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1.
A. S. Kostyukova 《Biochemistry. Biokhimii?a》2008,73(13):1467-1472
Actin filaments are polar; their barbed (fast-growing) and pointed (slow-growing) ends differ in structure and dynamic properties. The slow-growing end is regulated by tropomodulins, a family of capping proteins that require tropomyosins for optimal function. There are four tropomodulin isoforms; their distributions vary depending on tissue type and change during development. The C-terminal half of tropomodulin contains one compact domain represented by alternating α-helices and β-structures. The tropomyosin-independent actin-capping site is located at the C-terminus. The N-terminal half has no regular structure; however, it contains a tropomyosin-dependent actin-capping site and two tropomyosin-binding sites. One tropomodulin molecule can bind two tropomyosin molecules. Effectiveness of tropomodulin binding to tropomyosin depends on the tropomyosin isoform. Regulation of tropomodulin binding at the pointed end as well as capping effectiveness in the presence of specific tropomyosins may affect formation of local cytoskeleton and dynamics of actin filaments in cells. 相似文献
2.
《The Journal of cell biology》1994,127(6):1627-1635
Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells. 相似文献
3.
Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle 总被引:10,自引:5,他引:5
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《The Journal of cell biology》1993,120(2):411-420
The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length. 相似文献
4.
S Kimura A Ichikawa J Ishizuka S Ohkouchi T Kake K Maruyama 《European journal of biochemistry》1999,263(2):396-401
Tropomodulin is a tropomyosin-binding protein, originally isolated from human erythrocytes. Tropomodulin is currently regarded as the sole actin pointed-end capping protein [Weber, A., Pennise, C.R., Babcock, G.G. & Fowler, V.M. (1994) J. Cell Biol. 127, 1627-1635]. This work first describes a procedure for the purification of tropomodulin from rabbit skeletal muscle. Tropomodulin almost completely inhibited filament formation of actin in the presence of tropomyosin and troponin. For the maximal inhibition of actin polymerization, approximately 0.10, 0.12 and 0.003 mol of tropomyosin, troponin and tropomodulin per mol of actin were required, respectively. Fluorescence-intensity measurements, electron-microscopy and sedimentation experiments revealed that only very short fragments and amorphous aggregates, but not filaments, were formed when actin was copolymerized with tropomyosin, troponin and tropomodulin by the addition of 50 mM KCl at pH 8.0. The effects of tropomyosin, troponin and tropomodulin were more remarkable on Ca-actin than on Mg-actin. It appears that tropomodulin caps both the pointed and barbed ends of tropomyosin- and troponin-bound actin filaments. 相似文献
5.
Crystal structure of the C-terminal half of tropomodulin and structural basis of actin filament pointed-end capping
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Tropomodulin is the unique pointed-end capping protein of the actin-tropomyosin filament. By blocking elongation and depolymerization, tropomodulin regulates the architecture and the dynamics of the filament. Here we report the crystal structure at 1.45-A resolution of the C-terminal half of tropomodulin (C20), the actin-binding moiety of tropomodulin. C20 is a leucine-rich repeat domain, and this is the first actin-associated protein with a leucine-rich repeat. Binding assays suggested that C20 also interacts with the N-terminal fragment, M1-M2-M3, of nebulin. Based on the crystal structure, we propose a model for C20 docking to the actin subunit at the pointed end. Although speculative, the model is consistent with the idea that a tropomodulin molecule competes with an actin subunit for a pointed end. The model also suggests that interactions with tropomyosin, actin, and nebulin are all possible sources of influences on the dynamic properties of pointed-end capping by tropomodulin. 相似文献
6.
Tropomodulin 1 (Tmod1) is a approximately 40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (Kd approximately 0.4 microM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (Kd approximately 1.8 microM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity. 相似文献
7.
Structural requirements of tropomodulin for tropomyosin binding and actin filament capping 总被引:2,自引:0,他引:2
Kostyukova AS Rapp BA Choy A Greenfield NJ Hitchcock-DeGregori SE 《Biochemistry》2005,44(12):4905-4910
Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament. 相似文献
8.
Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin 总被引:11,自引:6,他引:5
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V M Fowler 《The Journal of cell biology》1990,111(2):471-481
Human erythrocytes contain a Mr 43,000 tropomyosin-binding protein that is unrelated to actin and that has been proposed to play a role in modulating the association of tropomyosin with spectrin-actin complexes based on its stoichiometry in the membrane skeleton of one Mr 43,000 monomer per short actin filament (Fowler, V. M. 1987. J. Biol. Chem. 262:12792-12800). Here, we describe an improved procedure to purify milligram quantities to 98% homogeneity and we show that this protein inhibits tropomyosin binding to actin by a novel mechanism. We have named this protein tropomodulin. Unlike other proteins that inhibit tropomyosin-actin interactions, tropomodulin itself does not bind to F-actin. EM of rotary-shadowed tropomodulin-tropomyosin complexes reveal that tropomodulin (14.5 +/- 2.4 nm [SD] in diameter) binds to one of the ends of the rod-like tropomyosin molecules (33 nm long). In agreement with this observation, Dixon plots of inhibition curves demonstrate that tropomodulin is a non-competitive inhibitor of tropomyosin binding to F-actin (Ki = 0.7 microM). Hill plots of the binding of the tropomodulin-tropomyosin complex to actin indicate that binding does not exhibit any positive cooperativity (n = 0.9), in contrast to tropomyosin (n = 1.9), and that the apparent affinity of the complex for actin is reduced 20-fold with respect to that of tropomyosin. These results suggest that binding of tropomodulin to tropomyosin may block the ability of tropomyosin to self-associate in a head-to-tail fashion along the actin filament, thereby weakening its binding to actin. Antibodies to tropomodulin cross-react strongly with striated muscle troponin I (but not with troponin T) as well as with a nontroponin Mr 43,000 polypeptide in muscle and in other nonerythroid cells and tissues, including brain, lens, neutrophils, and endothelial cells. Thus, erythrocyte tropomodulin may be one member of a family of tropomyosin-binding proteins that function to regulate tropomyosin-actin interactions in non-muscle cells and tissues. 相似文献
9.
Structure and tropomyosin binding properties of the N-terminal capping domain of tropomodulin 1
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Two families of actin regulatory proteins are the tropomodulins and tropomyosins. Tropomodulin binds to tropomyosin (TM) and to the pointed end of actin filaments and "caps" the pointed end (i.e., inhibits its polymerization and depolymerization). Tropomodulin 1 has two distinct actin-capping regions: a folded C-terminal domain (residues 160-359), which does not bind to TM, and a conserved, N-terminal region, within residues 1-92 that binds TM and requires TM for capping activity. NMR and circular dichroism were used to determine the structure of a peptide containing residues 1-92 of tropomodulin (Tmod1(1-92)) and to define its TM binding site. Tmod1(1-92) is mainly disordered with only one helical region, residues 24-35. This helix forms part of the TM binding domain, residues 1-35, which become more ordered upon binding a peptide containing the N-terminus of an alpha-TM. Mutation of L27 to E or G in the Tmod helix reduces TM affinity. Residues 49-92 are required for capping but do not bind TM. Of these, residues 67-75 have the sequence of an amphipathic helix, but are not helical. Residues 55-62 and 76-92 display negative 1H-15N heteronuclear Overhauser enhancements showing they are flexible. The conformational dynamics of these residues may be important for actin capping activity. 相似文献
10.
Tropomodulin is a human erythrocyte membrane cytoskeletal protein that binds to one end of tropomyosin molecules and inhibits tropomyosin binding to actin filaments [Fowler, V. M. (1990) J. Cell Biol. 111, 471-482]. We have characterized the interaction of erythroid and non-erythroid tropomyosins with tropomodulin by non-denaturing gel electrophoresis and by solid-phase binding assays using 125I-tropomyosin. Non-denaturing gel analysis demonstrates that all tropomodulin molecules are able to bind tropomyosin and that tropomodulin forms complexes with tropomyosin isoforms from erythrocyte, brain, platelet and skeletal muscle tissue. Scatchard analysis of binding data using tropomyosin isoforms from these tissues indicate that tropomodulin binds preferentially to erythrocyte tropomyosin. Specificity is manifested by decreases in the apparent affinity or the saturation binding capacity of tropomodulin for non-erythrocyte tropomyosins. Erythrocyte tropomyosin saturates tropomodulin at approximate stoichiometric ratios of 1:2 and 1:4 tropomyosin/tropomodulin (apparent Kd = 14 nM-1 and 5 nM-1, respectively). Brain tropomyosin saturates tropomodulin at a 1:2 ratio of tropomyosin/tropomodulin, but with a threefold lower affinity than erythrocyte tropomyosin. Platelet tropomyosin saturates tropomodulin at a tropomyosin/tropomodulin ratio of 1:4, but with a sevenfold lower affinity than erythrocyte tropomyosin at the 1:4 ratio. These results correlate with oxidative cross-linking data which indicate that tropomodulin can self-associate to form dimers and tetramers in solution. Since tropomodulin interacts with one of the ends of tropomyosin, varying interactions of tropomyosin isoforms with tropomodulin probably reflect the heterogeneity in N-terminal or C-terminal sequences characteristic of the different tropomyosin isoforms. Isoform-specific interactions of tropomodulin with tropomyosins may represent a novel mechanism for selective regulation of tropomyosin/actin interactions. 相似文献
11.
