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Timothy A Springer - One of the best experts on this subject based on the ideXlab platform.

Uyen T Phan - One of the best experts on this subject based on the ideXlab platform.

Johan Stenflo - One of the best experts on this subject based on the ideXlab platform.

  • SOLUTION STRUCTURE OF THE N-TERMINAL EGF-like Domain FROM HUMAN FACTOR VII
    Biochemistry, 1998
    Co-Authors: Andreas Muranyi, Johan Stenflo, Bryan E. Finn, Garry P. Gippert, Sture Forsén, Torbjörn Drakenberg
    Abstract:

    Blood coagulation is initiated by Ca2+-dependent binding of coagulation factor VIIa (FVIIa) to its cofactor, tissue factor (TF). The TF:FVIIa complex activates factors IX and X, ultimately leading to the formation of thrombin and the coagulation of blood. FVII consists of an N-terminal γ-carboxyglutamic-acid-containing (Gla) Domain followed by two epidermal growth factor (EGF) like Domains, the first of which can bind one Ca2+ ion (Kd ≈ 150 μM) and a C-terminal serine protease Domain. Using 1H nuclear magnetic resonance spectroscopy, we have determined the solution structure of a synthetic N-terminal EGF-like Domain (EGF1) of human FVII (residues 45−85) in the absence of Ca2+. A comparison of this structure of apo EGF1 with the Ca2+-bound EGF1 in the complex of FVIIa and TF [Banner, D. W., et al. (1996) Nature 380, 41−46] suggests that the structural changes in the EGF1 Domain upon Ca2+ binding are minor and are concentrated near the Ca2+-binding site, which is facing away from the TF interaction surface....

  • How an epidermal growth factor (EGF)-like Domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like Domain in coagulation factor X.
    The Journal of biological chemistry, 1992
    Co-Authors: Maria Selander-sunnerhagen, Egon Persson, Johan Stenflo, Magnus Ullner, Olle Teleman, Torbjörn Drakenberg
    Abstract:

    Domains homologous to the epidermal growth factor (EGF) are important building blocks for extracellular proteins. Proteins containing these Domains have been shown to function in such diverse biological processes as blood coagulation, complement activation, and the developmental determination of embryonic cell fates. Many of these proteins require calcium for their biological function. In the case of coagulation factors IX and X and anticoagulants proteins C and S, calcium has been found to bind to the EGF-like Domains. We have now determined the three-dimensional structure of the calcium-bound form of the NH2-terminal EGF-like Domain in coagulation factor X by two-dimensional NMR and simulated folding. Ligands to the calcium ion are the two backbone carbonyls in Gly-47 and Gly-64, as well as the side chains in Gln-49, erythro-beta-hydroxyaspartic acid (Hya) 63, and possibly Asp-46. The conserved Asp-48 is not a ligand in our present structures. The remaining ligands are assumed to be solvent molecules or, in the intact protein, ligands from neighboring Domains. Other proteins interacting in a calcium-dependent manner may also contribute ligands. A comparison with the calcium-free form shows that calcium binding induces strictly local structural changes in the Domain. Residues corresponding to the side chain ligands in factor X are conserved in many other proteins, such as the integral membrane protein TAN-1 of human lymphocytes and its developmentally important homolog, Notch, in Drosophila. Calcium binding to EGF-like Domains may be crucial for numerous protein-protein interactions involving EGF-like Domains in coagulation factors, plasma proteins, and membrane proteins. Therefore, there is reason to believe that this novel calcium site plays an important role in the biochemistry of extracellular proteins.

  • Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact Domains isolated from controlled proteolytic digests of bovine factor IX.
    Journal of Biological Chemistry, 1991
    Co-Authors: Jan Astermark, I Björk, Ann-kristin Öhlin, Johan Stenflo
    Abstract:

    Abstract Blood coagulation factor IX is composed of discrete Domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two Domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like Domain. To be able to determine the structure and function of the Gla- and EGF-like Domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact Domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like Domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like Domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like Domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Bjork, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42.

