Striated muscle

Striated muscle are assembled by bundles of muscle fibres composed of multi-nucleated cells which are typically 10 to 100µm in diameter and several centime-ters in length. The muscle fibres are composed by parallel myofibrils separated by the sarcoplasmic reticulum and transversely running tubules (T tubules).

A repetitive contractile unit in the myofibrils, called the sarcomere, packs end to end from one Z-disc to the next over a distance of 2.0-2.5µm. The repeti-tive unit emerges from the ordered arrays of myosin (thick) filaments and actin (thin) filaments. In the light microscope, the myofibrils display a character-istic pattern of alternating striation where the dark A-band (anisotrop) and the light I-band (isotrop) reflect the behaviour in polarised light (Figure 1.1).

Cross-sections along the sarcomere show the arrangement of thick and thin filaments. In the part of the A-band where both filaments overlap, the actin filaments adapt a trigonal arrangement to fit to the hexagonal array of myosin

1

2 Introduction

Figure 1.1: Striated muscle and the components of the sarcomere. Top panel:

electron micrograph of a longitudinal seciton of a skeletal muscle myofibril (adapted from Stryer, 1996). Bottom panel: schematic presentation of the sarcomere with its main components: the thin filament, the thick filament and titin, shown in blue (adapted from Tskhovrebova and Trinick, 2004).

filament, whereas their organisation towards the Z-disc gradually changes into a tetragonal array (Squire, 1997). During muscle contraction the length of the I-band changes, while that of the A-I-band remains the same. The sliding filament model, which was independently proposed by A. Huxley and R. Niedergerke (1954) and H. Huxley and J. Hanson (1954), explains the generation of force.

During muscle contraction the thick and thin filament slide past one another without changing the length of the filaments. Thereby, force is developed based on the interaction of the actin and myosin filaments during sliding. The mech-anism of the so-called cross-bridge cycle is ATP-dependent and describes the movement of the myosin-heads along the actin filament (Geeves and Holmes, 2005).

The thin filament comprises a number of proteins and the globular 42 kDa actin (G-actin) is a major component. Single G-actin (globular) molecules polymerise in an ATP-dependent manner into the double-helical (filament) F-actin. Tropomyosin and three troponin subunits, TnC (Ca²⁺-binding), TnI (inhibitory), and TnT (tropomyosin-binding), represent the other constituents

1.2 Titin 3

of the thin filament. Tropomyosin extends over seven actin subunits and tro-ponin regulates its binding to myosin. In the presence of Ca²⁺, TnC binds Ca²⁺ resulting in a conformational change and movement of tropomyosin into a position with exposure of the myosin-binding site.

The myosin molecule consists of a globular head region, the S1 fragment, which can interact with actin and possesses ATPase activity. The S2 frag-ment comprises a flexible region of regulatory and essential myosin light chains (MLC) and a tail which combines via a long coiled-coil region with tails of other molecules. A thick filament is assembled by approximately 300 myosin molecules with the myosin heads pointing away from the M-line where they are anchored.

Besides the thick and the thin filament, a third filament system exists. In contrast to the actin and myosin filament, it is constituted by a single protein called titin. This immensely large protein extends over half of the sarcomere length. Due to its size and location, titin can interact with numerous sarcomeric proteins existing in the different regions (Granzier and Labeit, 2004; Miller et al., 2004). Therefore, it is capable of regulating diverse processes. The giant protein is involved in muscle assembly and elasticity of the sarcomere as well as signalling pathways through interaction with binding partners. Since titin fulfills many diverse functions, it has been entitled a molecular ’control freak’ (Trinick and Tskhovrebova, 1999). Titin will be described in more detail in the following.

1.2 Titin

Titin, also known as connectin, has been identified as a large component of stri-ated muscle (Maruyama et al., 1977; Wang et al., 1979). The flexible molecule spans half of the sarcomere (Nave et al., 1989) from the Z-disc to the M-line and is more than 1µm long. With a molecular weight of up to 3.7 MDa in the skeletal N2A titin, the giant protein titin is the largest polypeptide known (Labeit and Kolmerer, 1995). In human, titin is encoded by a single gene on chromosome 2, region 2q31 (Labeit et al., 1990), containing 363 exons coding together for 38138 residues (GenBank accession code: AJ277892) (Bang et al., 2001).

