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William W. Kay - One of the best experts on this subject based on the ideXlab platform.
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Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.
Journal of molecular biology, 2000Co-Authors: Aaron White, S.karen Collinson, Pamela A. Banser, Daphne J Dolhaine, William W. KayAbstract:Abstract Two distinct Salmonella Fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an α-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA Fimbrins amenable to replacement with this epitope, PCR-generated chimeric Fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric Fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10 % of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75 % of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.
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Thin aggregative fimbriae from diarrheagenic Escherichia coli.
Journal of bacteriology, 1992Co-Authors: S K Collinson, L Emödy, Trevor J. Trust, William W. KayAbstract:Abstract Four strains of diarrheagenic Escherichia coli originally isolated from distinct geographic regions were found to produce unusual thin aggregative fimbriae requiring depolymerization in formic acid prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy of native fimbriae and Western blot (immunoblot) analysis of the corresponding 18-kDa Fimbrins showed that these E. coli fimbriae were serologically cross-reactive with SEF 17 (Salmonella enteritidis fimbriae with a Fimbrin molecular mass of 17 kDa). The E. coli and S. enteritidis Fimbrins had similar total amino acid compositions and highly conserved N-terminal amino acid sequences. These results indicate that E. coli and S. enteritidis produce biochemically related, aggregative fimbriae which constitute a new type of intergenerically distributed fimbriae for which we propose the descriptive name GVVPQ fimbriae on the basis of the conserved N-terminal amino acid sequence.
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Type 1 fimbriae of Salmonella enteritidis.
Journal of bacteriology, 1991Co-Authors: K H Müller, S K Collinson, Trevor J. Trust, William W. KayAbstract:Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) Fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr Fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the Fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 Fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 Fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr Fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 Fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other Fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 Fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21. Images
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Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.
Journal of bacteriology, 1991Co-Authors: S K Collinson, L Emödy, Trevor J. Trust, K H Müller, William W. KayAbstract:Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of Fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their Fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa Fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective Fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis. Images
Paul Matsudaira - One of the best experts on this subject based on the ideXlab platform.
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Antibodies to T- and L-Isoforms of the Cytoskeletal Protein, Fimbrin, in Patients with Systemic Lupus Erythematosus
2016Co-Authors: E C De Mendonca Neto, Paul Matsudaira, Nancy A. Shadick, A M Michon, Roger B. Eaton, Priti Kumar, Ashok Kumar, Peter H. SchurAbstract:The cytoskeleton is a complex network of proteins that main-tain cell shape, mobility, and organelle function. Its compo-nents can be divided into three distinct classes: microfilaments, microtubules, and intermediate filaments. Fimbrins are micro-filament proteins, a family of cytoplasmic phosphoproteins. Expression of the L-Fimbrin isoform is restricted to replicating blood cells and expression of the T-Fimbrin isoform to replicat-ing cells of solid tissues. Sera from normals and from patients with systemic lupus erythematosus (SLE), juvenile arthritis, rheumatoid arthritis, Sjogren's syndrome, osteoarthritis, va-sculitis, scleroderma, and mixed connective tissue disease were tested for the presence of antibodies to T- and L-Fimbrin by ELISA, using purified recombinant Fimbrin. The mean OD of sera from SLE patients was significantly higher than in nor-mals (T-Fimbrin, P < 0.0001; L-Fimbrin, P < 0.001). 48 of 98 SLE sera had antibodies to T-Fimbrin; 32 had antibodies to L-Fimbrin; 20 had antibodies to both; 28 had only anti-T, and 12 had only anti-L-Fimbrin. The mean OD for sera of the other rheumatic diseases was not significantly different from nor-mals. The presence ofeither L- or T-Fimbrin antibody was asso-ciated with pleuropericarditis (P = 0.015), photosensitivity (P = 0.011), and anti-Sm antibody (P = 0.010). Central nervous system SLE was associated with the presence of the L-Fimbrin antibody alone (P = 0.016). There was a strong association between DR7 (but not other MHC alleles) and anti-L-Fimbrin antibodies in SLE patients (chi square = 18; P < 0.00002). No MHC association was observed with anti-T-Fimbrin antibodies
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Ciba Foundation Symposium 95 - Brush Border Membranes - Structural and functional relationship between the membrane and the cytoskeleton in brush border microvilli.
Ciba Foundation symposium, 2008Co-Authors: Paul MatsudairaAbstract:Electron microscopic and biochemical studies have described the organization and composition of microvilli from chicken intestinal brush borders. An actin-based cytoskeleton, composed of a paracrystalline core of bundled microfilaments, maintains the finger-like shape of the membrane through a helical array of membrane-microfilament linkages. Two proteins, Fimbrin and villin, are components of the core bundle in situ and can independently bundle the actin filaments in vitro. Structural studies comparing microvillar core bundles with villin bundles and Fimbrin bundles suggest that Fimbrin, and not villin, is the major actin-filament-bundling protein in the microvillus core. These points, together with the capability of villin to sever actin filaments when activated by Ca2+, raise questions about villin's function in the microvillus. One possible explanation is that villin induces vesiculation of the membrane by disassembling the underlying cytoskeleton.
