Group:MUZIC:Titin

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Revision as of 15:03, 24 July 2011

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. 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. 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, 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) or fibronectin-type-III (FN3)-like domains ^[3].

Schematic domain structure

Concisely annotated schemes of titin's domain structure can also be found in Labeit et al., 2006 ^[4], domain arrangement and interacting proteins are concisely described in Kontrogianni-Konstantopoulos et al., 2009 ^[5] and Linke, 2007 ^[6].

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 ^[7] 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

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.
Atomic structure of Z1Z2 Ig-domain doublet of titin's N-terminus was determined by Zou et al., 2006.^[8]. Then it’s conformational features were thoroughly analyzed in a study that combined X-ray crystallography, SAXS, N15 relaxation NMR, residual dipolar couplings ^[9] . 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 ^[10] (also called “T-Cap”) which connects titin molecules from same half of the sarcomere into antiparallel “sandwich”. 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 helps to target other sarcomeric proteins (for example, FATZ, MLP, minK, PKD, MURF1 ). Domains Z1/Z2 are known to be connected to small-ankyrin-1 which is associated with spectrin, desmin and obscurin. Interaction with actin is reported for domains Z9-I1. Ig-like domains Z8/Z9 are bound to obscurin. Other proven partners 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 ^{[11] [12] [13]}. 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. 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.

^{[14] [15] [16] [17] [18] [19] [20] [21]}

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) ^[22] 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 ^[23] 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.^[24], propose that significant percent of cardiac diseases may be caused by titin mutations. 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 ^[25]. Missense mutation in Z4 (Trp930 to Arg) is predicted to destroy Ig-domain fold.

References:

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