Tsukada T Kotlyanskaya L Huynh R Desai B Novak SM Kajava AV Gregorio CC Kostyukova AS 《The Journal of biological chemistry》2011,286(3):2194-2204
Tropomodulin is a tropomyosin-dependent actin filament capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent actin-capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent actin-capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filament pointed ends, with site 2 acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the C-terminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent actin-capping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain. Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent actin-capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain. 相似文献
12.
The pointed end capping protein, tropomodulin, increases the critical concentration of barbed end capped actin, i.e. it lowers the apparent affinity of pointed ends for actin monomers. We show here that this is due to the conversion of pointed end ADP. P(i)-actin (low critical concentration) to ADP-actin (high critical concentration) when 70-98% of the ends are capped by tropomodulin. We propose that this is due to the low affinity of tropomodulin for pointed ends (K(d) approximately 0.3 microM), which allows tropomodulin to rapidly exchange binding sites and transiently block access of actin monomers to all pointed ends. This leaves time for ATP hydrolysis and phosphate release to go to completion between successive monomer additions to the pointed end. When the affinity of tropomodulin for pointed ends was increased about 1000-fold by the presence of tropomyosin (K(d) < 0.05 nM), capping of 95% of the ends by tropomodulin did not alter the critical concentration. However, the critical concentration did increase when the tropomodulin concentration was raised to the high values effective in the absence of tropomyosin. This may reflect transient tropomodulin binding to tropomyosin-free actin molecules at the pointed ends of the tropomyosin-actin filaments without a high affinity tropomodulin cap, i.e. the ends that determine the value of the actin critical concentration. 相似文献
13.
《European journal of cell biology》2022,101(2):151215
Tropomodulin and tropomyosin are important components of sarcomeric thin filaments in striated muscles. Tropomyosin decorates the side of actin filaments and enhances tropomodulin capping at the pointed ends of the filaments. Their functional relationship has been extensively characterized in vitro, but in vivo and cellular studies in mammals are often complicated by the presence of functionally redundant isoforms. Here, we used the nematode Caenorhabditis elegans, which has a relatively simple composition of tropomodulin and tropomyosin genes, and demonstrated that tropomodulin (unc-94) and tropomyosin (lev-11) are mutually dependent on each other in their sarcomere localization and regulation of sarcomeric actin assembly. Mutation of tropomodulin caused sarcomere disorganization with formation of actin aggregates. However, the actin aggregation was suppressed when tropomyosin was depleted in the tropomodulin mutant. Tropomyosin was mislocalized to the actin aggregates in the tropomodulin mutants, while sarcomere localization of tropomodulin was lost when tropomyosin was depleted. These results indicate that tropomodulin and tropomyosin are interdependent in the regulation of organized sarcomeric assembly of actin filaments in vivo. 相似文献
14.
Tropomodulin (Tmod) stabilizes the actin-tropomyosin filament by capping the slow-growing end (P-end). The N- and C-terminal halves play distinct roles; the N-terminal half interacts with the N-terminal region of tropomyosin, whereas the C-terminal half interacts with actin. Our previous study (A. Kostyukova, K. Maeda, E. Yamauchi, I. Krieger, and Y. Maéda Y., 2000, Eur. J. Biochem. 267:6470-6475) suggested that the two halves are also structurally distinct from each other. We have now studied the folding properties of the two halves, by circular dichroism spectroscopy and by differential scanning calorimetry of the expressed chicken E-type tropomodulin and its large fragments. The results showed that the C-terminal half represents a single, independently folded unit that melts cooperatively through a two-state transition. In contrast, the N-terminal half lacks a definite tertiary structure in solution. The binding of N11, a fragment that corresponds to the first 91 amino acids of the tropomodulin, to tropomyosin substantially stabilized the tropomyosin. This may indicate that the flexible structure of the N-terminal half of tropomodulin in solution is required for binding to tropomyosin and that the N-terminal half acquires its tertiary structure upon binding to tropomyosin. 相似文献
15.
Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly 总被引:5,自引:2,他引:3
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《The Journal of cell biology》1995,129(3):683-695
Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled. 相似文献
16.