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Macrophage podosomes assemble at the leading lamella by growth and fragmentation.
The Journal of cell biology, 2003Co-Authors: James G. Evans, Ivan Correia, Olga Krasavina, Nicki Watson, Paul MatsudairaAbstract:Podosomes are actin- and Fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with β-actin–ECFP and L-Fimbrin–EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, Fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.
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An atomic model of actin filaments cross-linked by Fimbrin and its implications for bundle assembly and function.
The Journal of cell biology, 2001Co-Authors: Niels Volkmann, Paul Matsudaira, David J. Derosier, Dorit HaneinAbstract:Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signaling. Fimbrin belongs to a large family of actin-bundling proteins and is involved in the formation of tightly ordered cross-linked bundles in the brush border microvilli and in the stereocilia of inner ear hair cells. Polymorphism in these three-dimensional (3D) bundles has prevented the detailed structural characterization required for in-depth understanding of their morphogenesis and function. Here, we describe the structural characterization of two-dimensional arrays of actin cross-linked with human T-Fimbrin. Structural information obtained by electron microscopy, x-ray crystallography, and homology modeling allowed us to build the first molecular model for the complete actin–Fimbrin cross-link. The restriction of the arrays to two dimensions allowed us to deduce the spatial relationship between the components, the mode of Fimbrin cross-linking, and the flexibility within the cross-link. The atomic model of the Fimbrin cross-link, the cross-linking rules deduced from the arrays, and the hexagonal packing of actin bundles in situ were all combined to generate an atomic model for 3D actin–Fimbrin bundles. Furthermore, the assembly of the actin–Fimbrin arrays suggests coupling between actin polymerization, Fimbrin binding, and crossbridge formation, presumably achieved by a feedback between conformational changes and changes in affinity.
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Integrating the Actin and Vimentin Cytoskeletons Adhesion-Dependent Formation of Fimbrin–Vimentin Complexes in Macrophages
The Journal of cell biology, 1999Co-Authors: Ivan Correia, Donald Chu, Ying Hao Chou, Robert D. Goldman, Paul MatsudairaAbstract:Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, Fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or Fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, Fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of Fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that Fimbrin interacts with a vimentin subunit. A Fimbrin-binding site was identified in the NH2-terminal domain of vimentin and the vimentin binding site at residues 143–188 in the CH1 domain of Fimbrin. Based on these observations, we propose that a Fimbrin–vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.
S K Collinson - One of the best experts on this subject based on the ideXlab platform.
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Periplasmic and fimbrial SefA from Salmonella enteritidis.
Biochimica et biophysica acta, 1998Co-Authors: S C Clouthier, S K Collinson, D Lippert, J Ausio, A P White, W W KayAbstract:Salmonella enteritidis produces thin, filamentous fimbriae composed of the Fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA Fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA Fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.
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Thin aggregative fimbriae from diarrheagenic Escherichia coli.
Journal of bacteriology, 1992Co-Authors: S K Collinson, L Emödy, Trevor J. Trust, William W. KayAbstract:Abstract Four strains of diarrheagenic Escherichia coli originally isolated from distinct geographic regions were found to produce unusual thin aggregative fimbriae requiring depolymerization in formic acid prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy of native fimbriae and Western blot (immunoblot) analysis of the corresponding 18-kDa Fimbrins showed that these E. coli fimbriae were serologically cross-reactive with SEF 17 (Salmonella enteritidis fimbriae with a Fimbrin molecular mass of 17 kDa). The E. coli and S. enteritidis Fimbrins had similar total amino acid compositions and highly conserved N-terminal amino acid sequences. These results indicate that E. coli and S. enteritidis produce biochemically related, aggregative fimbriae which constitute a new type of intergenerically distributed fimbriae for which we propose the descriptive name GVVPQ fimbriae on the basis of the conserved N-terminal amino acid sequence.
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Type 1 fimbriae of Salmonella enteritidis.
Journal of bacteriology, 1991Co-Authors: K H Müller, S K Collinson, Trevor J. Trust, William W. KayAbstract:Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) Fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr Fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the Fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 Fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 Fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr Fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 Fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other Fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 Fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21. Images
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Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.
Journal of bacteriology, 1991Co-Authors: S K Collinson, L Emödy, Trevor J. Trust, K H Müller, William W. KayAbstract:Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of Fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their Fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa Fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective Fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis. Images
Shinichi Kohsaka - One of the best experts on this subject based on the ideXlab platform.
