Group:MUZIC:Titin

From Proteopedia

proteopedia linkproteopedia link

Contents 1 Introduction 1.1 Sequence annotation 1.2 Schematic domain structure 2 Z-disc fragment of titin 3 Structures 4 Function and interactions 5 Pathology 6 Research groups working on titin: 7 References:

Introduction

Sequence annotation

The giant protein titin (also called „connectin“ by Maruyama et al., 1977 ^{[1] [2]}) is the largest known protein 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 TTN titin). Its molecular weight varies from 1,5 megaDalton to ~3,7 MegaDalton in different isoforms. These isoforms aree produced by alternative splicing mostly in the I-band region of titin. For example, 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 third most abundant protein in striated muscle cells after actin and myosin. It forms so called “third filament system”. Single titin macromolecules have length >1-μm and span from Z-disc to M-line through half-sarcomere. The main function of titin is to provide passive tension which helps to restore the length of resting sarcomere after contractile activity. However, titin has other important functions: it acts as a molecular ruler/scaffold, determining correct location of other muscular proteins. Titin also serves as a nodal point in signaling cascades within sarcomere, 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 not included). 90 % of titin is represented by immunoglobulin (Ig)^[3], PEVK motivs or fibronectin-type-III (FN3)-like ^[4] domains ^[5].
Ig-domain is ~ 100 amino acid residues large and consists of two β-sheets that create two-layered elongated fold. Majority of Ig-domains of muscular proteins belong to I-set subfamily. N- and C-termini of Ig-fold are located at opposite ends of structure and, thus, 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 domain type III has average size of ~100 amino acids residues and demonstrates a β-sandwich structure. Currently several structures of FN III domains from A-band part of titin are deposited in “Protein Data Bank”. They are: structure of domain A71 ^[8], structure of FnIII tandem A77-A78 ^[9], Ig(A168)-Ig(A169)-FnIII(A170) ^[10].
Ig-domains are most resistant to unfolding. Weaker FN III domains are positioned in the A-band portion of the titin which is additionally stabilized by the thick filaments. However, Ig-domains that are located in I-band differ in their stability. Domains, that are located closely to Z-line, have unfolding force of approximately150 pN, central Ig-domains have unfolding force of ~200 pN, Ig-domains, that are located closely to A-band, have unfolding force of ~ 250 pN. Thus, titin unfolding caused by muscle stretch, starts close to Z-line and continues towards A-band part of molecule. ^[11]
PEVK repeats are ~28-residue,proline, glutamine, valine, lysine reach motivs which are important for elasticity 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

---

Z-disc fragment of titin

Approximately 2000 residues part of titin’s amino-terminus, coded by exons 1–28, is localized within Z-disc. It consists of immunoglobulin-type domains and a variable number of unique 45-residue motifs called Z-repeats ^[15] Each of them share ~50% sequence homology. These repeats are located between Ig-domains 2 and 3. 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 due to differential splicing. Both repeats 1 and 7 are present in all isoforms except smooth muscle titin.

Structures

3D structures of titin or titin 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 is proven that Z1Z2 adopts semiextended conformation with certain rigidity and limited dynamics.
1YA5 - This structure shows the assembly of titin’s two N-terminal Ig-domains with Z-disc 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.

---

---

Function and interactions

Titin acts as the tension sensor in muscle cells. As it was mentioned before, titin molecules are extended within sarcomere, thus, have a proper position for detecting the sarcomere’s contraction and transfering correponding signals. N-termins of titin is attached to the actin filaments at the Z-disk and connected to myosin in the A-band/M-band. Given parts of titin sense tensile forces generated by 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, it interacts with α-actinin, nebulin, obscurin and γ-filamin within the Z disk, with obscurin with myosin heavy chain and myosin-binding protein-C in the A band and with myomesin in 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 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 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 telethonin ^{[18] [19]}(also called “T-Cap”) which connects titin molecules from same half of the sarcomere into antiparallel “sandwich” (given 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 Z-disc. 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. It proposed that indirect interaction of small ankyrin with titin amino-terminus promotes correct positioning of sarcoplasmic reticulum (SR) around Z-disc. 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 Z-disc and may influence functioning of potassium channel depending on myocyte stretch. Titin also interacts with growth factor myostatin and calsarcin-3 through T-Cap.In turn, the small-ankyrin-1 is associated with spectrin, desmin and obscurin. 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 Z-disc, however it is found also at costameres, in the I-band and in 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 hypertrophy. It is proposed that stress load of Z-disk activates this signaling cascade, however, precise mechanism of signal transduction and role of titin domains Z1/Z2 is to be investigated.Other proposed mechanisms of mechanosensing include MARP-Myopalladin complex interacting with the N2A-domain in the I-band the titin-kinase domain. Interaction with actin is reported for titin's domains Z9-I1. Other proven partners of inside Z-disk are nebulin and filamin C, that both interact with titin by their C-terminal parts.
Interaction of α-actinin and Z-repeats of titin within Z-disc was shown experimentally more than a decade ago ^{[22] [23] [24]}. 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 Ig-domain Z3 of titin and is contacting spectrin-like domains of α-actinin homodimer. Strong interactions between actin, α-actinin and titin form a spatial scaffold inside Z-disc, enabling correct placement of other protein components. In addition, it was also shown that 2 other proteins, LIM and FATZ, are interacting with both telethonin and α-actinin, reinforcing titin/telethonin and titin/ α-actinin networks. Z-disc 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. Thickness of Z-discs varies significantly between different types of muscles due to adaptation to different levels of mechanic stress. A hypothesis that ascribes titin, particularly it’s Z-repeats, role of the Z-disc thickness determinant, was proposed. It was grounded on the fact that number of repeats and layers in Z-disc correlate ( i.e. sarcomeres with full range of Z-repeats have the thickest disc). However, given idea remains unproven, since it has been found that length of a single repeat is less that thickness of single layer inside Z-disc (19 nm) and thus periodicity cannot be directly determined in proposed way.

^{[25] [26] [27] [28] [29] [30]}

---

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) ^[31] 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 ^[32] 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 Z-disc (decreased affinity to T-Cap and α-actinin, correspondingly). Studies of Kimura et al.^[33], 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 ^[34]. It is worth to mention specifically some mutations which are directly related to Z-disc 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 ^[35]. Missense mutation in Z4 (Trp930 to Arg) is predicted to destroy Ig-domain fold.

---

Research groups working on titin:

EMBL-Hamburg, Research group of Dr. Matthias Wilmanns: http://www.embl-hamburg.de/research/unit/wilmanns/index.html

University of Arisona, Departmant of Physiology and Molecular and Cellular Biology, Laboratory of Henk Granzier: http://www.mcrp.med.arizona.edu/html/henkgranzier/

Ruhr-Universitat Bochum, Faculty of medicine, Institute of Physiology, Cardiovascular Physiology, Laboratory of Prof. Dr. Wolfgang Linke: http://www.py.ruhr-uni-bochum.de/kardp/linke/Index.html.en

University of Leeds, Faculty of Biological Sciences, Institute of Molecular and Cellular Biology, Prof. John Trinick: http://www.astbury.leeds.ac.uk/people/staffpage.php?StaffID=JT

Univesitatsklinikum Mannheim, Klinik für Anästhesiologie und Operative Intensivmedizin, Group of Prof. Dr. med Siegfried Labeit: http://www.umm.de/2183.0.html

---

References:

---

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

---