Engineering of Single Ig Superfamily Domain of Intercellular Adhesion Molecule 1 (ICAM-1) for Native Fold and Function

Abstract

The immunoglobulin (Ig) superfamily is one of the largest families in the vertebrate genome, found most frequently in cell surface molecules. Intercellular adhesion molecule-1 (ICAM-1) contains five extracellular Ig superfamily domains (D1-D5) of which the first domain, D1, is the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1) and human rhinovirus. Despite the modular nature of many Ig superfamily domains with respect to domain folding and ligand recognition, D1 does not fold on its own due to the loss of its interaction with the second domain. The goal of this study was to engineer ICAM-1 D1 by introducing mutations that would stabilize the Ig superfamily domain fold while retaining its ability to bind to LFA-1 and rhinovirus. First, with a directed evolution approach, we isolated mutations in D1 that showed binding to conformation-specific antibodies and the ligand binding domain of LFA-1 called the inserted, or I, domain. Then, with a rational design approach we introduced mutations that contributed to the stability of ICAM-1 D1 in solution. The mutations that restored native folding of D1 in isolation were those that would convert hydrogen bond networks in buried regions into hydrophobic contacts. Notably, for most mutations, identical or similar types of substitutions were found in ICAM-1 molecules of different species and other ICAM family members. The systematic approach demonstrated in this study to engineer a single Ig superfamily fold in ICAM-1 can be broadly applicable to the engineering of modular Ig superfamily domains in other cell surface molecules.Intercellular adhesion molecule-1 (ICAM-1) 4 is an inducible transmembrane receptor expressed on the surface of several cell types including leukocytes and endothelial cells and is up-regulated by inflammatory mediators such as interleukin-1, interferon-␥, tumor necrosis factor, and lipopolysaccharide (1). ICAM-1 is the counter receptor for the leukocyte integrins, lymphocyte function-associated antigen-1 (LFA-1, ␣ L ␤ 2 ), Mac-1 (␣ M ␤ 2 ), and p150,95 (␣ x ␤ 2 ), and promotes cellular interactions important in immunity and inflammation (2-7). ICAM-1 is also used as a sequestration receptor for Plasmodium falciparum-infected erythrocytes (8) and has been subverted as a receptor for human rhinovirus (9, 10) and Coxsackievirus A21 (11). Structurally, ICAM-1 consists of five extracellular domains (D1-D5), a transmembrane spanning region, and a short cytoplasmic domain. The second, third, and fourth domains of ICAM-1 contain a number of glycan chains that appear to contribute to ICAM-1 stability and function (12-14). All five extracellular domains of ICAM-1 belong to the immunoglobulin (Ig) superfamily, a family that is the largest in the vertebrate genome (15) and found in many cell surface receptors (16). Ig superfamily domains frequently exist in tandem arrays in surface receptors, and each domain in one molecule may pair with different ligands.The Ig superfamily fold is characterized by a ␤-sandwich structure consisting ...

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“…Curiously, in agreement with our finding that D1 is expected to be one of the most disordered domains of iCAM-1, it was shown that this domain cannot fold on its own likely because of the loss of its interaction with the second domain. 88 Although Figures 1 and 2 show that human vCAM-1 (UniProt ID: P19320) is predicted to be the most ordered of the T2DM biomarkers analyzed in our study, this protein has several disordered and flexible regions characterized by the disorder scores exceeding 0. 5 Figure 1C and 2C by missing the 310-402 (which is substituted by a single alanine residue) and 52-113 regions, respectively.…”

“…The Ig-like domains are known to have different functions. For example, D1 serves as the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1) and human rhinovirus, 88 whereas D3 binds to the Mac-1 integrin. 89 Human iCAM-1 is predicted to have 6 disorder-based potential binding sites (molecular recognition features, MoRFs) and a highly disordered cytoplasmic C-tail, which is heavily decorated with multiple PTMs.…”

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

“…ICAM-1 is a member of the immunoglobulin (Ig) superfamily and the extracellular portion of the molecule has five Ig-like domains termed D1 as the most distal, to D5 as the most proximal to the cell membrane. HRV serotypes binding ICAM-1 have been reported to bind only to D1, while LFA-1 and Mac-1 bind to domains D1 and D3, respectively [21], [22], [23]. Thus antibodies specific to D1 of ICAM-1 would be needed to interfere with HRV infection; however they could potentially interfere with ICAM-1/LFA-1 interactions.…”

Complex Structure of Engineered Modular Domains Defining Molecular Interaction between ICAM-1 and Integrin LFA-1

“…Conformational variability of the loops in D1, analogous to the complementarity determining region (CDR) in antibodies [35], was also pronounced among the crystal structures of the wild-type ICAM-1 D1D2 with as much as 1.1, 2.9, and 4.8 Å RMSD between Cα distances in B–C, D–E, and F–G loops, respectively, whereas the rest of the domain differed by less than 0.6 Å RMSD. Although the stable D1 mutant retained its interaction with conformation-specific antibodies and the LFA-1 I domain, the full-length ICAM-1 D1-D5 containing the mutations found in D1 displayed lower binding to HRV [25]. In addition to the proposed role of charge complementarity at the interface between ICAM-1 and HRV [33], [36], the flexible nature of the loops [15], [24] and hydrogen bond network present in the N-terminal face of ICAM-1 may also be critical to recognition of over 90 different serotypes of rhinovirus by ICAM-1 [37].…”