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Paul A Mould - One of the best experts on this subject based on the ideXlab platform.
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distinct roles of β1 metal ion dependent Adhesion Site midas adjacent to midas admidas and ligand associated metal binding Site limbs cation binding Sites in ligand recognition by integrin α2β1
Journal of Biological Chemistry, 2008Co-Authors: Dimitra Valdramidou, Martin J Humphries, Paul A MouldAbstract:Abstract Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding Sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding Sites: the metal-ion dependent Adhesion Site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding Site). The role of these Sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+, and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent Site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding Site, occupancy of which increases the affinity of Mg2+ for the MIDAS.
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distinct roles of β1 metal ion dependent Adhesion Site midas adjacent to midas admidas and ligand associated metal binding Site limbs cation binding Sites in ligand recognition by integrin α2β1
Journal of Biological Chemistry, 2008Co-Authors: Dimitra Valdramidou, Martin J Humphries, Paul A MouldAbstract:Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding Sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding Sites: the metal-ion dependent Adhesion Site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding Site). The role of these Sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent Site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding Site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.
Robert C. Liddington - One of the best experts on this subject based on the ideXlab platform.
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cellular activation of leukocyte function associated antigen 1 and its affinity are regulated at the i domain allosteric Site
Journal of Immunology, 2001Co-Authors: Mark L Lupher, Robert C. Liddington, Edith A S Harris, Chan R Beals, Liming Sui, Donald E StauntonAbstract:The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent Adhesion Site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent Adhesion Site is regulated by the I domain allosteric Site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the K(d) of ICAM-1 binding. The K(d) of wild-type LFA-1 (1.2 x 10(-1) s(-1)) differed with that of the activating IDAS mutant (1.9 x 10(-2) s(-1)), but their K(a) values were identical (2.2 x 10(5) M(-1)s(-1)). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.
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interaction between collagen and the α2 i domain of integrin αβ1 critical role of conserved residues in the metal ion dependent Adhesion Site midas region
Journal of Biological Chemistry, 1999Co-Authors: Tetsuji Kamata, Robert C. Liddington, Yoshikazu TakadaAbstract:Abstract A docking model of the α2 I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512–28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154, Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of α2 (residues 284–288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted critical residues had no effect on collagen binding to whole α2β1, with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found that mutating the conserved surface-exposed residues in the metal ion-dependent Adhesion Site (MIDAS) (Tyr-157 and Gln-215) significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model, conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285 residues. These results suggest that the collagen-binding I-domains (e.g. α1, α2, and α10) bind to collagen in a similar fashion.
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Echovirus 1 interaction with the human very late antigen-2 (integrin α2β1) I domain: Identification of two independent virus contact Sites distinct from the metal ion-dependent Adhesion Site
The Journal of biological chemistry, 1997Co-Authors: Sandra L. King, Tetsuji Kamata, Jennifer A. Cunningham, Jonas Emsley, Robert C. Liddington, Yoshikazu Takada, Jeffrey M. BergelsonAbstract:Abstract The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding Sites for both collagen and echovirus 1 have been mapped to the I domain within the α2 subunit of the VLA-2 α2β1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199–201 and 212–216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent Adhesion Site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent Adhesion Site residues that abolish Adhesion to collagen had no effect on virus binding. These results confirm that different Sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands.
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The von willebrand factor A3 domain does not contain a metal ion-dependent Adhesion Site motif.
The Journal of biological chemistry, 1997Co-Authors: Jadwiga Bienkowska, Miguel A. Cruz, Andrew Atiemo, Robert I. Handin, Robert C. LiddingtonAbstract:Abstract von Willebrand factor (vWF) is a multimeric plasma protein that mediates platelet Adhesion to exposed subendothelium at Sites of vascular injury. The A3 domain of vWF (vWF-A3) forms the principal binding Site for collagens type I and III. We report here the crystal structure of the vWF-A3 domain at 2.2-A resolution. As expected, the structure is similar to the integrin I domain but with several novel features. Sequence alignments had suggested that the domain contained an integrin metal ion-dependent Adhesion Site (MIDAS) motif, but the crystal structure shows that the motif is modified and that no metal ion is bound. We have introduced mutations into the vestigial MIDAS motif and report that, unlike the I domain of integrin α2β1, vWF-A3 continues to bind collagen after disruption of the motif. We conclude that collagen recognition by vWF-A3 occurs by a mechanism different from that of the integrin α2β1.
