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

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 intermediateand 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 αL and β2 subunits, the ligand binding site is located exclusively in the inserted (I) domain of αL (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. SThe 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: [email protected].. 1Present address: Dept. of Biopharmaceutical Sciences and California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94017. 3The 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,N-bis(2-hydroxyethyl)glycine; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; HBS, Hanks’ balanced salt solution. NIH Public Access Author Manuscript J Biol Chem. Author manuscript; available in PMC 2006 May 2. Published in final edited form as: J Biol Chem. 2006 February 24; 281(8): 5042–5049. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript rejection in organ transplantation (5,6). Whereas all available small molecule antagonists for LFA-1 are allosteric inhibitors, many inhibitory antibodies directly block ligand binding to the I domain (5,7). We set out to explore a new class of competitive antagonists that mimic the native ligand, ICAM-1. Engineered high affinity ICAM-1 could serve as a biotherapeutic or a lead molecule in the development of competitive, small molecule agents for treatment of autoimmune diseases. Allostery of LFA-1 on the cell surface, regulated physiologically by inflammatory stimuli and signal transduction through the LFA-1 α and β subunit transmembrane domains, alters affinity for ICAM-1 (5). In the isolated I domain, mutationally introduced disulfide bonds have been used to stabilize the open conformation with high affinity, the intermediate conformation with intermediate affinity, or the closed conformation with low affinity for ICAM-1 (8,9). A recent crystal structure of the αL I domain in complex with ICAM-1 has revealed the binding interface between the αL I domain and ICAM-1 at 3.3-Å resolution (8). The I domain directly coordinates Glu-34 of ICAM-1 through a Mg2+, and a reorientation of Glu-241 of the I domain creates a critical salt bridge to Lys-39 of ICAM-1 (8). Further comparison of liganded and unliganded structures for both high affinity (HA) and intermediate affinity (IA) αL I domains reveals a shape-shifting pathway for integrin regulation by which allosteric signals convert the closed conformation to intermediate or open conformations. Binding of the IA I domain to ICAM-1 stabilizes the same open conformation as seen with the HA I domain (8). The affinity of the HA αL I domain for wild-type ICAM-1 is low (KD = 185 ± 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. 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 have taken multiple structure-based approaches to design ICAM-1 variants with enhanced affinity for αLβ2. Moreover, 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 αLβ2 and αLβ2-dependent adhesion. EXPERIMENTAL PROCEDURES Computational Design The crystal structure of the αL I domain in complex with ICAM-1 (PDB code 1MQ8) was used as the starting template for computational calculations (8). One of the variant libraries was designed using combined output from Protein Design Automation® (PDA) (13,17,18) and Sequence Prediction AlgorithmTM (SPA) (19) calculations. For PDA calculations, the conformations of amino acids at variable positions were represented as a set of backboneindependent side-chain rotamers derived from the rotamer library of Dunbrack and Cohen (20). The energies of all possible combinations of the considered amino acids at the chosen variable positions were calculated using a force field containing terms describing van der Waals, solvation, electrostatic, and hydrogen bond interactions. The optimal (ground state) sequence was determined using a Dead End Elimination algorithm, and a Monte Carlo algorithm was used to evaluate the energies of similar sequences around the predicted ground state. SPA calculations utilize a genetic algorithm to screen for low energy sequences, with energies being calculated during each round of “evolution” for those sequences being sampled. The conformations of amino acids were represented as a set of side-chain rotamers derived from a backbone-independent rotamer library using a flexible rotamer model (21). SPA calculations generated a list of 300 sequences that were subsequently clustered computationally Song et al. Page 2 J Biol Chem. Author manuscript; available in PMC 2006 May 2. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript into groups of similar sequences using a nearest neighbor single linkage hierarchical clustering algorithm. Parameters and other details for PDA and SPA calculations are described elsewhere (13,17–19) and in unpublished results. For these sets of calculations, critical contact residues Glu-34, Lys-39, Asn-68, and Gln-73 were fixed in both sequence and conformation. Calculations were carried out to evaluate single and combinatorial substitutions at variable residues Lys-29, Leu-30, Pro-38, Glu-41, Met-64, Tyr-66, and Thr-75. All residues in contact with these residues were floated, that is the amino acid conformation but not the amino acid identity was allowed to vary to allow for conformational adjustments. Final experimental substitutions, shown in Table 1, were chosen based on their predicted energies relative to wild type and their occupancy, that is the number times the substitution occurred in the set of 1000 Monte Carlo or 300 genetic algorithm sequences. Design calculations using Rosetta differed in the all-atom energy function (22,23), as well as in the amino acid side-chain sampling and optimization protocol. As above, two sets of design calculations were carried out to identify substitutions predicted to stabilize the ICAM-1-Idomain interface. In the first round, only single amino acid substitutions at positions Lys-29, Leu-30, Gly-32, Pro-36, Pro-38, Lys-39, Glu-41, Gln-62, Met-64, Tyr-66, Asn-68, Gly-72, Gln-73, Thr-75, and Lys-77 were modeled. Residue choices evaluated at each position were: Lys-29: Arg, Asp, Glu, and Ser; Leu-30: Asp, Glu, Arg, Lys, Tyr, Trp, and Phe; Gly-32: Ala and Ser; Pro-36: all amino acids; Pro-38: all amino acids; Lys-39: Arg; Glu-41: Arg, Lys, Asp, Ser, and Asn; Gln-62: Arg, Lys, Asp, Glu, Ser, and Asn; Met-64: Leu, Ile, Phe, Tyr, and Val; Tyr-66: Phe and His; Asn-68: Thr, Tyr, Arg, Asp, and Glu; Gly-72: Lys, Arg, Asp, Glu, Asn, Ser, Gln, and Pro; Gln-73: Thr, Asn, Ser, Arg, Asp, and Glu; Thr-75: all residues; and Lys-77: all residues. In a second round, 11 interface residues (Lys-29, Leu-30, Pro-36, Pro-38, Lys-39, Met-64, Tyr-66, Asn-68, Gln-73, Thr-75, and Lys-77) were designed (allowed to change to all 20 naturally occurring amino acids, including the native amino acid type but excluding cysteine) simultaneously. In each case, amino acid side chains contacting the substituted amino acid side chains were repacked (allowing all rotamers of the native amino acid type). Sequences and conformations with low energies were selected using a Monte-Carlo simulated annealing procedure as described previously (24). All resulting protein complex models (in the simultaneous design runs, several hundred models with similar energies were generated) were rescored by computing a predicted binding energy as described (22). Final sequences were selected for the lowest binding energy as described in the main text and are shown in Table 2. PARE (predicting association rate enhancement) mutations were kindly provided by Dr. Gideon Schreiber and Yossi Kuttner (Weizmann Institute of Science, Rehovot, Israel) to alter electrostatic interactions outside of the binding interface to enhance kon without affecting koff (12,25). Construction and Expression of Mutant Libraries cDNA of human ICAM-1 cloned in vector pAprM8 was used as the template. QuikChange (Stratagene) was used to generate single or multiple substitution mutations in the ICAM-1 D1 domain. Mutations were confirmed by DNA sequencing. Transient transfection of 293T cells was as described previously (26). Preparation of I Domain Tetramer A BirA enzyme recognition tag (LGGIFEAMKMELRD) was fused through a Gly-Gly-GlySer linker to the N terminus of the soluble HA mutant αL I domain (residues Gly-128 to Tyr-307, mutant K287C/K294C) (9,27–29). The cDNA was cloned into the NdeI and BamHI sites of the pET-20b vector. Protein was expressed in Escherichia coli BL21(DE3) (Novagen). The Song et al. Page 3 J Biol Chem. Author manuscript; available in PMC 2006 May 2. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript transformed bacteria were cultured in rich media (20 g/liter Tryptone, 10 g/liter yeast extract, 5 g/liter NaCl, 20 ml/liter glycerol, 50 mM K2HPO4, 10 mM MgCl2, 10 g/liter glucose, 100 mg/liter ampicillin). Expression was induced by addition of isopropyl 1-thio-β-Dgalactopyranoside (Invitrogen) to a final concentration of 1 mM when the A600 of cultures was 0.6–1.0. The I domain was refolded and purified as described (9) with some modifications in the step of refolding. Briefly, frozen cells were resuspended in 20 mM Tris (pH 8.0), 150 mM NaCl, with 1 mg/ml lysozyme at 37 °C for 15 min and then disrupted by ultrasonication. Inclusion bodies were harvested by centrifugation. After extensive washing with washing buffer (20 mM Tris (pH 8.0), 23% (w/v) sucrose, 0.5% (v/v) Triton X-100, 1 mM EDTA), the pellet was solubilized by adding 6 M guanidine HCl, 50 mM Tris (pH 8.0), 1 mM dithiothreitol. The I domain was rapidly diluted in a redox buffer (20 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, 5 mM cysteamine/2.5 mM cystamine) to a final concentration of 25 μg/ml and then incubated at 4 °C with slow stirring. Refolding was performed for 4–5 days until no free thiol group was detected with 5,5′-dithio-bis-(2-nitrobenzoic acid) (Pierce). The I domain was precipitated with ammonium sulfate and purified by Superdex S-200 gel-filtration in phosphate-buffered saline. Biotin was ligated to the tag with the BirA ligase (Avidity, Denver, CO). Typically, I domain at a