Dmitri Tolkatchev Balaganesh Kuruba Garry E. Smith Jr Kyle D. Swain Kaitlin A. Smith Natalia Moroz Trenton J. Williams Alla S. Kostyukova 《Protein science : a publication of the Protein Society》2021,30(2):423
Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1‐Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three‐dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin‐binding sites of tropomodulin and generated a three‐dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self‐consistent three‐dimensional model of tropomodulin assembly at the pointed end. The model of the pointed‐end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics. 相似文献
17.
Fischer RS Yarmola EG Weber KL Speicher KD Speicher DW Bubb MR Fowler VM 《The Journal of biological chemistry》2006,281(47):36454-36465
Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms. 相似文献
18.
A Almenar-Queralt A Lee C A Conley L Ribas de Pouplana V M Fowler 《The Journal of biological chemistry》1999,274(40):28466-28475
Tropomodulin (E-Tmod) is an actin filament pointed end capping protein that maintains the length of the sarcomeric actin filaments in striated muscle. Here, we describe the identification and characterization of a novel tropomodulin isoform, skeletal tropomodulin (Sk-Tmod) from chickens. Sk-Tmod is 62% identical in amino acid sequence to the previously described chicken E-Tmod and is the product of a different gene. Sk-Tmod isoform sequences are highly conserved across vertebrates and constitute an independent group in the tropomodulin family. In vitro, chicken Sk-Tmod caps actin and tropomyosin-actin filament pointed ends to the same extent as does chicken E-Tmod. However, E- and Sk-Tmods differ in their tissue distribution; Sk-Tmod predominates in fast skeletal muscle fibers, lens, and erythrocytes, while E-Tmod is found in heart and slow skeletal muscle fibers. Additionally, their expression is developmentally regulated during chicken breast muscle differentiation with Sk-Tmod replacing E-Tmod after hatching. Finally, in skeletal muscle fibers that coexpress both Sk- and E-Tmod, they are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively. All together, these observations support the hypothesis that vertebrates have acquired different tropomodulin isoforms that play distinct roles in vivo. 相似文献
19.
Vera C Sood A Gao KM Yee LJ Lin JJ Sung LA 《Archives of biochemistry and biophysics》2000,378(1):16-24
Tropomodulin is a globular protein that caps the pointed end of actin filaments by complexing with the N-terminus of a tropomyosin (TM) molecule. TM consists of coiled coils except for the N-terminus, which may be globular. Here we report that human TM isoform 5 (hTM5) lacking the N-terminal 18 residues lost its binding activity toward tropomodulin. We further characterized the tropomodulin-binding site by creating a series of deletion and missense mutations within this region, followed by a solid-phase binding assay. I7, V10, and I14, hydrophobic residues located at the a and d positions of N-terminal heptad repeats involving intertwine, are essential for tropomodulin binding. R12, a positively charged residue at the f position, is also involved in recognition. In contrast, A2R and G3Y mutations, each creating a bulky N-terminus, did not alter the binding. In addition, rat TM5b, which differs from hTM5 in residues 4-6, exhibits a similar binding affinity. The tropomodulin-binding site, therefore, is mapped to residues 7-14 at the beginning of the long heptad repeats. Column chromatography revealed that hTM5 mutants remained capable of dimerization. Results also suggest tropomodulin has a groove-type, rather than a cavity-type, binding site for hTM5. We also mapped the epitope of monoclonal antibody LC1 to residues 4-10 of hTM5 and showed the competition between mAb LC1 and tropomodulin in hTM5 binding. Since the N-terminal residues need to overlap with the C-terminus of TM in their head-to-tail association, this investigation elucidates the mechanisms by which the tropomodulin-hTM5 complex is formed and functions in regulating the actin filaments. 相似文献
20.
Tropomodulin, the P-end (slow-growing end)-capping protein of the actin-tropomyosin filament, and its fragment (C20) of the C-terminal half were studied by synchrotron small-angle X-ray scattering, restoring low-resolution shapes using an ab initio shape-determining procedure. Tropomodulin is elongated (115 A long) and consists of two domains, one of 65 A in length and the other being similar to C20 in shape and size if the long axes of the two are tilted by about 40 degrees relative to each other. We propose a model for tropomodulin in association with tropomyosin and actin: the N-terminal half of tropomodulin, a rod, binds to the N-terminus of tropomyosin and the C-terminal triangle domain protrudes from the P-end being slightly bent towards the actin subunit at the end, thereby blocking the P-end. 相似文献