The sequence of human titin was first published in 1995 (Labeit and Kolmerer, 1995) and was completed by additional PEVK exons and three unique I-band exons (termed novex-1 to -3) in 2001 (Bang et al., 2001). The sequence revealed several splicing alternatives leading to different lengths of the titin I-band. Hence, size differences in splice variants are manifested above all in the shorter cardiac titin isoforms with a molecular weight of approximately 3 MDa. The larger skeletal isoforms show sizes from 3.3 MDa in psoas muscle up to 3.7 MDa in soleus muscle (Maruyama, 1997).

Besides titin occurring in striated muscle of vertebrates, titin-like proteins are also present in invertebrate muscle, however, with a lower molecular weight.

These members of the titin family (Tskhovrebova and Trinick, 2003) are

4 Introduction

Figure1.2:Domainstructureofthegiantmuscleproteintitin,mainlycomposedofimmunoglobulin(Ig)domains(red)andfibronectintypeIII(FnIII)domains(white)(adaptedfromGregorioetal.,1999).

1.3 Structure and function of titin 5

known as I-connectin (from crayfish, 2 MDa; Fukuzawa et al., 2001), D-titin (from Drosophila melanogaster, 2 MDa; Machado and Andrew, 2000; Zhang et al., 2000), kettin (about 0.5-0.7 MDa; Lakey et al., 1993; Hakeda et al., 2000; Kolmerer et al., 2000), stretchin (Caenorhabditis elegans; Champagne et al., 2000), twitchin (Benian et al., 1989) and projectin (0.8-1.0 MDa; Ayme-Southgate et al., 1991).

Titin is a modular protein and 90 % of its length consists of two modules, each comprising about 90 to 100 residues. The type I are the fibronectin III (FnIII) and type II are the immunoglobulin (Ig) domains (Labeit et al., 1990). These form repetitive patterns, characteristic for their distinct occurrence within titin in the sarcomere. The designated unique sequences and other elements con-stitute the remaining 10 %, among it a single serine/threonine kinase domain (Figure 1.2).

1.3 Structure and function of titin

Titin can be divided functionally and structurally according to the location of its regions within the sarcomere.

1.3.1 Z-disc and anchoring of titin

The amino-terminal part of titin is anchored in the Z-disc by overlapping of molecules of two neighbouring sarcomeres. Titin in the Z-disc consists of Ig-like domains and a tissue-dependent number of the 45-residue motif called Z-repeat, which is differentially spliced (Sorimachi et al., 1997; Gautel et al., 1996). A characteristic protein of the Z-disc isα-actinin which cross-links actin-filaments of opposing polarity through its actin-binding domain (Schroeter et al., 1996; Blanchard et al., 1989) with titin binding to the Z-repeats via its C-terminal EF-hand (Sorimachi et al., 1997). Additionally, α-actinin inter-acts with other Z-disc proteins such as FATZ (Faulkner et al., 2000), actinin-associated LIM-protein (Xia et al., 1997) and ZASP (Faulkner et al., 1999).

Another protein which interacts with titin (Z1 and Z2, Ig domains) in the Z-disc is telethonin (Mues et al., 1998), also called T-cap (Gregorio et al., 1998), which presumably plays a role in the anchoring of the N-terminus of titin.

Functional connections beyond the contractile unit, laterally to the sarcomere or longitudinally to the neighbouring sarcomere, are transmitted via protein networks at the costameres and the intercalated discs, respectively, which are linked to the Z-disc (Miller et al., 2004).

1.3.2 I-band and elasticity of the sarcomere

The extensibility of the sarcomere is physiologically important for muscle con-traction. Therefore, an elastic connection between the thick and thin filaments is required, and this task is maintained by titin (Horowits et al., 1986). Together with the passive tension, a force that restores the sarcomere to its slack length, titin has been assigned as ’molecular spring’ with the basis in the I-band of titin.

6 Introduction

The I-band titin is composed of different segments: a constitutive I-band region consisting of tandem Ig domains, the N2-region, and the PEVK region, which is rich in proline (P), glutamic acid (E), valine (V) and lysine (K). The PEVK region itself comprises of pattern of the PPAK motifs (pI about 10) and polyE segments (pI about 3-4) which despite large differences, results in a pI of about 5 (Greaser, 2001). Tissue-specific alternative splicing leads to the large size differences in the titin isoforms, predominantly caused by a differing amount of tandem Ig domains in the non-constitutive region of up to 53 extra Ig domains in the skeletal muscle (Freiburg et al., 2000). Furthermore, the PEVK region in the stiffer cardiac muscle is much shorter (163 residues) than in the skele-tal muscle (up to 2174 residues), implying importance in elasticity and passive tension. Differential splicing results in the N2A, the N2B and the combination N2BA isoform. The N2A isoform usually occurs in skeletal muscle, whereas the N2B and N2BA isoforms are found exclusively in cardiac muscle (Labeit and Kolmerer, 1995; Freiburg et al., 2000). Coexpression of the cardiac N2A and N2BA isoforms at different ratios have been described to extend independently in the half-sarcomere (Trombit´as et al., 2001).