  • Protein structural requirements for Ca2+ binding to the light chain of factor X. Studies using isolated intact fragments containing the gamma-carboxyglutamic acid region and/or the epidermal growth factor-like Domains.
    Journal of Biological Chemistry, 1991
    Co-Authors: Egon Persson, I Björk, Johan Stenflo
    Abstract:

    Abstract Coagulation factor X is a multiDomain proenzyme of a serine protease. Calcium ions bind to the vitamin K-dependent gamma-carboxyglutamic acid (Gla) residues and to a site in the NH2-terminal of two epidermal growth factor (EGF)-like Domains. To study structure-function relationships in the NH2-terminal part of factor X and to determine the structure of isolated Domains, we have developed methods that allow the subsequent isolation of the first or both EGF-like Domains with or without an attached Gla Domain from controlled proteolytic digests of the protein. The Ca2(+)-induced changes of the intrinsic protein fluorescence were measured to elucidate whether the isolated fragments retain their native conformation. Changes in the fluorescence caused by Ca2+ binding were found to result from perturbations of the environment of the Trp residue in position 41. Calcium ion binding to the Gla-containing region linked to the NH2-terminal EGF-like Domain was identical with that to intact factor X, indicating a native orientation of the ligand binding groups in the fragment. In contrast, the isolated Gla peptide had a lower affinity for Ca2+, suggesting that the NH2-terminal EGF-like Domain serves as a scaffold for the folding of the Gla region. Similarly, the presence of the Gla region was found to increase the affinity of the Gla-independent site in the first EGF-like Domain for Ca2+. The metal ion-induced resistance against chymotryptic cleavage COOH-terminal of Tyr-44 in intact factor X is similar in the isolated fragment that contains the Gla region linked to one EGF-like Domain, indicating a native conformation of the fragment in the presence of Ca2+. Furthermore, the Gla-independent metal ion binding site binds Ca2+ but does not appear to bind Mg2+.

  • The epidermal growth factor-like Domains of factor IX. Effect on blood clotting and endothelial cell binding of a fragment containing the epidermal growth factor-like Domains linked to the gamma-carboxyglutamic acid region.
    Journal of Biological Chemistry, 1991
    Co-Authors: Jan Astermark, Johan Stenflo
    Abstract:

    The binding of factor IX to cultured bovine endothelial cells was characterized using isolated Domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like Domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like Domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like Domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like Domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like Domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like Domain(s) also contributes to the interaction of the enzyme with its cofactor.

J. E. Sadler - One of the best experts on this subject based on the ideXlab platform.

  • Thrombomodulin structure and function.
    Thrombosis and haemostasis, 1997
    Co-Authors: J. E. Sadler
    Abstract:

    Thrombomodulin is protein cofactor expressed on endothelial cell surfaces that modifies the substrate specificity of thrombin, apparently by an allosteric mechanism. The thrombin-thrombomodulin complex activates protein C, initiating an essential anticoagulant pathway. The cofactor function of membrane-associated thrombomodulin requires the last three of six tandemly repeated EGF-like Domains (numbers 4, 5, and 6), as well as a Ser/Thr-rich spacer between EGF-like Domain 6 and the transmembrane Domain. The Ser/Thr-rich Domain is variably modified with a chondroitin sulfate chain that influences the affinity of thrombin binding and the calcium ion dependence of cofactor function. The structure of EGF-like Domain 4 has been determined by NMR spectroscopy, and the structure of a complex between thrombin and a peptide from thrombomodulin EGF-like Domain 5 was determined by X-ray crystallography. These structures are small steps toward an understanding of how thrombomodulin regulates thrombin.

  • Sequences required for thrombomodulin cofactor activity within the fourth epidermal growth factor-like Domain of human thrombomodulin.
    The Journal of biological chemistry, 1993
    Co-Authors: Steven R. Lentz, Yan Chen, J. E. Sadler
    Abstract:

    Abstract Activation of protein C by thrombin is stimulated by the endothelial cell cofactor thrombomodulin. The structural regions of thrombomodulin necessary for cofactor activity have been localized to the fourth through sixth epidermal growth factor (EGF)-like Domains. The fourth EGF-like Domain is unnecessary for high affinity thrombin binding, but is required for cofactor activity. To identify essential sequences within the fourth EGF-like Domain, a series of recombinant human thrombomodulins consisting of EGF-like Domains four through six were expressed in human kidney cells. These mutants contain replacements of disulfide loops within the fourth EGF-like Domain, thereby conserving overall disulfide bond structure. All of the mutants bound to thrombin with high affinity, and inhibited the fibrinogen-clotting activity of thrombin to a similar extent. Two regions of the fourth EGF-like Domain were identified to be essential for cofactor activity: 1) the sequence consisting of amino acids Glu-357, Tyr-358, and Gln-359 shared by the overlapping first and second disulfide loops, and 2) the amino-terminal region of the third disulfide loop containing amino acids Glu-374, Gly-375, and Phe-376. These results suggest that amino acids critical for thrombomodulin cofactor activity are located near the junction between the two subDomains of the fourth EGF-like Domain.

