Group:MUZIC:Obscurin
From Proteopedia
Obscurin
IntroductionObscurin is a giant protein expressed in striated muscle cells. It`s one of the three giant scaffold proteins of the striated muscles (the others are titin and nebulin [1] the encoding gene is localized on chromosome 1 (1q42) and the protein product is about 800 kDa with multiple splice variants of different sizes. The protein architecture resembles the modular organization of titin and is composed of multiple Ig-like domains and Fibronectin-like domains arranged in tandem, with most of them encoded on single exons [2]. The obscurin gene (OBSCN) encodes for at least two splice variants, obscurin A and obscurin B[3] the common part of the two variants is composed of up to 67 Ig-like and fibronectin type III domains (Fn3). The COOH-terminal of the protein contains a calmodulin-binding IQ motif, an SH3 domain and a Rho guanine nucleotide exchange factor (GEF) domain followed by a non-modular sequence with multiple phosphorylation sites at the COOH-terminus. In the obscurin B variant, the skipping of exons 90 and 91 links the Ob67 domain to kinase exons leading to the formation of longer variants with two additional Ser/Thr kinase domains which can also be expressed separately from an internal promoter [4] [5]. As scaffold protein of the sarcomere obscurin has several binding partners, mechanical and signaling functions. Obscurin seems to be involved in the correct organization of myofibrils localized in different regions of the sarcomere at different stages of myofibrillogenesis, starting with the subsarcolemmal sarcomeres in early developing hearts [6]. In developing muscles, obscurin is predominantly found at the Z-disk. Later in development, several lines of evidence showed the predominant localization of the protein in the M-band of mature striated muscles. Both localizations seem predominantly determined by the interaction with titin [7]. The presence of the GEF domain at the COOH-terminus suggests a role as a linker between sarcomeric proteins and the G-protein regulated pathways in myofibrils. Moreover, an involvement of obscurin in the lateral alignment of myofibrils and the M-band associated Sarcoplasmic Reticulum (SR) has been suggested [8] due to the highly stable complex formed by the N-terminal domains of obscurin, the C-terminal M10 domain of titin and a linker region of myomesin, which drives the correct alignment of the M-band and SR [9] [10]. Immunofluorescence analysis demonstrated a peripheral localization with respect to the myofibrillar proteins, and therefore a transverse orientation of obscurin with respect to the filament axis, which can then form a link between the myofibril and the SR lipid membranes [11] [12]. Sequence annotationThe aminoacid sequences of human obscurin are deposited in the UniProt database (Q5VST9) and GenBank . The sequence provided in Figure 1 is based on the analysis of the human splice variants [13]. Based on these different splice pathways, at least 4 isoforms are predicted: 2 long isoforms, two shorter isoforms in the extremely C-terminal part are possible but currently uncharacterized. Cardiac obscurin schematic structure
Protein structureObscurin is a ~800kDa protein expressed in skeletal and cardiac muscle. Its modular structure resembles the architecture of titin, indeed the NH2-terminal half of the protein consists of 49 Ig-like domains (with the characteristic β-sandwich shape) and 2 Fn3 domains, each one with a length of 88-92 amino acids residues and no linker sequences between subsequent domains (except between Ob2-Ob3 and Ob24-Ob25). Two clusters of these Ig domains show 70-90% homology at the protein level; these are Ob36-42 and Ob9-18. As in other sarcomeric proteins, the Ig domains present a highly stable structural scaffold that is necessary for mechanical stability as well as versatile surface that fits well with the role of binding site for other proteins [15]. The COOH-terminus shows a less ordered architecture with a non-modular 417 amino acid sequence with several consensus phosphorylation motifs for ERK-kinase (SPXR) [16]. A PH domain is located upstream of the last tandem Ig domains Ob56-57, preceding the DH/RhoGEF domain. After the Ob51 Ig domain a non-modular structure includes an IQ domain, so called for the conserved Ile-Gln that is part of a well-characterized binding motif for calmodulin or calmodulin-like protein like myosin light chains. In the isoform obscurin B, an additional COOH-terminal part includes two more Ser/Thr Kinase domains, 2 Ig like domains and an Fn3 domain [17]. This isoform does not include the extremely COOH-terminal non-modular structure that characterizes the isoform A and contains the Ank1.5 binding site. PDB entries for solved obscurin domain structures1v1c - Solution structure os SH3 domain of obscurin, residues 5601-5668 2cr6 – Solution structure of the Ig domain (2998-3100) of human obscurin 2dku – Solution structure of the third Ig-like domain of human KIAA1556 protein 2dm7 – Solution structure of the 14th Ig-like domain of human KIAA1556 protein 2e7b – Solution structure of the 6th Ig-like domain from human KIAA1556 2gqh – Solution structure of the 15th Ig-like domain of human KIAA1556 protein 2edf – Solution structure of the second Ig-like domain (2826-2915) from human obscurin 2edh – Solution structure of the PDz domain (3614-3713) from human obscurin 2edl – Solution structure of the Ig-like domain (3801-3897) of human obscurin 2edq – Solution structure of the Ig-like domain (3713-3806) of human obscurin 2edr – Solution structure of the Ig-like domain (3361-3449) of