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J. Biol. Chem., Vol. 277, Issue 21, 18769-18776, May 24, 2002

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Delineation of the Key Amino Acids Involved in Neutrophil Inhibitory Factor Binding to the I-domain Supports a Mosaic Model for the Capacity of Integrin _M2 to Recognize Multiple Ligands^*

Valentin A. Ustinov and Edward F. Plow^§

From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, and Department of Molecular Cardiology/NB50, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, October 24, 2001, and in revised form, February 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To gain insight into the mechanism by which the _MI-domain of integrin _M2 interacts with multiple and unrelated ligands, the identity of the neutrophil inhibitory factor (NIF) recognition site was sought. A systematic strategy in which individual amino acid residues within three previously implicated segments were changed to those in the _LI-domain, which is structurally very similar but does not bind NIF, was implemented. The capacity of the resulting mutants, expressed as glutathione S-transferase fusion proteins, to recognize NIF was assessed. These analyses ultimately identified Asp¹⁴⁹, Arg¹⁵¹, Gly²⁰⁷, Tyr²⁵², and Glu²⁵⁸ as critical for NIF binding. Cation binding, a function of the metal ion-dependent adhesion site (MIDAS) motif, was assessed by terbium luminescence to evaluate conformational perturbations induced by the mutations. All five mutants bound terbium with unaltered affinities. When the five residues were inserted into the _LI-domain, the chimera bound NIF with high affinity. Another ligand of _M2, C3bi, which is known to use the same segments of the _MI-domain in engaging the receptor, failed to bind to the chimeric _LI-domain. Thus, the _MI-domain appears to present a mosaic of exposed amino acids within surface loops on its MIDAS face, and different ligands interact with different residues to attain high affinity binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrins are cell surface glycoprotein receptors that are major contributors to cell-cell and cell-matrix interactions, cell migration, growth, differentiation, and extracellular matrix assembly (1-4). Members of the integrin family are noncovalent / heterodimers (5-7). Each  and  subunit is composed of a short cytoplasmic tail, a single transmembrane domain, and a large extracellular domain (several hundred amino acids) to which numerous ligands bind. _M2 is a member of the ₂ integrin subfamily, which also includes _L2, _X2, and _D2. These integrins share a common  subunit (CD18) of 95 kDa, which is noncovalently linked to distinct but homologous ~150-kDa  subunits (8-11). Numerous physiological functions have ascribed to _M2, including adhesion and transmigration of leukocytes through endothelium (11-13), phagocytosis of foreign materials (14), activation of neutrophils and monocytes (15), and apoptosis of neutrophils (16). Excessive activation of _M2 contributes to sustained inflammation, reperfusion injury, and tissue damage (17). The importance of this integrin subfamily is underscored by the severe phenotype of human patients congenitally deficient in the ₂ integrins (18, 19).

_M2 resides primarily on neutrophils and monocytes and can recognize a wide variety of ligands, including C3bi (20), neutrophil inhibitory factor (NIF)¹ (21, 22), ICAM-1 (23), fibrinogen (24, 25), factor X (26), lipopolysaccharide (27), zymosan (28), gp63 (29), and denatured proteins (30). These ligands are not recognized by _L2 (11, 31), and this extensive repertoire of _M2 may explain why neutrophils adhere to such a broad range of immobilized substrates, including fibronectin (32, 33), vitronectin (33), fibrinogen (32, 33), thrombospondin (33), laminin (32, 33), and collagen (33), in an _M2-dependent manner. The molecular mechanism by which _M2 can interact with so many structurally unrelated ligands and yet exhibit high affinity for each is unknown. Even more confounding is the fact that many ligands of _M2 interact with a relatively small segment of the integrin, the I-domain within its _M subunit (22, 34-39).

