Rational Design of Intercellular Adhesion Molecule-1 (ICAM-1) Variants for Antagonizing Integrin Lymphocyte Function-associated Antigen-1-dependent Adhesion

Abstract

The interaction between integrin lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is critical in immunological and inflammatory reactions but, like other adhesive interactions, is of low affinity. Here, multiple rational design methods were used to engineer ICAM-1 mutants with enhanced affinity for LFA-1. Five amino acid substitutions 1) enhance the hydrophobicity and packing of residues surrounding Glu-34 of ICAM-1, which coordinates to a Mg 2+ in the LFA-1 I domain, and 2) alter associations at the edges of the binding interface. The affinity of the most improved ICAM-1 mutant for intermediate-and high-affinity LFA-1 I domains was increased by 19-fold and 22-fold, respectively, relative to wild type. Moreover, potency was similarly enhanced for inhibition of LFA-1-dependent ligand binding and cell adhesion. Thus, rational design can be used to engineer novel adhesion molecules with high monomeric affinity; furthermore, the ICAM-1 mutant holds promise for targeting LFA-1-ICAM-1 interaction for biological studies and therapeutic purposes.Intercellular adhesion molecule-1 (ICAM-1) 3 is a cell surface ligand for lymphocyte functionassociated antigen-1 (LFA-1), a member of the integrin family of adhesion receptors (1,2). The interaction of LFA-1 and ICAM-1 is critical to many immunological reactions, including T lymphocyte antigen-specific responses and leukocyte accumulation in inflamed tissues (3). Although the extracellular domains of LFA-1 are composed of the large and complex α L and β 2 subunits, the ligand binding site is located exclusively in the inserted (I) domain of α L (4). Many antagonists of this interaction, including monoclonal antibodies to the I domain of LFA-1 and small molecules, have been developed to treat autoimmune diseases and prevent immune * This work was supported by National Institutes of Health Grant CA31798.S The on-line version of this article (available at http://www.jbc.org) contains Fig. S1.2 To whom correspondence should be addressed: CBR Institute for Biochemical Research, 200 Longwood Ave., Boston, MA 02115.Tel.: 617-278-3225; Fax: 617-278-3232; E-mail: springeroffice@cbr.med.harvard.edu.. 1 Present address: Dept. of Biopharmaceutical Sciences and California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94017. 3 The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; LFA-1, lymphocyte function-associated antigen-1; PDA, protein design automation; SPA, sequence prediction algorithm; SPR, surface plasmon resonance; PARE, predicting association rate enhancement; MFI, mean fluorescence intensity; HA, high affinity; IA, intermediate affinity; Bicine, N, Recent advances in computational protein design algorithms (10-12) have markedly improved capabilities for generating novel proteins with optimized properties, including enhanced stability (13), altered substrate specificity (10), improved binding affinity (14,15), and optimized pharmacokinetics (16). We hav...

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