Group:MUZIC:Titin

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Contents 1 Introduction 1.1 Sequence annotation 1.2 Schematic domain structure 2 The Z-disk fragment of titin 3 Structures 4 Function and interactions 5 Pathology 6 References:

Introduction

Sequence annotation

The giant protein titin (also called „connectin“ by Maruyama et al., 1977 ^{[1] [2]}) is the largest known protein which is composed of 38138 amino acid residues (see Uniprot for Q8WZ42). The human titin gene is located on chromosome 2q31, is 294 kilobases large and contains 363 exons (see gen information at TTN titin). Its molecular weight varies from 1,5 MegaDalton to ~3,7 MegaDalton in numerous isoforms. These isoforms are produced by alternative splicing which occurs mostly in the I-band region of titin. For example, the following predominant titin isoforms are found in human heart : the N2B-isoform (3,000 kDa) – 65%, which contains N2B element composed of 3 Ig domains and a central ~570 a.a. unique sequence, and N2BA-isoforms (~3,200–3,700 kDa) - 35%. Titin is the third most abundant protein in striated muscle cells after actin and myosin. It forms the so called “third filament system”. Single titin macromolecules have a length >1-μm and span from the Z-disk to M-line through the half-sarcomere. The main function of titin is to provide passive tension which helps to restore the length of the resting sarcomere after contractile activity. However, titin has other important functions: it acts as a molecular ruler/scaffold, determining the correct location of various muscle proteins. Titin also serves as a nodal point in signaling cascades within thesarcomere, takes part in sarcomere formation and maintenance. It is also worth mentioning that set of titin-like proteins is expressed in non-muscular tissues and a distinct titin isoform of ~1 MDa can be found in human smooth muscle tissues (280 of 363 existing exons are excluded ). 90 % of titin is composed of immunoglobulin (Ig) domains ^[3], PEVK motifs and fibronectin-type-III (FN3)-like ^[4] domains ^[5].
The Ig-domain is usually ~ 100 amino acid residues large and consists of two β-sheets that create a two-layered elongated fold. Majority of Ig-domains of muscle proteins belong to so calles I-set subfamily. N- and C-termini of the Ig-fold are located at the opposite ends of structure and, thus, a protein, composed mainly of Ig-domains resembles beads on a string. Molecular elasticity of such proteins is created by stretching of interdomain linkers or by unfolding of domains themselves ^{[6] [7]}.
The fibronectin type III domain has an average size of ~100 amino acids residues and adopts a β-sandwich structure. Currently several structures of FN III domains from the A-band part of titin are deposited in the “Protein Data Bank”. They are: structure of domain A71 ^[8], structure of FnIII tandem A77-A78 ^[9], Ig(A168)-Ig(A169)-FnIII(A170) ^[10].
Among the structural elements of titin, the Ig-domains are most resistant to mechanical unfolding. The slightly weaker FN III domains are located in the A-band portion of the titin, which is stabilized additionally by interactions with the thick filaments. Of note, Ig-domains that are located in the I-band differ in their stability. Domains that are located close to the Z-disk show unfolding forces of approximately 150 pN, central Ig-domains unfold around ~200 pN, while Ig-domains that are located closely to the A-bandunfold around ~ 250 pN. Thus, titin unfolding caused by muscle stretch should start close to the Z-disk and continue towards the A-band part of molecule. ^[11]
PEVK repeats are ~28-residues disordered motifs enriched in proline, glutamine, valine, lysine which are important for the entropic (rubber-like) elasticity of titin. Due to the large size of titin, this entry is mainly focused on the Z-disk portion of titin.