Martin J Humphries - One of the best experts on this subject based on the ideXlab platform.
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distinct roles of β1 metal ion dependent Adhesion Site midas adjacent to midas admidas and ligand associated metal binding Site limbs cation binding Sites in ligand recognition by integrin α2β1
Journal of Biological Chemistry, 2008Co-Authors: Dimitra Valdramidou, Martin J Humphries, Paul A MouldAbstract:Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding Sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding Sites: the metal-ion dependent Adhesion Site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding Site). The role of these Sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent Site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding Site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.
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distinct roles of β1 metal ion dependent Adhesion Site midas adjacent to midas admidas and ligand associated metal binding Site limbs cation binding Sites in ligand recognition by integrin α2β1
Journal of Biological Chemistry, 2008Co-Authors: Dimitra Valdramidou, Martin J Humphries, Paul A MouldAbstract:Abstract Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding Sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding Sites: the metal-ion dependent Adhesion Site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding Site). The role of these Sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+, and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent Site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding Site, occupancy of which increases the affinity of Mg2+ for the MIDAS.
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Bamacan BM-CSPG
The Extracellular Matrix FactsBook, 1998Co-Authors: Shirley Ayad, Martin J Humphries, Ray Boot-handford, Karl E. Kadler, Adrian ShuttleworthAbstract:The chapter discusses the structural and functional Sites of bamacan. Bamacan is a chondroitin sulfate proteoglycan distinct from perlecan and agrin and present in a number of basement membranes. The protein's structural features are in common with proteins that stabilize the chromosomal scaffold at mitosis. As deduced from complementary DNA (cDNA) sequences, the protein's predicted molecular weight is 138,000. The predicted protein is divided into five domains. The first domain—165 amino acids—lack cysteine and are hydrophilic. Domains II and IV contain 335 and 364 amino acids, respectively, and have high potentials for coiled-coil structure. Domain III lies between two coiled-coil regions and is comprised of 165 amino acids. It contains four cysteines and a potential cell Adhesion Site. The chapter also presents the primary structure and isolation methods of bamacan.
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the minimal essential sequence for a major cell type specific Adhesion Site cs1 within the alternatively spliced type iii connecting segment domain of fibronectin is leucine aspartic acid valine
Journal of Biological Chemistry, 1991Co-Authors: A Komoriya, Linda J Green, M Mervic, S S Yamada, Kenneth M Yamada, Martin J HumphriesAbstract:Abstract Fibronectin contains at least two major domains that support cell Adhesion. One is the central cell-binding domain that is recognized by a variety of cell types via the integrin alpha 5 beta 1. The second, originally identified by its ability to support melanoma cell Adhesion, is located in the alternatively spliced type III connecting segment (IIICS). A dominant cell type-specific Adhesion Site within the IIICS has been localized to a peptide designated as CS1 comprising its amino-terminal 25 residues. The receptor for CS1 is the integrin alpha 4 beta 1. We have synthesized a variety of peptides with overlapping sequences in order to identify the minimum active amino acid sequence of this major cell Adhesion Site. A peptide comprising the carboxyl-terminal 8 amino acids of CS1, EILDVPST, was found to support melanoma cell spreading, while all peptides without this sequence had little or no activity. Two smaller overlapping pentapeptides, EILDV and LDVPS, were also active, whereas EILEV, containing a conservative substitution of Glu for Asp, was inactive. These data suggested that the minimum sequence for cell Adhesion activity is Leu-Asp-Val, the tripeptide sequence common to both active peptides. This prediction was confirmed by the observed ability of the Leu-Asp-Val peptide itself to block spreading on fibronectin, whereas Leu-Glu-Val was inactive. Interspecies amino acid sequence comparison also supports the importance of the LDV sequence, since it is completely conserved in the IIICS regions of human, rat, bovine, and avian fibronectins.
Dimitra Valdramidou - One of the best experts on this subject based on the ideXlab platform.