  • Functional Domains of membrane-bound human thrombomodulin. EGF-like Domains four to six and the serine/threonine-rich Domain are required for cofactor activity.
    The Journal of biological chemistry, 1992
    Co-Authors: M Tsiang, Steven R. Lentz, J. E. Sadler
    Abstract:

    Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like Domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like Domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like Domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like Domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich Domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this Domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich Domain as an important spacer, in addition to EGF-like Domains 4-6, for efficient protein C activation.

Penny A Handford - One of the best experts on this subject based on the ideXlab platform.

  • calcium binding properties of an epidermal growth factor like Domain pair from human fibrillin 1
    Journal of Molecular Biology, 1996
    Co-Authors: Vroni Knott, Caroline M. Cardy, A.k. Downing, Penny A Handford
    Abstract:

    Ca2+binding epidermal growth factor-like (EGF-like) Domains are found in a large number of extracellular proteins with diverse functions, including those involved in blood coagulation, determination of cell fate, cell adhesion and connective tissue architecture. Their importance is emphasised by the identification of mutations in these Domains in patients with haemophilia B (defective in coagulation factor IX) and the Marfan syndrome (defective in the connective tissue protein fibrillin-1). The X-ray crystal structure of a single Ca2 +binding EGF-like Domain from human coagulation factor IX has recently been solved. It shows that the Ca2+ligands form a pentagonal bipyramid, where one ligand is provided by an adjacent (N-terminal) EGF-like Domain in the crystal. The N and C termini of the neighbouring Domains are only|similar|4 A apart, hence the crystal packing has been proposed as a model for the association of contiguous EGF-like Domains in proteins. Since the adjacent EGF-like Domain in the crystal, although close, is not covalently linked to its neighbour, this model requires verification. In this study we have expressed and purified a Ca2+binding EGF-like Domain pair from human fibrillin-1 and used anin vitrorefolding system to obtain protein with the correct EGF fold. The Ca2+binding properties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal Domain for Ca2 +is |similar|25-fold higher than that of the N-terminal Domain, consistent with the two Ca2+binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca2+plays a major role in defining the interDomain linkage in multiple repeats of Ca2+binding EGF-like Domains.

  • CALCIUM BINDING PROPERTIES OF AN EPIDERMAL GROWTH FACTOR-LIKE Domain PAIR FROM HUMAN FIBRILLIN-1
    Journal of molecular biology, 1996
    Co-Authors: Vroni Knott, Caroline M. Cardy, A.k. Downing, Penny A Handford
    Abstract:

    Ca2+ binding epidermal growth factor-like (EGF-like) Domains are found in a large number of extracellular proteins with diverse functions, including those involved in blood coagulation, determination of cell fate, cell adhesion and connective tissue architecture. Their importance is emphasised by the identification of mutations in these Domains in patients with haemophilia B (defective in coagulation factor IX) and the Marfan syndrome (defective in the connective tissue protein fibrillin-1). The X-ray crystal structure of a single Ca2+ binding EGF-like Domain from human coagulation factor IX has recently been solved. It shows that the Ca2+ ligands form a pentagonal bipyramid, where one ligand is provided by an adjacent (N-terminal) EGF-like Domain in the crystal. The N and C termini of the neighbouring Domains are only approximately 4 angstrum apart, hence the crystal packing has been proposed as a model for the association of contiguous EGF-like Domains in proteins. Since the adjacent EGF-like Domain in the crystal, although close, is not covalently linked to its neighbour, this model requires verification. In this study we have expressed and purified a Ca2+ binding EGF-like Domain pair from human fibrillin-1 and used an in vitro refolding system to obtain protein with the correct EGF fold. The Ca2+ binding properties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal Domain for Ca2+ is approximately 25-fold higher than that of the N-terminal Domain, consistent with the two Ca2+ binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca2+ plays a major role in defining the interDomain linkage in multiple repeats of Ca2+ binding EGF-like Domains.

