scholarly journals A novel region of the α4 integrin subunit with a modulatory role in VLA-4-mediated cell adhesion to fibronectin

1997 ◽  
Vol 327 (3) ◽  
pp. 727-733 ◽  
Author(s):  
Marisa MUÑOZ ◽  
Juan SERRADOR ◽  
Marta NIETO ◽  
Alfonso LUQUE ◽  
Francisco SÁNCHEZ-MADRID ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Binding Sites ◽  
Close Association ◽  
Cell Aggregation ◽  
Integrin Subunit ◽  
Vascular Cell Adhesion ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Cell Adhesion Molecule 1

The integrin VLA-4 (α4β1) is a receptor for fibronectin and vascular cell-adhesion molecule 1 (VCAM-1). Four functionally different epitopes, designated A, B1, B2 and C, have previously been defined on the α4 subunit. Using K562 α4 mutant transfectants we found that α4 amino acids Tyr151, Gln152, Asp153, Tyr154 and Val155 are important for the structure of the epitope B2. Mutations at α4 Gln152 substantially impaired the transfectant adhesion to a CS-1-containing fragment of fibronectin (FN-H89), whereas this adhesion was not affected on the other α4 mutant transfectants. None of the α4 mutations significantly altered the adhesion of the different α4 transfectants to VCAM-1. In addition, we have identified residues Gln152, Asp153 and Tyr154 as part of the α4 epitope B2 involved in homotypic cell aggregation. The decrease in adhesion to FN-H89 shown by Gln152 α4 mutant transfectants was the result of an inefficient binding of FN-H89 by VLA-4 mutated at this residue. Also, mutant VLA-4 displayed an altered reactivity with HUTS-21, an anti-β1 monoclonal antibody that reacts with functionally active VLA integrins. Adhesion to FN-H89 was not restored unless stimuli that increase the ligand-binding affinity of VLA heterodimers were added, suggesting that cell adhesion was affected in the initial phases. These results indicate that α4 Gln152 modulates cell adhesion to FN-H89 by playing important roles in the maintenance and/or the acquisition of an active state of VLA-4, an integrin that is normally expressed on the cell surface in a range of multiple activation states. The location of the α4 Gln152 residue on a loop of the upper surface of the proposed β-propeller structure suggests a close association with potential ligand-binding sites.

scholarly journals Identification of putative ligand-binding sites of the integrin α4β1 (VLA-4, CD49d/CD29)

1995 ◽  
Vol 305 (3) ◽  
pp. 945-951 ◽  
Author(s):  
T Kamata ◽  
W Puzon ◽  
Y Takada
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Adhesion Molecule ◽  
Cho Cells ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Cell Adhesion Molecule ◽  
Chinese Hamster ◽  
Ligand Binding Sites ◽  
Cell Adhesion Molecule 1

Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].

A naturally occurring Tyr143HisαIIb mutation abolishes αIIbβ3 function for soluble ligands but retains its ability for mediating cell adhesion and clot retraction: comparison with other mutations causing ligand-binding defects

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3485-3491 ◽  
Author(s):  
Teruo Kiyoi ◽  
Yoshiaki Tomiyama ◽  
Shigenori Honda ◽  
Seiji Tadokoro ◽  
Morio Arai ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Binding Sites ◽  
Clot Retraction ◽  
Naturally Occurring ◽  
Binding Function ◽  
293 Cells ◽  
Ligand Binding Sites ◽  
Functional Defect ◽  
And Function

The molecular basis for the interaction between a prototypic non–I-domain integrin, αIIbβ3, and its ligands remains to be determined. In this study, we have characterized a novel missense mutation (Tyr143His) in αIIb associated with a variant of Glanzmann thrombasthenia. Osaka-12 platelets expressed a substantial amount of αIIbβ3(36%-41% of control) but failed to bind soluble ligands, including a high-affinity αIIbβ3-specific peptidomimetic antagonist. Sequence analysis revealed that Osaka-12 is a compound heterozygote for a single 521T>C substitution leading to a Tyr143His substitution in αIIb and for the null expression of αIIb mRNA from the maternal allele. Given that Tyr143 is located in the W3 4-1 loop of the β-propeller domain of αIIb, we examined the effects of Tyr143His or Tyr143Ala substitution on the expression and function of αIIbβ3 and compared them with KO (Arg-Thr insertion between 160 and 161 residues of αIIb) and with the Asp163Ala mutation located in the same loop by using 293 cells. Each of them abolished the binding function of αIIbβ3 for soluble ligands without disturbing αIIbβ3 expression. Because immobilized fibrinogen and fibrin are higher affinity/avidity ligands for αIIbβ3, we performed cell adhesion and clot retraction assays. In sharp contrast to KO mutation and Asp163AlaαIIbβ3, Tyr143HisαIIbβ3-expressing cells still had some ability for cell adhesion and clot retraction. Thus, the functional defect induced by Tyr143HisαIIb is likely caused by its allosteric effect rather than by a defect in the ligand-binding site itself. These detailed structure–function analyses provide better understanding of the ligand-binding sites in integrins.