human obscurin 2edw – Solution structure of the I-set domain (3537-3630) of human obscurin 2eny – Solution structure of the Ig-like domain (2735-2825) of human obscurin 2eo1 – Solution structure of the Ig-like domain of human OBSCN protein 2yz8 – Crystal structure of the 32th Ig-like domain of human obscurin (KIAA1556) Function and interactionsObscurin binding partners in the sarcomereThe main feature of the giant scaffold proteins of the sarcomere is to be involved in several interactions with the other proteins that form the muscle cytoskeleton, these networks are essential to obtain the mechanical stability and strength resistance required in muscle contractions. The first documented interaction of obscurin was detected in the Z-disk, between Titin Ig domains Z9/Z10 and obscurin Ig domains Ob 58/59 [18] (in agreement with the new nomenclature proposed by Fukuzawa et al. [19]); this observation seemed in contrast with the prevalent M-band localization in adult muscle and the predicted maximum extension achieved by the complete elongation of the protein, which is not sufficient to cover the distance between Z-disk and M-band. Despite that, the obscurin-titin complex in the Z-disk is formed during myofibrillogenesis and may be one of the key events for the correct assembly of the thin filaments. The same binding site is able to interact with the Ig-21 domain of the smaller titin isoform Novex-3 [20]; this protein span from the Z-disk to the I-band and seems to form a dynamic complex with obscurin that moves out of the Z-disk with the increasing length of the sarcomere. In adult myocytes obscurin has a prominent localization at the M-band [21]; the three extremely NH2-terminal Ig domain of obscurin binds the titin COOH-terminal domain M10 while Ob-Ig3 interact with the linker region between myomesin Fn3 domains My4 and My5 [22]. The correct targeting of obscurin at the M-band seems to depend on these interactions, in agreement with the structurally stabilizing role of the entire M-bands complex. The interactions with different isoforms of membrane-associated proteins ankyrin (Ank1.5, Ank1.9, Ank2.2) suggest a key role for obscurin in the linkage of the SR to the myofibrils cytoskeleton [23] [24]; the correct alignment of these crucial structures in myocytes has been an intriguing challenge, and obscurin seems to play a pivotal role [25]. Showing the direct binding of integral membrane proteins with a sarcomeric component, a bridge has been provided for the linkage between lipid membranes and mechanical apparatus during myofibrillogenesis. Obscurin is the major ligand of the cytoplasmic domain of Ank1.5 although different ankyrin proteins are able to bind with different affinities to the C-terminal region of obscurin. Ank1.5 shows the strongest obscurin binding capacity with an affinity of 130 nM, however controversial data on the binding region due to the presence of multiple ankyrin binding sites on obscurin are discussed [26] [27]. The obscurin GEF domain is one of the most intriguing features of this protein; GEF proteins have the ability to activate small GTPases involved in strictly regulated signaling pathways, suggesting an obscurin role as a signal transduction protein. This domain can bind the protein RanBP9 that has also been suggested to interact with two NH2-terminal domains of titin [28]. This interaction would take place in the Z-disk, and although there is no clear evidence, the interaction between titin and obscurin might be involved in the correct integration and maintenance of the Z-disk during myofibrillogenesis. This suggested role is in contrast, however, to the apparently normal myofibrillogenesis in obscurin KO mice (see below). The PH domain can also have a role in the GTP pathway regulation; this domain is able to bind the mammalian protein RhoA in tandem with DH domain. This interaction has been proposed to take place in the M-band to activate the effector Rho kinase (ROCKs) [29]; this is not the only binding partner of the obscurin GEF domain, indeed it seems also to interact with the small GTPase TC10/RhoQ [30]. Obscurin knockout mice and proposed physiological rolesIn order to fully investigate the role of obscurin in vivo, knockout mice for the OBSCN gene have been generated [31]. Despite the intriguing expression pattern of this protein during myofibrillogenesis, with the protein localizing to both Z-disks and M-bands, the lack of obscurin doesn’t affect correct Z-disk or M-band organization. This might be due to the redundant function between the N-terminal Ig domain of obscurin and obscurin like-1 [32]. On the other hand, Kontrogianni-Kostantopoulos et al. proposed an essential role for obscurin not only in the SR anchorage to the sarcomere periphery via the interaction with ankyrin protein family [33], but also in the M-band organization and the myosin filament assembly in the A-band region [34]. PathologyTo date, the involvement if obscurin in myopathies has been reported in hereditary cardiac and skeletal muscle myopathies in human [35] [36], even though the knock-out mice failed to show typical signs of cardiomyophaties. Again, this might be because the myopathies affect only the obscurin binding sites on titin, but not those for myomesin, possibly retaining some anchorage and function. However, misaligned obscurin was shown in two hereditary titinopathies, LGMD2J and Salih myopathy [37]. No myopathies have so far been identified as being caused by mutations in the obscurin gene. References
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Andrea Ghisleni 17:09, 23 November 2012 (IST)