I-domains (also known as A-domains), are inserted domains of ~200 amino acids, which are found in nine integrin -subunits as well as other proteins (e.g. von Willebrand factor) and have been implicated in mediating a variety of protein-protein interactions, including ligand binding to integrins. Integrin  subunits, including ₂, may also contain an I-like domain (40). The _MI-domain and _LI-domain were the first of several I-domains to have their three-dimensional structures solved by x-ray crystallography (41, 42). They are all similar, being composed of six or seven -helices and five -sheets connected by flexible loops. A cation-binding site is formed at the vertex of the -sheets, and the bound cation is coordinated in a MIDAS motif (41). Point mutations at residues involved in coordination of cation within the _MI-domain destroy C3bi binding to the _M2 receptor (34). Using blocking monoclonal antibodies, which map to the I-domain or recombinant _MI-domains, the MIDAS face of the _MI-domain has been implicated in the binding of ICAM-1 and fibrinogen (36, 39, 43) as well as C3bi (38, 44) and NIF (45, 46). Taken together, these data suggest that the _MI-domain can fold and function as an independent structural unit, capable of interacting with many different proteins. Nevertheless, the ligand binding function of _M2 is not merely a property of its I-domain. The ₂ subunit and the EF-hand-like cation binding domain adjacent to the I-domain in the  subunit also may contribute to ligand recognition (47).

NIF was originally identified as an activity within the extracts of canine hookworms that inhibited many neutrophil functions, such as adhesion to endothelial cells and the respiratory burst (21). These effects arose from its specific binding to _M2 but not to other ₂ integrins. Subsequently, it was shown that NIF completely blocked _M2-mediated binding of C3bi (38) and ICAM-1 (22) and adhesion to protein-coated surfaces (46) and partially blocked fibrinogen binding (37) to _M2-bearing cells. As an antagonist, NIF has been shown effective in attenuating the deleterious effects of excessive neutrophil activation, such as tissue damage and ischemia-reperfusion injury in an animal model (48, 49), and, hence, it is a potential anti-inflammatory drug. Three specific segments, Pro¹⁴⁷-Arg¹⁵², Pro²⁰¹-Lys²¹⁷, and Asp²⁴⁸-Arg²⁶¹, on the face of the _MI-domain containing the MIDAS were implicated in NIF binding by a homologous scanning mutagenesis approach (50). In this approach, small structural units (helices, -sheets, and loops) in the _MI-domain were replaced by the homologous sequences from the _LI-domain, and the segments noted above were identified as being necessary for NIF binding. In the present report, we have sought to precisely define the amino acids within these segments that are required for high affinity recognition of NIF by _MI-domain, thereby defining the molecular basis for binding of this model ligand to _M2. Ultimately, five individual amino acid residues are identified within the _MI-domain as being critical to NIF binding. The placement of these five residues and our knowledge of the contact sites for other residues in the _MI-domain support a new molecular explanation for the capacity of this integrin to recognize multiple ligands.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- NIF was kindly provided by Corvas International (San Diego, CA). The NIF used in this study was a recombinant protein expressed in Pichia pastoris and was purified to homogeneity as described Muchowski et al. (37). Monoclonal antibodies 44, IB4, and CBL 189 were obtained from Sigma, the American Tissue Culture Collection (Manassas, VA), and Chemicon (Temecula, CA), respectively.