Schematic domain structure

Concisely annotated schemes of titin's domain structure can also be found in Labeit et al., 2006 ^[12], domain arrangement and interacting proteins are concisely described in Kontrogianni-Konstantopoulos et al., 2009 ^[13] and Linke, 2007 ^[14].

spinqualitylabelsall models Structure of full length telethonin in complex with the N-terminus of titin. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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The Z-disk fragment of titin

Approximately 2000 residues of titin’s amino-terminus, coded by exons 1–28, is localized within the Z-disk. This region consists of immunoglobulin-type domains, proline-rich ZIS region and a variable number of unique 45-residue motifs called Z-repeats ^[15] These repeats are located between Ig-domains 2 and 3. Each of them share ~50% sequence homology. N-terminal immunoglobulin domains Z1-Z4 are present in all isoforms of titin, whereas number of Z-repeats varies from 2 to 7 in different types of striated muscles because of differential splicing. Both repeats 1 and 7 are present in all isoforms except smooth muscle titin.

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Structures

3D structures of N-terminal part of titin or its complexes, deposited in "Protein Data Bank"

PDB ID	Structure	PubMed link
2F8V	Structure of full length telethonin in complex with the N-terminus of titin	PMID 16713295
2A38	Crystal structure of the N-Terminus of titin	PMID 16962974
1YA5	Crystal structure of the titin domains z1z2 in complex with telethonin	PMID 16407954
1H8B	EF-hands 3,4 from alpha-actinin / Z-repeat 7 from titin	PMID 11573089

2F8V -This structure shows a complex of titin N-terminus with full-length telethonin at 2.75 Ǻ resolution. Data, complementary to the structure, show formation of a dimer of 2 titin/telethonin complexes and possibly formation of higher oligomers.
2A38 - This structure shows two Ig-domains, Z1Z2, from amino-terminus of titin at 2 Ǻ resolution. It proves that Z1Z2 moiety adopts semiextended conformation with certain rigidity and limited dynamics.
1YA5 - This structure shows the assembly of titin’s two N-terminal Ig-domains with the Z-disk protein telethonin (residues 1 to 90) at 2.44 Ǻ resolution. It also proposes a model for crosslinking of actin filaments.
1H8B - This structure shows a complex of Calmoduline-like calcium insensitive EF-hand domain of α-actinin and Z-repeat 7 of titin solved by solution NMR.

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Function and interactions

Titin acts as the tension sensor in muscle cells. As it was mentioned in the "Sequence annotation", titin molecules are extended through whole sarcomere, thus, they have a proper position for detecting the sarcomere’s contraction and transfering correponding signals.The N-termins of titin is attached to the actin filaments at the Z-disk and connected to the myosin in the A-band/M-band. Given parts of titin sense tensile forces generated by a sarcomere during stretch/contraction. Transmission of these signals is possible because of titin’s interactions with other sarcomeric proteins. Up to date approximately 20 different proteins are known to interact with titin at so called “hot spots” along the entire molecule and to participate in signal transduction. For example, titin interacts with α-actinin, nebulin, obscurin and γ-filamin within the Z-disk, with obscurin and with myosin heavy chain and myosin-binding protein-C at the A band and with myomesin at the M band.
Atomic structure of Z1Z2 Ig-domain doublet of titin's N-terminus was determined by Zou et al., 2006.^[16]. Then it’s conformational features were thoroughly analyzed in a comprehensive study that combined X-ray crystallography, SAXS, N15 relaxation NMR, residual dipolar couplings ^[17] . It was show that Z1Z2 adopts semiextended conformation in solution which is in agreement with crystallographic data. Surprisingly, it was elucidated that dynamics of Ig-doublet is rather restricted despite the presence of long interdomain linker and absence of contacts between Ig domains. NMR experiments further shown absence of movements of the linker moiety and overall semirigid state of given structure. These data agree with NMR studies of I91–I92 and may be considered as a general model of conformation state of Ig-doublets along titin fiber.

