ICAM-1 Peptide Inhibitors of T-cell Adhesion bind to the allosteric site of LFA-1. An NMR Characterization (original) (raw)
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Journal of Biological Chemistry, 1998
On T cells the leukocyte integrin leukocyte functionassociated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg 2؉ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 ␣ subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg 2؉-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg 2؉activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg 2؉-activated LFA-1 to ICAM-1 is blocked by peptides covering the ␣4-3 loop, the 3-␣5 loop, and the ␣5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the -propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.
Journal of Biological Chemistry, 1998
By extensive mutagenic analysis of the inserted domain (I-domain) of the ␣-chain (CD11a) of the leukocyte function-associated antigen-1 (LFA-1), we have defined a putative binding surface for intercellular adhesion molecules 1 and 2 (ICAM-1 and-2). This analysis showed that individually mutating Leu-205 or Glu-241 to alanine completely abolished LFA-1 binding to ICAM-1 or-2 without affecting I-domain structure, as assayed by antibody binding. Mutating Thr-243 to alanine also had a profound effect on LFA-1 binding to ICAM-1 and-2, as seen by complete loss of binding to ICAM-1 and a significant reduction (70% decrease) in binding to ICAM-2. Mutating Glu-146 to alanine reduced LFA-1 binding to ICAM-1 or-2 by 70%, and mutating His-264 or Glu-293 to alanine reduced binding to ICAM-1 or-2 by about 30-40%. Mutating Thr-175 to alanine reduced binding to ICAM-1 by about 30% and binding to ICAM-2 by about 70%. Interestingly, mutating Lys-263 to alanine preferentially abolished LFA-1 binding to ICAM-2. Using these data, we have generated a model of the interface between the LFA-1 I-domain and residues in the first domain of ICAM-1 that have been shown to be critical for this interaction. In addition, this model, together with the ICAM-2 crystal structure, has been used to map residues that are likely to mediate LFA-1 I-domain binding to ICAM-2. Leukocyte function-associated antigen-1 (LFA-1) 1 is a member of the leukocyte integrin family. Like other members of this family, it is expressed exclusively on leukocytes and shares a common -chain ( 2 , CD18). The four members of this family that differ in their ␣-chains are LFA-1 (CD11a, ␣L), Mac-1 (CD11b, ␣M), p150,95 (CD11c, ␣X), and ␣d 2 (CD11d, ␣d). LFA-1 is constitutively expressed on the cell surface and is thought to undergo a conformational change that renders it capable of high affinity ligand binding. The primary ligands for LFA-1 are intercellular adhesion molecules-1,-2, and-3 (ICAM-1,-2, and-3). The interaction of ICAM-3 with LFA-1 is of considerably lower affinity than ICAM-1 or-2 binding to
Proceedings of The National Academy of Sciences, 1993
The signaling that causes the leukocyte integrin lymphocyte function-associated molecule (LFA-1) to bind firmly to its ligand intercellular adhesion molecule 1 (ICAM-1) is transduced indirectly through other T-cell receptors and is termed inside-out signaling. We show here that the highaffinity state of LFA-1 is characterized by expression of the LFA-1 epitope detected by monoclonal antibody 24. This epitope is expressed not in response to the initial agonistmediated signal but when LFA-1 binds to ICAM-1, indicating that ligand binding induces an alteration in LFA-1. As would be predicted, the monoclonal antibody 24 epitope is confined to the LFA-1, which is located at the site ofcontact between T cells and ICAM-1-expressing transfectants. When a fixation protocol for "freezing" receptors is used, only T cells that are fixed after prior exposure to ICAM-1 bind frmly to ICAM-1 a
Febs Letters, 1991
The adhesion of human T lymphoblasts to ICAM-l-expressing normal dermal fibroblasts has been assessed as a sensitive model system for the analysis of the interaction of the leucocyte integrin LFA-I with its counter-receptor ICAM-I. Using this model system, the effects of factors known to regulate the activity of LFA-I have been quantitated: temperature; concentration of divalent cations; and exposure to phorbol esters. We show here that under the appropriate assay conditions, this model system represents a useful and simple alternative to the detection of leucocyte binding to purified ICAM-I and also has the additional advantage of permitting more sensitive quantification than is possible using the homotypic adhesion assay.
