution of immunoglobulin and fibronectin domains in titin and related muscle proteins.

The family of regulatory and structural muscle proteins, which includes the giant kinases titin, twitchin and projectin, has sequences composed predominantly of serially linked immunoglobulin I set (Ig) and fibronectin type III (FN3) domains. This paper explores the evolutionary relationships between 16 members of this family. In titin, groups of Ig and FN3 domains are arranged in a regularly repeating pattern of seven and 11 domains. The 11-domain super-repeat has its origins in the seven-domain super-repeat and a model for the duplications which gave rise to this super-repeat is proposed. A super-repeat composed solely of immunoglobulin domains is found in the skeletal muscle isoform of titin. Twitchin and projectin, which are presumed to be orthologs, have undergone significant insertion/deletion of domains since their divergence. The common ancestry of myomesin, skelemin and M-protein is shown. The relationship between myosin binding proteins (MyBPs) C and H is confirmed, and MyBP-H is proposed to have given rise to MyBP-C by the acquisition of some titin domains.     

Molecular structure of the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of alpha-actinin.

The sarcomeric Z-disk, the anchoring plane of thin (actin) filaments, links titin (also called connectin) and actin filaments from opposing sarcomere halves in a lattice connected by alpha-actinin. We demonstrate by protein interaction analysis that two types of titin interactions are involved in the assembly of alpha-actinin into the Z-disk. Titin interacts via a single binding site with the two central spectrin-like repeats of the outermost pair of alpha-actinin molecules. In the central Z-disk, titin can interact with multiple alpha-actinin molecules via their C-terminal domains. These interactions allow the assembly of a ternary complex of titin, actin and alpha-actinin in vitro, and are expected to constrain the path of titin in the Z-disk. In thick skeletal muscle Z-disks, titin filaments cross over the Z-disk centre by approximately 30 nm, suggesting that their alpha-actinin-binding sites overlap in an antiparallel fashion. The combination of our biochemical and ultrastructural data now allows a molecular model of the sarcomeric Z-disk, where overlapping titin filaments and their interactions with the alpha-actinin rod and C-terminal domain can account for the essential ultrastructural features.     

Molecular structure of the sarcomeric M band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin.

The M band of sarcomeric muscle is a highly complex structure which contributes to the maintenance of the regular lattice of thick filaments. We propose that the spatial coordination of this assembly is regulated by specific interactions of myosin filaments, the M band protein myomesin and the large carboxy-terminal region of titin. Corresponding binding sites between these proteins were identified. Myomesin binds myosin in the central region of light meromyosin (LMM, myosin residues 1506-1674) by its unique amino-terminal domain My1. A single titin immunoglobulin domain, m4, interacts with a myomesin fragment spanning domains My4-My6. This interaction is regulated by phosphorylation of Ser482 in the linker between myomesin domains My4 and My5. Myomesin phosphorylation at this site by cAMP-dependent kinase and similar or identical activities in muscle extracts block the association with titin. We propose that this demonstration of a phosphorylation-controlled interaction in the sarcomeric cytoskeleton is of potential relevance for sarcomere formation and/or turnover. It also reveals how binding affinities of modular proteins can be regulated by modifications of inter-domain linkers.     

Titins in C.elegans with unusual features: coiled-coil domains,novel regulation of kinase activity and two new possible elastic regions

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism.     

Titin and the sarcomere symmetry paradox

Titin is thought to play a major role in myofibril assembly, elasticity and stability. A single molecule spans half the sarcomere and makes interactions with both a thick filament and the Z-line. In the unit cell structure of each half sarcomere there is one thick filament with 3-fold symmetry and two thin filaments with approximately 2-fold symmetry. The minimum number of titin molecules that could satisfy both these symmetries is 12. We determined the actual number of titin molecules in a unit cell from scanning transmission electron microscopy mass measurements of end-filaments. One of these emerges from each tip of the thick filament and is thought to be the in-register aggregate of the titin molecules associated with the filament. The mass per unit length of the end-filament (17.1 kDa/nm) is consistent with six titin molecules not 12. Thus the number of titin molecules present is insufficient to satisfy both symmetries. We suggest a novel solution to this paradox in which four of the six titin molecules interact with the two thin filaments in the unit cell, while the remaining two interact with the two thin filaments that enter the unit cell from the adjacent sarcomere. This arrangement would augment mechanical stability in the sarcomere.     

Two immunoglobulin tandem proteins with a linking β-strand reveal unexpected differences in cooperativity and folding pathways

The study of the folding of single domains, in the context of their multidomain environment, is important because more than 70% of eukaryotic proteins are composed of multiple domains. The structures of the tandem immunoglobulin (Ig) domain pairs A164-A165 and A168-A169, from the A-band of the giant muscle protein titin, reveal that they form tightly associated domain arrangements, connected by a continuous β-strand. We investigate the thermodynamic and kinetic properties of these tandem domain pairs. While A164-A165 apparently behaves as a single cooperative unit at equilibrium, unfolding without the accumulation of a large population of intermediates, domains in A168-A169 behave independently. Although A169 appears to be stabilized in the tandem protein, we show that this is due to nonspecific stabilization by extension. We elucidate the folding and unfolding pathways of both tandem pairs and show that cooperativity in A164-A165 is a manifestation of the relative refolding and unfolding rate constants of each individual domain. We infer that the differences between the two tandem pairs result from a different pattern of interactions at the domain/domain interface.     

Titin: a molecular control freak.