At the N-terminal end of titin Ig-domains Z1/Z2 interact with small 19 kDa protein, called telethonin ^{[18] [19]}(also called “T-Cap”). It connects titin molecules from same half of the sarcomere into antiparallel “sandwich”. This complex has 2:1 stoichiometry which means that one telethonin molecule allows an antiparallel arrangement of two titin molecules from neighbouring sarcomeres. Due to numerous hydrogen bonds which connect β-strands of two molecules titin-telethonin complex is extremely resistant to stretching. Telethonin is proved to be responsible for anchoring of titin molecules in the Z-disk. It also plays a role of mechanosensor and participates in targeting of other sarcomeric proteins. For example, T-Cap is connected to membrane-associated proteins small ankyrin-1 (sANK1) and the potassium channel subunit minK ^[20], that are localized in the sarcoplasmic reticulum and T-tubules, correspondingly. In turn, the small-ankyrin-1 is associated with spectrin, desmin and obscurin. It proposed that indirect interaction of small ankyrin with titin amino-terminus promotes correct positioning of sarcoplasmic reticulum (SR) around the Z-disk. Titin could have impact on SR organization also via interaction of Ig-like domains Z8/Z9 with obscurin ^[21]. Binding of telethonin to minK places T-tubules in proximity of the Z-disk and may influence functioning of potassium channel depending on myocyte stretch. Titin also interacts with growth factor myostatin and calsarcin-3 through T-Cap. It is also worth mentioning that NH2-terminus of titin is connected via telethonin to muscle LIM protein (MLP). In striated muscles, MLP localizes mainly in the Z-disk, however it is found also at costameres, in the I-band and in the nucleus. Transport of MLP to the nucleus may activate transcription factors and upregulate protein expression. Other related cascade, namely, the “Telethonin-MLP-calcineurin-nuclear factor of activated T cells (NFAT)” signaling pathway is involved in mechanosensing and leads to physiological hypertrophy. It is proposed that stress load of the Z-disk activates this signaling cascade, however, precise mechanism of signal transduction and role of titin domains Z1/Z2 is to be investigated further. Another important telethonin mediated interaction of titin’s amino terminus is with MDM2 (mouse double minute-2). This protein is an E3 ubiquitin ligase that performs ubiqutination of tumor suppressor p53/TP53 which leads to proteasomal degradation. Other mechanisms of mechanosensing, not allocated to the Z-disk, include MARP-Myopalladin complex which interacts with the titin's N2A-domain in the I-band and the titin-kinase domain. Interaction with actin is reported for titin's domains Z9-I1. Additional proven partners of titin within the Z-disk are nebulin and filamin C which both interact direct with titin by their carboxy-terminal parts.
Essential interaction of α-actinin and Z-repeats of titin within the Z-disk was shown experimentally more than a decade ago ^{[22] [23] [24]}. The binding was reported for Z-repeats 1 and 7 and calmodulin-like domains (syn. EF-hands) at C-terminus of α-actinin. Third putative point of interaction is located between Z-repeat 7 and adjacent Ig-domain of titin and may involve spectrin-like domains of α-actinin homodimer. Strong interactions between actin, α-actinin and titin form a spatial scaffold , thus enabling correct placement of other proteins inside the Z-disk. In addition, it was also shown that 2 muscular proteins, LIM and FATZ, are interacting with both telethonin and α-actinin, reinforcing titin/telethonin and titin/ α-actinin networks. The Z-disk connects all elastic and contractile components of sarcomere and enables transduction of tensile forces. Some of these components take part in different signaling pathways, others are responsible for direct mechanosensing. Within the Z-disk several layers of actin crosslinked by α-actinin are usually visible on electron microscopy images. The thicker is the Z-disk, the more layers it has. Thickness of the Z-disks varies significantly between different types of muscles due to adaptation to variable levels of mechanic stress. A hypothesis that ascribes titin, particularly it’s Z-repeats, role of the Z-disk thickness determinant, was proposed. It was grounded on the fact that number of repeats and layers in the Z-disk correlate ( i.e. sarcomeres with full range of Z-repeats have the thickest disk). However, given idea remains dubious, since it has been found that length of a single repeat is less that thickness of single layer inside the Z-disk (19 nm) and thus periodicity cannot be directly determined in proposed way.
It is also necessary to mention additional noncanonical function of NH2-terminus of titin that was discovered recently. Comprehensive studies of mammalian non-muscular cell cultures provided facts that give a solid proof of nuclear localization of amino-terminal region of titin. Immunostaining coupled with fluorescence microscopy has shown that Z1-Z2-Zr moiety of titin is, indeed, transported into the nucleus. A functional nuclear localization signal (NLS) 200-PAKKTKT-206 was discovered by screening of titin constructs of various length. Given NLS enables transport of titin N-terminal domain to the nucleus. This finding was confirmed in following cell lines: human MG-63 and BHK-21, mouse MC3T3-E1, COS-7. It was shown that mutation (Lysin203 to Alanine) in this region leads to the loss of NLS’ function and results in cytoplasmic localization of Z1-Z2-Zr.
In contrast to sarcomeres, within non-muscle cells titin doesn’t form an ordered network of fibrils, but has rather a “punctate pattern” of distribution both in the nucleus and cytoplasm. Using human osteoblast cells, MG-63, it was shown that overexpression of titin Z1-Z2-Zr domain leads to change of cell shape (from spindle-like to rounded), decreases contact inhibition of cells and facilitates cell proliferation. Proposed mechanism of action involves activation of Wnt/β-catenin pathway. This signaling cascade is important for proper bone maintenance, Z1-Z2-Zr part of titin may participate in remodeling of bone tissue. ^[25] A list of proteins that directly interact with the Z-disk portion of titin and their functions are given in the table below (cited from minireview of Kruger and Linke, 2011. ^[26])