Protein Science, 2006
The lymphocyte function-associated antigen-1 (LFA-1) binding of a unique class of small-molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule-1 (sICAM-1) and A-286982, which respectively define direct and allosteric competitive binding sites within LFA-1's inserted (I) domain. All three molecules antagonized LFA-1 binding to ICAM-1-Immunoglobulin G fusion (ICAM-1-Ig) in a competition ELISA, but only compound 3 and sICAM-1 inhibited the binding of a fluorescein-labeled analog of compound 3 to LFA-1. Compound 3 and sICAM-1 displayed classical direct competitive binding behavior with ICAM-1. Their antagonism of LFA-1 was surmountable by both ICAM-1-Ig and a fluoresceinlabeled compound 3 analog. The competition of both sICAM-1 and compound 3 with ICAM-1-Ig for LFA-1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross-linking studies with a photoactivated analog of compound 3 localized the high-affinity smallmolecule binding site to the N-terminal 507 amino acid segment of the a chain of LFA-1, a region that includes the I domain. In addition, cells transfected with a variant of LFA-1 lacking this I domain showed no significant binding of a fluorescein-labeled analog of compound 3 or ICAM-1-Ig. These results demonstrate that compound 3 inhibits the LFA-1/ICAM-1 binding interaction in a directly competitive manner by binding to a high-affinity site on LFA-1. This binding site overlaps with the ICAM-1 binding site on the a subunit of LFA-1, which has previously been localized to the I domain. ; fax: (650) 225-5337.
Journal of Biological Chemistry, 1996
Intercellular adhesion molecule 3 (ICAM-3; CD50) is the predominant counter-receptor on resting T cells and monocytes for the leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18), and may play an important role in the initial stages of the T cell-dependent immune response. Deletion of individual immunoglobulin superfamily (IgSF) domains of ICAM-3 and ICAM-3 IgSF domain chimeras with CD21 showed there is a single LFA-1 binding site in ICAM-3 and that IgSF domain 1 is necessary and sufficient for LFA-1 binding. Epitope mapping and functional studies performed with 17 anti-ICAM-3 monoclonal antibodies demonstrated that only some monoclonal antibodies, with epitopes wholly within domain 1 of ICAM-3, were able to block binding of ICAM-3 bearing cells to purified LFA-1, in agreement with the data obtained from the domain deletion mutants and CD21 chimeras. Analysis of a panel of 45 point mutants of domain 1 of ICAM-3 identified five residues that may contact LFA-1 as part of the binding site, Asn 23 , Ser 25 , Glu 37 , Phe 54 , and Gln 75. These five residues are predicted by molecular modeling, based on the structure of vascular cell adhesion molecule 1 (VCAM-1), to cluster in two distinct locations on domain 1 of ICAM-3 on the BED face (Asn 23 and Ser 25) and on the C strand or CD loop (E37), the E strand (F54), and the FG loop (Q75). The residues, Asn 23 and Ser 25 , comprise a consensus N-linked glycosylation site.
?L?2 Integrin/LFA-1 Binding to ICAM-1 Induced by Cytohesin-1, a Cytoplasmic Regulatory Molecule
Cell, 1996
Schwartz, 1995; Pasqualini and Hemler, 1994). and Brian Seed † ‡ The most prominent integrins in the immune system *Laboratorium fü r Molekulare Biologie form a family of three members, each consisting of a Genzentrum der Universitä t Mü nchen common  subunit (2) paired with a different ␣ subunit. D-81375 Mü nchen By convention, the different integrins are called ␣L2 Federal Republic of Germany (LFA-1, CD11a/CD18); ␣M2 (Mac-1, CD11b/CD18; † Department of Genetics CR3); and ␣X2 (p150,95, CD11c/CD18). All three are Harvard Medical School expressed by neutrophils, whereas lymphocytes and Boston, Massachusetts 02115 natural killer cells typically express only ␣L2. The physi- ‡ Department of Molecular Biology ological importance of the leukocyte integrins is under-Massachusetts General Hospital scored by the finding that individuals lacking the 2 Boston, Massachusetts 02114 subunit suffer from a severe syndrome, leukocyte adhe- § Institute of Immunology-VIRCC sion deficiency, characterized by an inability to clear University of Vienna pathogens, recurrent infections, and, frequently, death A-1235 Vienna at an early age (reviewed by Springer, 1994). Austria