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distinct roles of β1 metal ion dependent Adhesion Site midas adjacent to midas admidas and ligand associated metal binding Site limbs cation binding Sites in ligand recognition by integrin α2β1
Journal of Biological Chemistry, 2008Co-Authors: Dimitra Valdramidou, Martin J Humphries, Paul A MouldAbstract:Abstract Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding Sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding Sites: the metal-ion dependent Adhesion Site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding Site). The role of these Sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+, and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent Site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding Site, occupancy of which increases the affinity of Mg2+ for the MIDAS.
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distinct roles of β1 metal ion dependent Adhesion Site midas adjacent to midas admidas and ligand associated metal binding Site limbs cation binding Sites in ligand recognition by integrin α2β1
Journal of Biological Chemistry, 2008Co-Authors: Dimitra Valdramidou, Martin J Humphries, Paul A MouldAbstract:Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding Sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding Sites: the metal-ion dependent Adhesion Site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding Site). The role of these Sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent Site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding Site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.
Timothy A Springer - One of the best experts on this subject based on the ideXlab platform.
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the relative influence of metal ion binding Sites in the i like domain and the interface with the hybrid domain on rolling and firm Adhesion by integrin α4β7
Journal of Biological Chemistry, 2004Co-Authors: Jianfeng Chen, Junichi Takagi, T Xiao, Timothy A SpringerAbstract:We examined the effect of conformational change at the 7 I-like/hybrid domain interface on regulating the transition between rolling and firm Adhesion by integrin 47 .A nN-glycosylation Site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the 47 headpiece. Wild-type 47 mediates rolling Adhesion in Ca 2 and Ca 2 /Mg 2 but firm Adhesion in Mg 2 and Mn 2 . Stabilizing the open headpiece resulted in firm Adhesion in all divalent cations. The interaction between metal binding Sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two Sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding Site (LIMBS) and activating wedge mutations could still be activated by Mn 2 , confirming the importance of the adjacent to metal ion-dependent Adhesion Site (ADMIDAS) in integrin activation by Mn 2 . Overall,
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bistable regulation of integrin adhesiveness by a bipolar metal ion cluster
Nature Structural & Molecular Biology, 2003Co-Authors: Jianfeng Chen, Azucena Salas, Timothy A SpringerAbstract:Integrin α4β7 mediates rolling Adhesion in Ca2+ and Ca2+ + Mg2+, and firm Adhesion in Mg2+ and Mn2+, mimicking the two key steps in leukocyte accumulation in inflamed vasculature. We mutated an interlinked linear array of three divalent cation-binding Sites present in integrin β-subunit I-like domains. The middle, metal ion–dependent Adhesion Site (MIDAS) is required for both rolling and firm Adhesion. One polar Site, that adjacent to MIDAS (ADMIDAS), is required for rolling because its mutation results in firm Adhesion. The other polar Site, the ligand-induced metal binding Site (LIMBS), is required for firm Adhesion because its mutation results in rolling. The LIMBS mediates the positive regulatory effects of low Ca2+ concentrations, whereas the ADMIDAS mediates the negative regulatory effects of higher Ca2+ concentrations, which are competed by Mn2+. The bipolar Sites thus stabilize two alternative phases of Adhesion.
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small molecule integrin antagonists that bind to the β2 subunit i like domain and activate signals in one direction and block them in the other
Immunity, 2003Co-Authors: Motomu Shimaoka, Azucena Salas, Wei Yang, Gabriele Weitzschmidt, Timothy A SpringerAbstract:Abstract Leukocyte integrins contain an inserted (I) domain in their α subunits and an I-like domain in their β 2 subunit, which directly bind ligand and regulate ligand binding, respectively. We describe a novel mechanistic class of integrin inhibitors that bind to the metal ion-dependent Adhesion Site of the β 2 I-like domain and prevent its interaction with and activation of the α L I domain. The inhibitors do not bind to the α L I domain but stabilize α/β subunit association and can show selectivity for α L β 2 compared to α M β 2 . The inhibitors reveal a crucial intersection for relaying conformational signals within integrin extracellular domains. While blocking signals in one direction to the I domain, the antagonists induce the active conformation of the I-like domain and stalk domains, and thus transmit conformational signals in the other direction toward the transmembrane domains.