Binding partners of titin within the Z-disk

Titin region	Interacting partner	Suggested function
Z1-Z2	Telethonin (Tcap)	Connects NH2 termini of two titin filaments from same half-sarcomere; forms putative stretch sensor complex with MLP
Z1-Z2	Small Ankyrin-1	May help position the sarcoplasmic reticulum near the Z-disk region
Z2-Zis-1	γ-Filamin	Links titin to integrin and focal adhesion complex
Z2-Zis-1	Nebulin/Nebulette	Stabilization of cytoskeletal linkages to the Z-disk
Zis-1; Z-repeats; Zis-2	α-Actinin	Anchors titin's NH2-terminus in the Z-disk;provides Z-disk stability
Z8/Z9	Obscurin	Unknown
Z9/I1	Actin	Connects titin to thin filaments at the N1-line of the sarcomere

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Pathology

Titin is a subject of mutations that cause various muscle pathologies. Detailed information about titin’s gene structure and massive sequencing approach allows to to link some alterations with their phenotypical consequences. For example, presence of mutations that cause dilated cardiomyopathy (DCM) ^[27] was shown for exons 18 and 326. The mutation in exon 326 leads to expression of truncated form of titin (~2 mDa) which is sensitive to proteolysis. Mutation in exon 18 causes disruption of normal fold of encoded Ig-domain which in turn affects function of entire titin.
Recent studies have shown connection between tibial muscular dystrophy ^[28] and a mutation in exon 363. This mutation also affects natural fold of Ig-domain. Moreover, disease-causing mutations in titin’s exon 2, exon 14 and exon 49 were identified by massive sequencing approach. The first and the second mutation severely affect interaction between titin and it’s ligands inside the Z-disk (decreased affinity to T-Cap and α-actinin, correspondingly). Studies of Kimura et al.^[29], propose that significant percent of cardiac diseases may be caused by titin mutations. It is worth to mention that microscopy studies of cardiac hypertrophy and degeneration have shown significant downregulation of titin expression. Shortage of titin may cause to decreased elasticity of cells in failing hearts ^[30]. It is worth to mention specifically some mutations which are directly related to the Z-disk moiety of titin. Val54Met point mutation in domain Z1 leads to decreased binding to telethonin. Z-repeat 7 Ala743Val point mutation affects interaction with α-actinin. Point mutation of Ala740 to Leu has opposite effect ^[31]. Missense mutation in Z4 (Trp930 to Arg) is predicted to destroy Ig-domain fold.

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References:

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Third filament diseases. 19181097 Zaspopathy in a large classic late-onset distal myopathy family. 17337483 The Z-disk diseases. 19181098

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