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microglia macrophage specific protein iba1 binds to Fimbrin and enhances its actin bundling activity
Journal of Neurochemistry, 2004Co-Authors: Keiko Ohsawa, Yoshinori Imai, Yo Sasaki, Shinichi KohsakaAbstract:Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage-specific calcium-binding protein. Iba1 has the actin-bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1-related intracellular signalling pathway in greater detail, we employed a yeast two-hybrid screen to isolate an Iba1-interacting molecule and identified another actin-bundling protein, L-Fimbrin. In response to stimulation, L-Fimbrin accumulated and co-localized with Iba1 in membrane ruffles induced by M-CSF-stimulation and phagocytic cups formed by IgG-opsonized beads in microglial cell line MG5. L-Fimbrin was shown to associate with Iba1 in cell lysate of COS-7 expressing L-Fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L-Fimbrin was demonstrated by immunoprecipitation, glutathione S-transferase pull-down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin-bundling activity of L-Fimbrin. These results indicate that Iba1 forms complexes with L-Fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co-operates with L-Fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.
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Microglia/macrophage‐specific protein Iba1 binds to Fimbrin and enhances its actin‐bundling activity
Journal of neurochemistry, 2004Co-Authors: Keiko Ohsawa, Yoshinori Imai, Yo Sasaki, Shinichi KohsakaAbstract:Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage-specific calcium-binding protein. Iba1 has the actin-bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1-related intracellular signalling pathway in greater detail, we employed a yeast two-hybrid screen to isolate an Iba1-interacting molecule and identified another actin-bundling protein, L-Fimbrin. In response to stimulation, L-Fimbrin accumulated and co-localized with Iba1 in membrane ruffles induced by M-CSF-stimulation and phagocytic cups formed by IgG-opsonized beads in microglial cell line MG5. L-Fimbrin was shown to associate with Iba1 in cell lysate of COS-7 expressing L-Fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L-Fimbrin was demonstrated by immunoprecipitation, glutathione S-transferase pull-down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin-bundling activity of L-Fimbrin. These results indicate that Iba1 forms complexes with L-Fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co-operates with L-Fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.
Edward H Egelman - One of the best experts on this subject based on the ideXlab platform.
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High-resolution cryo-EM structure of the F-actin-Fimbrin/plastin ABD2 complex.
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Vitold E Galkin, Albina Orlova, Olga Cherepanova, Marie-christine Lebart, Edward H EgelmanAbstract:Many actin binding proteins have a modular architecture, and calponin-homology (CH) domains are one such structurally conserved module found in numerous proteins that interact with F-actin. The manner in which CH-domains bind F-actin has been controversial. Using cryo-EM and a single-particle approach to helical reconstruction, we have generated 12-A-resolution maps of F-actin alone and F-actin decorated with a fragment of human Fimbrin (L-plastin) containing tandem CH-domains. The high resolution allows an unambiguous fit of the crystal structure of Fimbrin into the map. The interaction between Fimbrin ABD2 (actin binding domain 2) and F-actin is different from any interaction previously observed or proposed for tandem CH-domain proteins, showing that the structural conservation of the CH-domains does not lead to a conserved mode of interaction with F-actin. Both the stapling of adjacent actin protomers and the additional closure of the nucleotide binding cleft in F-actin when the Fimbrin fragment binds may explain how Fimbrin can stabilize actin filaments. A mechanism is proposed where ABD1 of Fimbrin becomes activated for binding a second actin filament after ABD2 is bound to a first filament, and this can explain how mutations of residues buried in the interface between ABD2 and ABD1 can rescue temperature-sensitive defects in actin.
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high resolution cryo em structure of the f actin Fimbrin plastin abd2 complex
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Vitold E Galkin, Albina Orlova, Olga Cherepanova, Marie-christine Lebart, Edward H EgelmanAbstract:Many actin binding proteins have a modular architecture, and calponin-homology (CH) domains are one such structurally conserved module found in numerous proteins that interact with F-actin. The manner in which CH-domains bind F-actin has been controversial. Using cryo-EM and a single-particle approach to helical reconstruction, we have generated 12-A-resolution maps of F-actin alone and F-actin decorated with a fragment of human Fimbrin (L-plastin) containing tandem CH-domains. The high resolution allows an unambiguous fit of the crystal structure of Fimbrin into the map. The interaction between Fimbrin ABD2 (actin binding domain 2) and F-actin is different from any interaction previously observed or proposed for tandem CH-domain proteins, showing that the structural conservation of the CH-domains does not lead to a conserved mode of interaction with F-actin. Both the stapling of adjacent actin protomers and the additional closure of the nucleotide binding cleft in F-actin when the Fimbrin fragment binds may explain how Fimbrin can stabilize actin filaments. A mechanism is proposed where ABD1 of Fimbrin becomes activated for binding a second actin filament after ABD2 is bound to a first filament, and this can explain how mutations of residues buried in the interface between ABD2 and ABD1 can rescue temperature-sensitive defects in actin.