Recent studies of the giant protein titin have shed light on its roles in muscle assembly and elasticity and include the surprising findings described here. We now know that the titin kinase domain, which has long been a puzzle, has a novel regulation mechanism. A substrate, telethonin, has been identified that is located over one micron away from the kinase domain in mature muscle. Single-molecule studies have demonstrated the fascinating process of reversible mechanical unfolding of titin. Lastly, and most surprisingly, it has been claimed that titin controls assembly and elasticity in chromosomes.     

Stretching molecular springs: elasticity of titin filaments in vertebrate striated muscle

Titin, the giant protein of striated muscle, provides a continuous link between the Z-disk and the M-line of a sarcomere. The elastic I-band section of titin comprises two main structural elements, stretches of immunoglobulin-like domains and a unique sequence, the PEVK segment. Both elements contribute to the extensibility and passive force development of nonactivated muscle. Extensibility of the titin segments in skeletal muscle has been determined by immunofluorescence/immunoelectron microscopy of sarcomeres stained with sequence-assigned titin antibodies. The force developed upon stretch of titin has been measured on isolated molecules or recombinant titin fragments with the help of optical tweezers and the atomic force microscope. Force has also been measured in single isolated myofibrils. The force-extension relation of titin could be readily fitted with models of biopolymer elasticity. For physiologically relevant extensions, the elasticity of the titin segments was largely explainable by an entropic-spring mechanism. The modelling explains why during stretch of titin, the Ig-domain regions (with folded modules) extend before the PEVK domain. In cardiac muscle, I-band titin is expressed in different isoforms, termed N2-A and N2-B. The N2-A isoform resembles that of skeletal muscle, whereas N2-B titin is shorter and is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. Examination of N2-B titin extensibility revealed that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at lower stretch and, in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains. However, unfolding of a very small number of Ig domains remains a possibility.     

Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin

Cardiac myofibrillogenesis was examined in cultured chick cardiac cells by immunofluorescence using antibodies against titin, actin, tropomyosin, and myosin. Primitive cardiomyocytes initially contained stress fiber-like structures (SFLS) that stained positively for alpha actin and/or muscle tropomyosin. In some cases the staining for muscle tropomyosin and alpha actin was disproportionate; this suggests that the synthesis and/or assembly of these two isoforms into the SFLS may not be stoichiometric. The alpha actin containing SFLS in these myocytes could be classified as either central or peripheral; central SFLS showed developing sarcomeric titin while peripheral SFLS had weak titin fluorescence and a more uniform stain distribution. Sarcomeric patterns of titin and myosin were present at multiple sites on these structures. A pair of titin staining bands was clearly associated with each developing A band even at the two or three sarcomere stage, although occasional examples of a titin band being associated with a half sarcomere were noted. The appearance of sarcomeric titin patterns coincided or preceded sarcomere periodicity of either alpha actin or muscle tropomyosin. The early appearance of titin in myofibrillogenesis suggests it may have a role in filament alignment during sarcomere assembly.     

I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure.

In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment.We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.     

The titin A-band rod domain is dispensable for initial thick filament assembly in zebrafish

The sarcomeres of skeletal and cardiac muscle are highly structured protein arrays, consisting of thick and thin filaments aligned precisely to one another and to their surrounding matrix. The contractile mechanisms of sarcomeres are generally well understood, but how the patterning of sarcomeres is initiated during early skeletal muscle and cardiac development remains uncertain. Two of the most widely accepted hypotheses for this process include the “molecular ruler” model, in which the massive protein titin defines the length of the sarcomere and provides a scaffold along which the myosin thick filament is assembled, and the “premyofibril” model, which proposes that thick filament formation does not require titin, but that a “premyofibril” consisting of non-muscle myosin, α-actinin and cytoskeletal actin is used as a template. Each model posits a different order of necessity of the various components, but these have been difficult to test in vivo. Zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms, and here we report the analysis of the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning is still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with the previously noted role of titin in maintaining the contractile integrity of mature sarcomeres. This conflicts with the “molecular ruler” model of early sarcomere patterning and supports a titin-independent model of thick filament organization during sarcomerogenesis. These findings are also consistent with the symptoms of human titin myopathies that exhibit a late onset, such as tibial muscular dystrophy.     

Structural and functional studies of titin's fn3 modules reveal conserved surface patterns and binding to myosin S1--a possible role in the Frank-Starling mechanism of the heart.

The A-band part of titin, a striated-muscle specific protein spanning from the Z-line to the M-line, mainly consists of a well-ordered super-repeat array of immunoglobulin-like and fibronectin-type III (fn3)-like domains. Since it has been suspected that the fn3 domains might represent titin's binding sites to myosin, we have developed structural models for all of titin's 132 fn3-like domains. A subset of eight experimentally determined fn3 structures from a range of proteins, including titin itself, was used as homology templates. After grouping the models according to their position within the super-repeat segment of the central A-band titin region, we analyzed the models with respect to side-chain conservation. This showed that conserved residues form an extensive surface pattern predominantly at one side of the domains, whereas domains outside the central C-zone super-repeat region show generally less conserved surfaces. Since the conserved surface residues may function as protein-binding sites, we experimentally studied the binding properties of expressed multi-domain fn3 fragments. This revealed that fn3 fragments specifically bind to the sub-fragment 1 of myosin. We also measured the effect of fn3 fragments on the contractile properties of single cardiac myocytes. At sub-maximal Ca(2+) concentrations, fn3 fragments significantly enhance active tension. This effect is most pronounced at short sarcomere length, and as a result the length-dependence of Ca(2+) activation is reduced. A model of how titin's fn3-like domains may influence actomyosin interaction is proposed.     

Substructure and accessory proteins in scallop myosin filaments

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.