  • The structure of a Ca(2+)-binding epidermal growth factor-like Domain: its role in protein-protein interactions.
    Cell, 1995
    Co-Authors: Zihe Rao, Vroni Knott, George G. Brownlee, Penny A Handford, M. Mayhew, David Stuartz
    Abstract:

    Abstract Various diverse extracellular proteins possess Ca 2+ -binding epidermal growth factor (EGF)-like Domains, the function of which remains uncertain. We have determined, at high resolution (1.5 A), the crystal structure of such a Domain, from human clotting factor IX, as a complex with Ca 2+ . The Ca 2+ ligands form a classic pentagonal bipyramid with six ligands contributed by one polypeptide chain and the seventh supplied by a neighboring EGF-like Domain. The crystal structure identifies the role of Ca 2+ in maintaining the conformation of the N-terminal region of the Domain, but more importantly demonstrates that Ca 2+ can directly mediate protein-protein contacts. The observed crystal packing of the Domains provides a plausible model for the association of multiple tandemly linked EGF-like Domains in proteins such as fibrillin-1, Notch, and protein S. This model is consistent with the known functional data and suggests a general biological role for these Domains.

  • CRYSTALLIZATION OF A CALCIUM-BINDING EGF-like Domain
    Acta Crystallographica Section D Biological Crystallography, 1995
    Co-Authors: Zihe Rao, Vroni Knott, Penny A Handford, M. Mayhew, G. G. Brownlee, David I. Stuart
    Abstract:

    Crystals of a calcium-binding epidermal growth factor (EGF)-like Domain of human clotting factor IX suitable for X-ray diffraction analysis have been obtained by vapour diffusion (sitting drop) against 48% PEG 400. The crystals belong to the tetragonal space group P43212, with unit-cell dimensions a = b = 40.3, c = 98.2 A. The crystals diffract beyond 1.5 A resolution and are relatively stable in the X-ray beam. This is the first reported crystallization of a calcium-binding EGF-like Domain.

  • the calcium binding properties and molecular organization of epidermal growth factor like Domains in human fibrillin 1
    Journal of Biological Chemistry, 1995
    Co-Authors: Penny A Handford, A.k. Downing, D R Hewett, B C Sykes, Cay M Kielty
    Abstract:

    Abstract Human fibrillin-1 is a 350-kDa glycoprotein found in 10-nm connective tissue microfibrils. Mutations in the gene encoding this protein cause the Marfan syndrome, a disease characterized by cardiovascular, ocular, and skeletal abnormalities. Fibrillin-1 has a modular structure that includes 47 epidermal growth factor-like (EGF-like) Domains, 43 of which contain a consensus sequence associated with calcium binding. A mutation causing an Asn-2144 Ser amino acid change in one of the potential calcium binding residues has been described in a patient with the Marfan syndrome. We have chemically synthesized a wild-type EGF-like Domain (residues 2126-2165 of human fibrillin-1) and a mutant EGF-like Domain containing the Asn-2144 Ser amino acid change and measured calcium binding to each using 1H-NMR spectroscopy. The wild-type Domain binds calcium with a similar affinity to isolated EGF-like Domains from coagulation factors IX and X; however, the mutant Domain exhibits >5-fold reduction in affinity. Rotary shadowing of fibrillin-containing microfibrils, isolated from dermal fibroblast cultures obtained from the Marfan patient, shows that the mutation does not prevent assembly of fibrillin into microfibrils but does alter the appearance of the interbead region. We have modeled a region of fibrillin-1 (residues 2126-2331) encompassing five calcium binding EGF-like Domains, using data derived from the recently determined crystal structure of a calcium binding EGF-like Domain from human factor IX. Our model suggests that these fibrillin-1 EGF-like Domains adopt a helical arrangement stabilized by calcium and that defective calcium binding to a single EGF-like Domain results in distortion of the helix. We propose a mechanism for the interaction of contiguous arrays of calcium binding EGF-like Domains within the microfibril.