concentration of 1–2 mg/ml was incubated with BirA (15–20 μg/ml) at room temperature overnight in a buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, 10 mM magnesium acetate, 50 mM Bicine (pH 8.3), 10 mM ATP, and 50 μM biotin. The unbound biotin was removed by passing the sample through a Superdex S-200 column (Amersham Biosciences). The biotinylated I domain was mixed with streptavidin-FITC (BIOSOURCE International) or streptavidin (BIO-SOURCE International) at a molar ratio of 8:1 for 2 h at 21 °C. The mixture was then subjected to Superdex S-200 chromatography in TBS (20 mM Tris·HCl, pH 8.0, 150 mM NaCl) to separate the tetrameric complex peak from free I domain. Immunofluorescent Flow Cytometry Plasmids encoding full-length ICAM-1 were transfected into 293T cells using calcium phosphate precipitation (26). The mAbs RR1/1, CBR IC1/11, CBR IC1/12, and CA-7 were used to stain transfected cells as described (30). For soluble I domain tetramer binding, the ICAM-1-transfected cells were washed once with Hanks’ Balanced Salt solution (Invitrogen), 10 mM EDTA, and twice with 20 mM Hepes (pH 7.4), 150 mM NaCl (HBS)/5.5% glucose/1% bovine serum albumin. Cells were incubated with streptavidin-I domain tetramer (2 μg/ml)/ HBS/5.5% glucose/1% bovine serum albumin in the presence of 1 mM Mg2+ at room temperature for 1 h, and washed three times with HBS/1 mM Mg2+. FITC-goat anti-streptavidin (10 μg/ml, Vector Laboratories) and Cy3-labeled CBR IC1/11 (10 μg/ml), a nonblocking antibody to LFA-1 mapped to domain 3 of ICAM-1 (31), were added and incubated with the cells on ice for 15 min. After washing three times with HBS/1 mM Mg2+, cells were subjected to fluorescence flow cytometry. Mean FITC fluorescence intensity (MFI) of Cy3-positive cells was determined. In all experiments, background CBR IC1/11 binding to mock transfected cells and background tetramer binding to ICAM-1-transfected cells in the presence of 5 mM EDTA were subtracted to obtain specific ICAM-1 expression and ligand binding, respectively: % binding = (mutant MFI − background MFI)/(wild-type MFI − background MFI) × 100. Preparation of Soluble ICAM-1 cDNA of the extracellular domain of wild-type or mutant ICAM-1 was fused to pEF-Fc vector (32,33) with a thrombin cleavage site (LVPRGS) between ICAM-1 and human IgG Fc portions. Culture supernatants from GnTI− HEK293S cells (34,35) were collected 5–7 days after transient transfection. ICAM-1 Fc fusion proteins were purified from supernatants on a protein G column (Invitrogen). Contaminating IgGs were removed by gel filtration on a Superdex 200 column (Amersham Biosciences) in TBS (20 mM Tris·HCl, pH 8.0, 150 mM NaCl). ICAM-1 Fc eluted earlier than IgGs. After thrombin cleavage (1 mg of protein/20 units of thrombin, Amersham Biosciences) at room temperature overnight, soluble ICAM-1 D1–5 was further Song et al. Page 4 J Biol Chem. Author manuscript; available in PMC 2006 May 2. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript purified by gel filtration to remove the Fc portion and trace amounts of thrombin. Typical yield was 5 mg/liter. Antibody Binding Assay Antibody-binding assay for soluble ICAM-1-Fc was performed as described (36) except using goat anti-human IgG (I-3391, Sigma) as the capture antibody. Surface Plasmon Resonance ICAM surfaces or a control surface were prepared by injecting ICAMs (20 μg/ml) in 10 mM sodium acetate buffer (pH 4.0) or buffer only (control) over the flow cells activated with Nethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide and then blocking these surfaces with ethanolamine. To prepare I domain surfaces, biotinylated HA I domain (20 μg/ml) or 5 μM biotin as control in 10 mM sodium acetate buffer (pH 4.0) was directly captured on a streptavidin-conjugated sensor chip (Biacore) using 10 mM HEPES (pH 7.4), 150 mM NaCl (HBS-N, Biacore) as running buffer. I domains or ICAMs were infused in 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM MgCl2, and regeneration was done in 20 mM Tris (pH 8.0), 0.3 M NaCl, 20 mM EDTA. The KD value was calculated by curve fitting with Langmuir 1:1 binding model or Scatchard analysis if the binding reached steady-state. koff was derived from curve fitting on the dissociation phases. kon was calculated as koff/KD. Soluble ICAM-1 Binding Binding of soluble, multimeric ICAM-1-IgA-Fc/FITC-anti-IgA to K562 cells expressing wildtype LFA-1 was as described previously (37). For competition assay, the Hi3-ICAM-1 mutant or wild-type ICAM-1 D1–D5 was mixed with ICAM-1-IgA-Fc (5 μg/ml)/FITC-anti-IgA (25 μg/ml) at a series of concentrations and then incubated with the cells at room temperature for 1 h. Cell Adhesion to Immobilized ICAM-1 Interleukin-15 cultured peripheral blood lymphocytes were prepared and maintained as described (38). Adhesion of peripheral blood lymphocytes in the presence of phorbol 12myristate 13-acetate (1 μM) to wild-type ICAM-1 was performed in the presence of the indicated concentration of ICAM-1 D1–D5 as described (36). Cell adhesion in the presence of ICAM-1blocking mAb was <5% of input cells.