scholarly journals Identification of Iron-Sulfur (Fe-S) and Zn-binding Sites Within Proteomes Predicted by DeepMind′s AlphaFold2 Program Dramatically Expands the Metalloproteome

2021 ◽  
Author(s):  
Zachary J Wehrspan ◽  
Robert T McDonnell ◽  
Adrian Elcock
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Structure Prediction ◽  
Disulfide Bonds ◽  
3D Protein Structure ◽  
E Coli ◽  
Iron Sulfur ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Binding Orientations

DeepMind′s AlphaFold2 software has ushered in a revolution in high quality, 3D protein structure prediction. In very recent work by the DeepMind team, structure predictions have been made for entire proteomes of twenty-one organisms, with >360,000 structures made available for download. Here we show that thousands of novel binding sites for iron-sulfur (Fe-S) clusters and zinc ions can be identified within these predicted structures by exhaustive enumeration of all potential ligand-binding orientations. We demonstrate that AlphaFold2 routinely makes highly specific predictions of ligand binding sites: for example, binding sites that are comprised exclusively of four cysteine sidechains fall into three clusters, representing binding sites for 4Fe-4S clusters, 2Fe-2S clusters, or individual Zn ions. We show further: (a) that the majority of known Fe-S cluster and Zn-binding sites documented in UniProt are recovered by the AlphaFold2 structures, (b) that there are occasional disputes between AlphaFold2 and UniProt with AlphaFold2 predicting highly plausible alternative binding sites, (c) that the Fe-S cluster binding sites that we identify in E. coli agree well with previous bioinformatics predictions, (d) that cysteines predicted here to be part of Fe-S cluster or Zn-binding sites show little overlap with those shown via chemoproteomics techniques to be highly reactive, and (e) that AlphaFold2 occasionally appears to build erroneous disulfide bonds between cysteines that should instead coordinate a ligand. These results suggest that AlphaFold2 could be an important tool for the functional annotation of proteomes, and the methodology presented here is likely to be useful for predicting other ligand-binding sites.

Activation Dependent and Independent VLA-4 Binding Sites on Vascular Cell Adhesion Molecule-1

1994 ◽  
Vol 2 (2) ◽  
pp. 87-99 ◽  
Author(s):  
Lindsey A. Needham ◽  
Sonja Van Dijk ◽  
Rod Pigott ◽  
R. Mark Edwards ◽  
Maura Shepherd ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Binding Sites ◽  
Cell Adhesion Molecule ◽  
Vascular Cell Adhesion ◽  
Vascular Cell ◽  
Vascular Cell Adhesion Molecule ◽  
Cell Adhesion Molecule 1

The Heterodimeric Sweet Taste Receptor has Multiple Potential Ligand Binding Sites

2006 ◽  
Vol 12 (35) ◽  
pp. 4591-4600 ◽  
Author(s):  
Meng Cui ◽  
Peihua Jiang ◽  
Emeline Maillet ◽  
Marianna Max ◽  
Robert Margolskee ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Taste Receptor ◽  
Sweet Taste ◽  
Sweet Taste Receptor ◽  
Ligand Binding Sites ◽  
Potential Ligand

scholarly journals Docking Studies on Cyclooxygenases-2 Inhibitors based On Potential Ligand Binding Sites

2014 ◽  
Vol 87 (1) ◽  
pp. 27-34
Author(s):  
Karima Nekkaz ◽  
Ismail Daoud ◽  
Kawther Younes ◽  
Salima Merghache ◽  
Naouel Khebichat ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Docking Studies ◽  
Ligand Binding Sites ◽  
Potential Ligand

scholarly journals Quantification of allosteric communications in matrix metalloprotease-1 on alpha-synuclein aggregates and substrate-dependent virtual screening