The components of the I-band act as a molecular spring (Labeit and Kolmerer, 1995; Linke and Granzier, 1998; Granzier and Labeit, 2002) gen-erating passive tension during extension. Extension beyond the resting length entails first in a straightening of the tilted tandem Ig domains, followed by an unfolding of the PEVK sequence and N2 regions. The PEVK region has long been assumed to present a coiled unstructured conformation. However, some evidence of a preferred conformation of repeats of a 28-residue motif within the PEVK, which adapt a polyproline II helix, has been provided (Greaser, 2001;

Ma et al., 2001).

1.3.3 A-band and the thick filament

In the A-band, titin is involved in the control of the thick filament assembly and its centering in the A-band (Whiting et al., 1989). Hence, it has been ascribed to act as a template in the sarcomere assembly. The A-band constitutes the largest part of titin with almost 2 MDa and a length of approximately 0.8µm (Labeit and Kolmerer, 1995). Notably, this is the only part in titin, where the FnIII domains are found. The FnIII domains are embedded in two types of super-repeats together with the Ig-like domains (Gautel et al., 1996; Gautel, 1996).

In the D-zone (Figure 1.2) the super-repeat is composed of 5 FnIII and 2 Ig-like domains with a pattern of Ig-FnIII-FnIII-Ig-FnIII-FnIII-FnIII-FnIII. This pattern is repeated seven times (Gautel, 1996; Gregorio et al., 1999).

In the C-zone (Figure 1.2) of the A-band (Labeit and Kolmerer, 1995), how-ever, the eleven times repeated pattern Ig-FnIII-FnIII-Ig-FnIII-FnIII-FnIII-Ig-FnIII-FnIII-FnIII consists of 11 domains, seven FnIII and four Ig-like domains, resulting in a length of 43 nm as evidenced by electron microscopy (EM) and immunofluorenscence. The myosin binding proteins (MyBP) as ’accessory’ pro-teins of myosin show eleven repeats with a periodicity of about 43 nm (F¨urst

1.4 Modules in titin: Ig and FnIII domains 7

et al., 1989; Trinick, 1996). The correlation with titin A-band super-repeats suggests a role as a scaffold involved in arrangement of the thick filaments and definition of their length (Trinick, 1996). Therefore, titin was attributed as a ’molecular ruler’ or template in the sarcomere (Whiting et al., 1989). The interaction of titin and MyBP-C is directed by titin Ig domains (Freiburg and Gautel, 1996). The P-zone (Figure 1.2) comprises of a unique patch of Ig-Ig-FnIII-FnIII-Ig-Ig-FnIII and a kinase domain. Together with the M-line region of titin, the D-zone, C-zone, P-zone reflect the zones of myosin in the A-band.

1.3.4 M-line and anchoring function

The M-line region corresponds to the carboxy-terminal part of titin with a kinase domain at its periphery to the A-band (Obermann et al., 1996). Besides, the M-line titin consists predominantly of Ig-like domains interspersed with unique sequences such as the KSP-repeats between Ig modules M5 and M6, phosphorylated by titin KSP kinase during muscle differentiation (Gautel et al., 1993).

Comparable to the anchoring of titin in the Z-disc, the titin filaments also overlap in the M-line with those titin filaments from opposite sarcomeres mann et al., 1997). The M-line Ig domain M5 interacts with myomesin (Ober-mann et al., 1997), a protein mainly comprised of Ig-like and FnIII domains.

Myomesin has been suggested to crosslink myosin, as an antiparallel dimer, with titin by binding to myosin with its N-terminal domain. Furthermore, myosin was shown to interact with its central part with two antiparallel over-lapping titin molecules (Lange et al., 2005b; Agarkova and Perriard, 2005).

