Structural plasticity in Ig superfamily domain 4 of ICAM-1 mediates cell surface dimerization

Abstract

The Ig superfamily (IgSF) intercellular adhesion molecule-1 (ICAM-1) equilibrates between monomeric and dimeric forms on the cell surface, and dimerization enhances cell adhesion. A crystal structure of ICAM-1 IgSF domains (D) 3-5 revealed a unique dimerization interface in which D4s of two protomers fuse through edge ␤-strands to form a single super ␤-sandwich domain. Here, we describe a crystal structure at 2.7-Å resolution of monomeric ICAM-1 D3-D5, stabilized by the monomer-specific Fab CA7. CA7 binds to D5 in a region that is buried in the dimeric interface and is distal from the dimerization site in D4. In monomeric ICAM-1 D3-D5, a 16-residue loop in D4 that is disordered in the dimeric structure could clearly be traced as a BC loop, a short C strand, and a CE meander with a cis-Pro followed by a solvent-exposed, flexible four-residue region. Deletions of 6 or 10 residues showed that the C-strand is essential for monomer stability, whereas a distinct six-residue deletion showed little contribution of the CE meander. Mutation of two inward-pointing Leu residues in edge ␤-strand E to Lys increased monomer stability, confirming the hypothesis that inward-pointing charged side chains on edge ␤-strands are an important design feature to prevent ␤-supersheet formation. Overall, the studies reveal that monomer-dimer transition is associated with a surprisingly large, physiologically relevant, IgSF domain rearrangement.crystal structure ͉ leukocytes ͉ flow cytometry ͉ mutagenesis I ntercellular adhesion molecule-1 (ICAM-1; CD54) is perhaps the most important member of a family of related Ig superfamily (IgSF) molecules that serve as ligands for the integrins ␣ L ␤ 2 , ␣ M ␤ 2 , and ␣ X ␤ 2 (1). ICAM-1 is expressed on the surface of cells important in immune responses. Inflammatory mediators further enhance expression of ICAM-1 on these cells and induce it on other cell types, including endothelial, epithelial, and fibroblastic cells. Increased ICAM-1 expression augments immune responses and leukocyte accumulation in inflamed tissues.ICAM-1 consists of five extracellular IgSF domains (D1-D5), a hydrophobic transmembrane domain, and a short cytoplasmic domain (1). In its native state on the cell surface ICAM-1 is in equilibrium between a monomeric and dimeric state (2, 3). Chemical cross-linking reveals a substantial proportion of dimeric material (2, 3). The fraction of monomeric cell-surface ICAM-1 can be estimated by using a mAb termed CA7 that binds to D5 of ICAM-1 (4) and binds much better to monomeric than dimeric ICAM-1 (2). The transmembrane domain of ICAM-1 stabilizes dimerization. ICAM-1 with the transmembrane domain replaced with a glycosylphosphatidylinositol (GPI) anchor is largely monomeric, whereas wild-type ICAM-1 is largely dimeric (2, 3). Recombinant soluble ICAM-1, lacking the transmembrane and cytoplasmic domains, exists as a monomer in solution (2, 4, 5).Crystal structures of D1-D2 and D3-D5 fragments of ICAM-1 have revealed two sites for dimerization, in D1 and D4. A dimerization site on the BE...

Transmigration of Neutrophils across Inflamed Endothelium Is Signaled through LFA-1 and Src Family Kinase

“…We discovered that at a minimum, formation of LFA-1 dimers formed by binding to ICAM-1 dimers, or cross-linked by mAb TS2/4, was sufficient to initiate outside-in signaling. These data led us to speculate that the distance (e.g., ~100 Å) between respective ICAM-1 molecules presented on the recombinant IgG construct attached to our beads, as well as the native homodimer up-regulated on in-flamed endothelium (12, 39), is a strategic one for conducting the outside-in signal via LFA-1. This spatial acuity between ligated pairs of high-affinity LFA-1 may facilitate linkage with accessory molecules such as talin (e.g., cross-sectional area of ~60 nm) that exist as elongated flexible antiparallel dimers with binding sites for several β integrin cytodomains (40, 41).…”

“…For more than a decade it has been known that native ICAM-1 up-regulated on inflamed endothelium exists in equilibrium between the monomeric and dimeric states (10, 11). More recent data suggest that structural rearrangement of the Ig domain supports a transition to dimeric ICAM-1 that may optimally orient the D1 binding site for adhesion via leukocyte LFA-1 (12). Taken together, these data suggest that both LFA-1 conformation and valence in binding ICAM-1 under shear stress can dramatically alter the adhesive dynamics.…”

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“…1B). The binding between subunits in the homodimer occurs through residues exposed on structural rearrangement of Ig domain four (22, 23). Dimerization of ICAM-1 subunits significantly increases the affinity for LFA-1, which may impact the ligand's ability to promote intracellular signaling (24).…”

Cell-stiffness-induced mechanosignaling – a key driver of leukocyte transendothelial migration

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