2021 ◽  
Author(s):  
Sumaer Kamboj ◽  
Chase Harms ◽  
Derek Wright ◽  
Anthony Nash ◽  
Lokender Kumar ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Ligand Binding ◽  
Poisson Process ◽  
Binding Sites ◽  
Conformational Dynamics ◽  
Alpha Synuclein ◽  
Two Dimensional ◽  
Kinetic Rates ◽  
Ligand Binding Sites ◽  
Potential Ligand

AbstractAlpha-synuclein (aSyn) has implications in pathological protein aggregations observed in neurodegenerative disorders, including Parkinson’s and Alzheimer’s diseases. There are currently no approved prevention and cure for these diseases. In this context, matrix metalloproteases (MMPs) provide an opportunity because MMPs are broad-spectrum proteases and cleave aSyn. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, MMP1 activity, and ligands. However, allosteric communications in MMP1 on aSyn-induced aggregates have not been explored. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations, with the two domains well-separated. These open conformations are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics. The best-fit parameters for a Gaussian fit to the distributions of FRET values provide the two states. The ratio of the kinetic rates between the two states comes from the ratio of fitted areas around the two states. The decay rate of an exponential fit to the correlations between FRET values provides the sum of the kinetic rates. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to determine the most appropriate pose. We performed virtual screening against the potential ligand-binding sites on the appropriate aSyn-MMP1 binding pose and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading pathological aggregates in neurodegeneration by targeting MMPs.SignificanceWe have quantified MMP1 interdomain dynamics on aSyn-induced aggregates by a two-state Poisson process. Histograms and correlations of FRET values determine the kinetic rates of interconversion between the two states. We quantify the conformational dynamics of the whole MMP1 and allosteric communications by the two-dimensional matrix of correlations between every pair of amino acids from experimentally-validated all-atom simulations. The two-dimensional correlations lead to a Gray Level Co-occurrence Matrix and a measure of Shannon entropy describing the conformational fluctuations. As such, we address the quantification of allosteric communications, a leading challenge in defining allostery. We report that the potential ligand-binding sites and lead molecules change for MMP1 upon binding alpha-synuclein and depend on the binding pose selected. This suggests that one needs to take the substrate into account while targeting MMPs.

scholarly journals Activated endothelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1.

1992 ◽  
Vol 176 (1) ◽  
pp. 99-107 ◽  
Author(s):  
L Osborn ◽  
C Vassallo ◽  
C D Benjamin
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Binding Sites ◽  
Cell Adhesion Molecule ◽  
Vascular Cell Adhesion ◽  
Vascular Cell ◽  
Vascular Cell Adhesion Molecule ◽  
Cell Adhesion Molecule 1

Vascular cell adhesion molecule-1 (VCAM-1) is induced on endothelial cells by inflammatory cytokines, and binds mononuclear leukocytes through the integrin very late antigen-4 (VLA-4) (alpha 4 beta 1). This adhesion pathway has been implicated in a diverse group of physiological and pathological processes, including B cell development, leukocyte activation and recruitment to sites of inflammation, atherosclerosis, and tumor cell metastasis. The major form of VCAM-1 (VCAM-7D) has seven extracellular immunoglobulin (Ig)-like domains, of which the three NH2-terminal domains (domains 1-3) are similar in amino acid sequence to domains 4-6. By functional analysis of VCAM-7D relative to VCAM-6D (a minor 6-domain form of VCAM-1 in which domain 4 is deleted because of alternative splicing), and chimeric constructs between VCAM-1 and its structural relative intercellular adhesion molecule-1 (ICAM-1), we show that either the first or the homologous fourth domain of VCAM-1 is required for VLA-4-dependent adhesion. Either of these binding sites can function in the absence of the other. When both are present, cell binding activity is increased relative to monovalent forms of the molecule. The homologous binding regions appear to have originated by internal duplication of a portion of a monovalent ancestral gene, before the mammalian radiation. Thus VCAM-1 exemplifies evolution of a functionally bivalent cell-cell adhesion molecule by intergenic duplication. We have also produced a new class of anti-VCAM-1 monoclonal antibodies that block domain 4-dependent adhesion, and demonstrate that both binding sites participate in the adhesion function of VCAM-1 on endothelial cells in vitro. Therefore both sites must be blocked in clinical, animal, or in vitro studies depending on the use of anti-VCAM-1 antibodies to inactivate the VCAM-1/VLA-4 adhesion pathway.

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