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

Gang Song,Greg A Lazar,Tanja Kortemme,Motomu Shimaoka,John R Desjarlais,David Baker,Timothy A Springer

doi:10.1074/jbc.m510454200

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 Mg2+ 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.

Highlights

Intercellular adhesion molecule-1 (ICAM-1)3 is a cell surface ligand for lymphocyte function-associated antigen-1 (LFA-1), a member of the integrin family of adhesion receptors [1, 2]
One set of variants was designed using Protein Design Automation (PDA) [13, 17, 18] and Sequence Prediction Algorithm (SPA) [19] calculations to optimize the free energy of the ICAM-11⁄7LFA-1 complex
In a number of cases, similar residues were substituted for functional or biophysical reasons: Gln was used instead of Glu (%Occ Sequence Prediction AlgorithmTM (SPA) ϭ 8) at position 64 due to the lack of a visible interacting residue on LFA-1 yet in proximity to the negative and critical Glu-34 on ICAM-1; Val was included at position 75 because of similarity to the isosteric Thr and high occupancy of Ile (PDA and SPA); Phe was included at position 75 because of a preference for hydrophobics in the calculations; Val was used at position 66 instead of the isosteric Thr, because of the otherwise complete preference for hydrophobics despite the fact that the predicted ␥-hydroxyl H-bond was intramolecular

Summary

Introduction

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface ligand for lymphocyte function-associated antigen-1 (LFA-1), a member of the integrin family of adhesion receptors [1, 2]. The extracellular domains of LFA-1 are composed of the large and complex ␣L and ␤2 subunits, the ligand binding site is located exclusively in the inserted (I) domain of ␣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 rejection in organ transplantation [5, 6]. The affinity of the HA ␣L I domain for wild-type ICAM-1 is low (KD ϭ 185 Ϯ 12 nM) [9] compared with many other protein-protein interactions Enhancement of this affinity is essential for therapeutic applications or for accurate measurement of physiologically induced increase in affinity of LFA-1 on the cell surface. We have measured the kinetics and affinity of I domains stabilized in different conformations for high affinity ICAM-1 mutants and investigated the inhibitory effects of our most improved variant on binding of ICAM-1 to cell surface ␣L␤2 and ␣L␤2-dependent adhesion

Methods

Results

Conclusion