Site-directed Mutagenesis, Expression, and Purification of I-domain Fusion Proteins-- The cDNAs of _MI-domain (675 nucleotides, Arg¹¹⁵-Ser³⁴⁰) and _LI-domain (630 nucleotides, Pro¹²⁰-Ser³³⁰) were cloned and inserted into the pGEX-5X-3 expression vector (37). All wild-type and mutant I-domains were expressed as GST fusion proteins. Mutations were created in these I-domains by oligonucleotide-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA). The selective introduction of the desired mutations into the I-domains was confirmed by DNA sequence analyses. I-domain expression plasmids were used to transform competent Escherichia coli cells (BL21(DE3)pLysS (Stratagene). One liter of L broth (Bio 101, Carlsbad, CA), containing 100 µg/ml ampicillin (Sigma), was inoculated with 1 ml of an overnight culture of transformed E. coli, and cells were grown at 37 °C with vigorous shaking until an absorbance of 0.6-1 OD at 600 nm was attained. Expression was induced by the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (Amersham Biosciences). Four hours after induction, cells were harvested by centrifugation and resuspended in 20 ml of sonication buffer (PBS containing 100 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (Calbiochem) and 50 µg/ml leupeptin (Calbiochem)). Cells were disrupted with four 30-s cycles at 70 watts in a Branson Ultrasonics power sonicator. Insoluble cellular membranes were removed by centrifugation at 25,000 × g for 15 min at 4 °C. The glutathione S-transferase (GST)-I-domain fusion proteins were present in the supernatant and were purified by adsorption onto glutathione-Sepharose 4B (Amersham Biosciences) and elution with elution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM glutathione, 2.5 mM CaCl₂). Such preparations of the fusion proteins were ~90% pure as assessed by SDS-PAGE. Concentrations of purified proteins were determined using the BCA Protein Assay (Pierce) and by spectrophotometry at 280 nm.

Radiolabeling and NIF Binding Assays-- NIF (100 µg) was radiolabeled with 0.7-1.0 mCi of Na¹²⁵I (Amersham Biosciences) using the IODO-BEADS iodination reagent from Pierce. The reaction mix was incubated for 15 min at 22 °C. Radiolabeled protein was separated from free iodine by gel filtration through a PD10-DG column (Bio-Rad) using PBS containing 1% BSA (Calbiochem) as the elution buffer. The specific activity of radiolabeled protein was ~12 µCi/µg.

To assess NIF binding to the recombinant I-domains, ¹²⁵I-NIF (from 66 pM to 330 nM) was mixed with 1 µg of _MI-domain (or _LI-domain) fusion proteins bound to glutathione-Sepharose 4B in TBS (20 mM Tris-HCl, pH 7.6, 250 mM NaCl) containing 1 mM Ca²⁺ in a final volume of 50 µl. The mixture was incubated overnight with agitation at 4 °C. The resin then was washed three times with TBS, and bound ¹²⁵I-NIF was measured with a  counter. Based upon the previous evidence that NIF binds to a single high affinity binding site in the _MI-domain, NIF titration data were fit to a single site binding model using the following equation: [NIF]_bound = B_max × [NIF]/(K_d + [NIF]), where B_max represents the maximal binding and K_d is its dissociation constant, using the SigmaPlot program (Jandel Co., San Rafael, CA). K_d values in Tables I-III are presented as means ± S.D.

For selected experiments, the I-domain was cleaved from its GST fusion partner by incubation of the recombinant protein adsorbed onto glutathione-Sepharose 4B with factor Xa (10 µg/ml factor Xa per 200 µg/ml I-domain) overnight at room temperature with agitation. The cleaved _MI-domain was recovered by washing the resin with 3 column volumes of TBS, and the eluate was concentrated by centrifugation through a Millipore membrane. For solid phase binding assays with the isolated _MI-domain, 96-well microtiter plates (Costar, Cambridge, MA) were coated with the _MI-domain at 50 µg/ml overnight at 4 °C and postcoated with 3% BSA. ¹²⁵I-NIF was added, and binding was measured as described above.