An additional M-band component, the M-protein, is related with myomesin in domain composition of Ig-like and FnIII-domains. In contrast to myomesin, it is not equally expressed in different muscle types (Agarkova et al., 2003). M-protein as well as myomesin bind to myosin filaments (Bahler et al., 1985) and to titin (Nave et al., 1989) in isolated muscle. The M-protein bridges myosin filaments at the M-line (Obermann et al., 1996). The recent three-dimensional model of the M-band network presents the arrangement of titin, myosin and myomesin within the M-band (Lange et al., 2005b).

Expression of a truncated titin devoid of the kinase domain and the MURF-1 binding sites, resulted in sarcomere disassembly (Gotthardt et al., 2003). In-terestingly, in the truncated M-line titin, expression of CARP (cardiac ankyrin repeat protein) and ankrd2, a CARP-like protein, are upregulated. Both have been implicated in linking between the sarcomere and the nucleus (Miller et al., 2003; Kojic et al., 2004).

1.4 Modules in titin: Ig and FnIII domains

The Ig-like domains and the FnIII domains appear like beads on a string in the electron micrograph images of the sarcomere (Trinick et al., 1984). The number of Ig- and FnIII domains varies from 244 to 297 (Witt et al., 1998),

8 Introduction

due to alternative splicing in different muscle tissue. The Ig-like domains make up about 112 to 166 of the titin domains.

According to differences in sequence similarities and a resulting variable num-ber and length of the strands in Ig domain structures, Ig domains are classified in the V-(variable), C1-, C2-(constant) and I-(intermediate) set. It is still un-clear, whether the Ig domains evolved by convergence to a stable fold, or by divergence from one ancestral domain (Williams and Barclay, 1988; Bork et al., 1994; Kenny et al., 1999). Assuming a common ancestor within the Ig domain sets, diverse options of the primordial domain have been discussed (Williams and Barclay, 1988; Hunkapiller and Hood, 1989; Smith and Xue, 1997).

The I-set of Ig domain was introduced based on sequence comparisons and the structure of telokin, a muscle member of the immunoglobulin superfam-ily (Harpaz and Chothia, 1994). Accordingly, Ig domains of sarcomeric pro-teins were predicted to belong to the I-set (Kenny et al., 1999; Improta et al., 1996). The first structure of a titin Ig domain, M5 of the M-band, confirmed its classification as an I-set Ig domain (Pfuhl and Pastore, 1995). Although the sequence identity of the overall Ig domain is about 20-35 % and the simi-larity about 50 % (Fraternali and Pastore, 1999), some subgroups with a high similarity of about 90 % exist (Tskhovrebova and Trinick, 2004). Among the different Ig-like domains of titin, there are groups of closer related domains. In the I-band, where the Ig-like domains are arranged mainly in tandem domains,

”proximal” and ”distal” to the PEVK segment differences in their consensus sequence exist. In the A-band, the FnIII and Ig-like domains are assembled in the 7- and 11-domain super-repeats, repeated six and eleven times, respec-tively (Gautel, 1996), in which domains at similar positions have higher se-quence homology (Amodeo et al., 2001a; Muhle-Goll et al., 1998; Fraternali and Pastore, 1999).

In titin, FnIII domains are exclusively found in the A-band arranged in the super-repeats (Tskhovrebova and Trinick, 2004). Similarly to Ig domains, the FnIII domains in titin are less than 50 % conserved within the family. The atomic structures of FnIII and Ig domains are similar; both share a greek-key superfold with a two β-sheet sandwich structure. Of the seven strands in the FnIII domains, strands A, B, E compose one β-sheet and strands G, F, C and C’ the other. Based on the structural similarity of the overall fold with the different types of Ig domains, these domains were all joint in the immunoglobulin fold family IgFF, including the FnIII domains (Halaby et al., 1999). Superimposition of Ig and FnIII domains reveals that strand C’ is on two differentβ-sheets on the two domain types (Erickson, 1994). Despite their overall structural similarity, the consensus sequence of the Ig and FnIII domains is different. The structure of the first intracellular FnIII domain, A71 of the titin A-band, confirmed the characteristic FnIII fold (Muhle-Goll et al., 1998).

1.5 Titin kinase 9

1.5 Titin kinase

The catalytic domain of titin, a serine/threonine kinase termed titin kinase, is located in the transition between the A-band and the M-line of titin (Labeit et al., 1992). In general protein kinases are involved in diverse, important bio-logical functions where they control the reversible process of phosporylation.