C3bi Binding to the I-domains-- C3bi binding was performed as described by Bilsland et al. (51) with certain modifications. Sheep erythrocytes (Colorado Serum Co., Denver, CO) at 7 × 10⁸ were washed twice in HBSS, containing 5 mM HEPES and 1 mM Mg²⁺, and treated with anti-sheep erythrocyte IgM antibody M1/87 (Accurate Chemical and Scientific Co., Westbury, NY) and human C5-deficient serum (Sigma). The coated erythrocytes were surface-labeled with biotin using 1 mg of sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce) at 37 °C for 20 min. The biotinylated cells were resuspended in 0.9 ml of HBSS with 5 mM HEPES, 1 mM Ca²⁺, and 1 mM Mg²⁺; mixed with 100 µl of C5-deficient serum; and incubated at 37 °C for 60 min. After washing twice, the resulting erythrocytes were resuspended in 2 ml of the above solution, and the cells were disrupted in a sonicator (Branson Ultrasonics, VWR Scientific) with one 3-s cycle at 70 watts. The insoluble cellular material recovered by centrifugation at 25,000 × g for 15 min at 4 °C was used as a C3bi source. To test the interaction of the wild-type _M, _L, and chimeric I-domains with C3bi, 96-well plates (Immulon 4BX; Dynex Technologies Inc., Chantilly, VA) were coated with the I-domains at 50 µg/ml for 1 h at 37 °C and postcoated with 2% BSA for 1 h at 37 °C. After washing three times, the C3bi in 20 mM Tris-HCl, pH 7.6, containing 100 mM NaCl and 2 mM CaCl₂, was added to the wells and incubated for 1 h at 37 °C. After washing three times, the bound C3bi was detected using avidin-alkaline phosphatase and p-nitrophenyl phosphate. As a control, the background reaction on BSA-coated wells was subtracted.

Luminescence Emission Spectroscopy-- Tb³⁺ luminescence experiments were performed as previously described (52). The excitation wavelength used was 285 nm, and Tb³⁺ emission was measured at 545 nm. Emission spectra were corrected for the blank contribution, and the instrument response and normalized to an absorbance of 0.100 at 285 nm in a quartz cell of 0.5- or 1-cm path length. All measurements were made in an FP-777 spectrofluorometer (Jaco, Hauppauge, NY).

SDS-PAGE-- Gel electrophoresis was performed according to the method of Laemmli (53) in 8% acrylamide gels. Proteins were detected in the gels by staining with 0.04% Coomassie G-250 (Bio-Rad).

Molecular Modeling-- The model building of the _MI-domain based upon its crystal structure (54), Protein Data Bank code 1JLM, was carried out using InsightII (MSI, San Diego, CA) using the program defaults, unless otherwise stated, on a Silicon Graphics Indigo work station. The charges on the amino acids of _MI-domain were adjusted to pH 7.2. The I-domain was then soaked with five layers of water, and the minimized energy conformation was obtained by gradually annealing the protein water as previously described (55). The resulting minimized structure was then subjected to a series of molecular dynamic simulations and energy minimizations, referred to as the molecular simulation procedure. Docking was performed manually with a distance cut-off of 15 Å using MSI's docking program. Final van der Waals and electrostatic energies of interaction were obtained using a distance cut-off of 100 Å.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification and NIF-binding Function of Recombinant _MI-domains-- Previous studies have established that the _MI-domain is sufficient to account for the high affinity and specific binding of NIF to integrin _M2 (21, 22, 37). Furthermore, based upon a homolog mutagenesis strategy, in which homologous small structural elements, -helixes, -sheets, and connecting loops of the _MI-domain were swapped into the _LI-domain, three segments, corresponding to connecting loops on the cation-binding MIDAS face of the I-domain were specifically implicated in NIF recognition by _M2 (50). These segments were Pro¹⁴⁷-Arg¹⁵², Pro²⁰¹-Lys²¹⁷, or Asp²⁴⁸-Arg²⁶¹ (numbering based on the amino acid sequence of intact _M; Pro²⁰¹-Lys²¹⁷ actually consisted of two contiguous segments, Pro²⁰¹-Gly²⁰⁷ and Arg²⁰⁸-Lys²¹⁷, but are combined for simplicity). Based upon these results, we sought to localize the specific amino acid residues within these segments of the _MI-domain that are required for the high affinity recognition of NIF, thereby providing a molecular basis for recognition of this by _M2.