The reaction which they catalyse is the transfer of the γ-phosphate of ATP to the hydroxyl group of a serine, threonine, or tyrosine within the substrate protein. Phosphorylation can evoke distinct responses, which are comparable to molecular switches, where an on or off signal results in a reverse molec-ular reaction such as conversion of enzyme activation to inhibition (Johnson and O’Reilly, 1996). The class of serine/threonine kinases is a well conserved superfamily which is reflected both in the sequence similarity and in the over-all structural fold of the activated kinase owing to chemical constraints of the catalysed reaction (Huse and Kuriyan, 2002). The kinases can exhibit confor-mational differences between the active or ’on’ state and the inactive, also called

’off’ state. The regulation mechanism of the kinases which can be determined by interaction with specific regulatory domains or proteins, e.g. in response to second messengers (Johnson et al., 1996), generates a conformational plasticity of the inactive kinases (Huse and Kuriyan, 2002).

Comparison with other kinases and biochemical assays (Mayans et al., 1998) revealed an assignment of titin kinase to the subfamily of Ca²⁺/calmodulin (cal-cium modulating protein, abbreviated as CaM) regulated kinases. Regulation of the activation of the kinase by binding of Ca²⁺/CaM or the related protein S100 is typical for kinases like the CaMK family (Ca²⁺-calmodulin-dependent protein kinase (CaMK) family), the MLCK (myosin light chain kinase), and the twitchin kinase, another muscle kinase but found in invertebrate smooth muscle. In absence of calmodulin, the substrate binding site is blocked by an intrinsic autoinhibition mechanism (Kobe et al., 1996). Calmodulin is an ubiquitously expressed, highly conserved protein with four binding sites for cal-cium. Two EF-hands at each end which are connected by a short linker can each bind a calcium ion. Upon calcium binding, calmodulin undergoes a confor-mational change resulting in a dumbbell-shaped structure. When calmodulin binds to a synthetic calmodulin recognition peptide (Vetter and Leclerc, 2003;

Hoeflich and Ikura, 2002) which covers the calmodulin binding site of for ex-ample, titin kinase (Amodeo et al., 2001b), smMLCK (Meador et al., 1992), CaMKIIα (Meador et al., 1993), or CaMKK (Kurokawa et al., 2001), it forms a globular structure wrapped around the substrate helix by bending the cen-tral linker. However, in complex with a Ca²⁺-pump (Elshorst et al., 1999), a K⁺-channel (Schumacher et al., 2001) and the anthrax adenylate cyclase exo-toxin (Drum et al., 2002) calmodulin shows an extended conformation.

Structurally, protein kinases present the typical bilobal fold, with a smaller N lobe consisting of a five-stranded β-sheet and one prominent α-helix αC, and a larger predominately helical C lobe. The ATP binding site is situated in the cleft between the two lobes beneath the conserved phosphate binding P

10 Introduction

Figure 1.3: The ribbon presentation of titin kinase (Mayans et al., 1998) illus-trates the typical protein kinase fold of a bilobal structure. The regulatory tail in the C-terminal lobe is shown in red (with the helices αR1 and αR2 and the strand βR1), the catalytic loop in violet and the activation segment in green which is including the P+1 loop in yellow. The catalytic residue D127 and residue Y170, which is phosphorylated in the activation process, are displayed in grey.

loop (GXGXΦG with Φ=Y or F) connecting β1 and β2 of the N lobe. The substrate binds to the activation loop or activation segment. In many kinases, the activation segment needs to be activated by phosphorylation. In the C lobe, the catalytic loop with the catalytic site, an invariant aspartate, is found.

Kinases that are regulated by phosphorylation in the activation segment are all so-called RD-kinases, in which the catalytic aspartate is preceded by an arginine residue (Johnson et al., 1996). Titin kinase, however, with a phenylalanine instead of the arginine, was the first described non-RD kinase which is activated by phosphorylation (Mayans et al., 1998).

Studies on titin kinase revealed that both phosphorylation of tyrosine 170 (Y170) in the P+1 loop and the binding of Ca²⁺/CaM are required to acti-vate the kinase (Mayans et al., 1998). The structure of titin’s kinase domain

1.6 Titin kinase signalling pathway 11

has been solved (Mayans et al., 1998) and it revealed an autoinhibited

has been solved (Mayans et al., 1998) and it revealed an autoinhibited

Im Dokument Structural insight into the environment of the serine/threonine protein kinase domain of titin (Seite 17-0)