To begin this analysis, wild-type _MI-domain (Arg¹¹⁵-Ser³⁴⁰), wild-type _LI-domain (Pro¹²⁰-Ser³³⁰), and the three homolog scanning mutants were expressed as GST fusion proteins in E. coli. The recombinant proteins were purified from the bacterial lysates on glutathione-Sepharose. The purified I-domains migrated as major bands of ~48 and ~52 kDa for _LI-domain and _MI-domain, respectively, when analyzed by SDS-PAGE (Fig. 1, lanes 1 and 2), consistent with their predicted molecular weights. The three homolog scanning mutants also migrated as single bands on the gels (Fig. 1, lanes 3-5).

View larger version (84K): [in this window] [in a new window]   Fig. 1.   Characterization of representative I-domains by SDS-PAGE. I-domains, expressed as GST fusion proteins, were purified on glutathione-Sepharose, electrophoresed on 7% acrylamide gels under reducing conditions, and stained with Coomassie G-250. Lane 1, affinity-purified LI-domain; lane 2, MI-domain; lane 3, Pro147-Arg152 homolog scanning mutant; lane 4, Pro201-Lys217 homolog scanning mutant; lane 5, Asp248-Arg261 homolog scanning mutant.

A facile binding assay for quantifying NIF binding to the recombinant I-domains was developed by measuring the binding of ¹²⁵I-NIF to the recombinant I-domains captured onto glutathione-Sepharose. 1 mM Ca²⁺ was used as the cation. After incubation for 2 h at 37 °C, the beads were washed by centrifugation and counted. As shown in Fig. 2A, ¹²⁵I-NIF exhibited minimal reaction with glutathione-Sepharose. Minimal reaction also was observed between the _LI-domain fusion protein and radiolabeled NIF (Fig. 2A). With the _MI-domain, binding was observed, which was dependent on the concentration of ¹²⁵I-NIF added; 50% saturation was attained at 170 pM added. The K_d of the interaction of ¹²⁵I-NIF with the _MI-domain GST fusion protein was estimated from a Scatchard plot to be 5 nM, consistent with the reported K_d of NIF for the _MI-domain (22) and for intact _M2 (50). A similar interaction of ¹²⁵I-NIF with the _MI-domain that was cleaved from the GST fusion protein and immobilized directly to the microtiter plates was also observed (see Fig. 2A). The K_d of this interaction was estimated to be 4 nM, indicating that the GST fusion partner did not influence NIF binding to the _MI-domain.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   NIF binding to recombinant I-domains. A, 125I-NIF was added at the indicated concentrations to glutathione-Sepharose (in a final reaction volume of 50 µl) armed with the MI-domain GST fusion proteins (1 µg) (), the LI-domain GST fusion protein (), GST alone (), or unarmed glutathione-Sepharose (). Alternatively, the 125I-NIF was incubated with microtiter wells coated with purified MI-domain (50 µg/ml) cleaved from its GST fusion partner (). The incubation was performed in the presence of 1 mM Ca2+, and samples were processed as indicated under "Experimental Procedures." B, MI-domain GST fusion protein bound to glutathione-Sepharose was incubated with 125I-NIF (132 ng) in the presence of the different following inhibitors: control (no addition); nonlabeled NIF (528 ng); EDTA (10 mM); monoclonal antibody 44 (100 ng) to the MI-domain; and monoclonal antibody IB4 (100 ng) to the 2 subunit. Values are presented as the means ± S.E. of at least three experiments.

The specificity of the interaction of radiolabeled NIF with the _MI-domain was indicated by the ability of nonlabeled NIF and EDTA to block binding (Fig. 2B). The inhibition produced by unlabeled NIF at 5 nM was 80% and did not increase further when the concentration of unlabeled NIF was increased by 20-fold, indicating that ~20% of the binding of the radiolabeled ligand was nonspecific. In separate experiments, we found that the capacity of unlabeled NIF to inhibit the binding of ¹²⁵I-NIF was dose-dependent. Half-maximal inhibition of the specific binding of ¹²⁵I-NIF added at 170 pM binding occurred at 150 pM unlabeled NIF (37). Also, as shown in Fig. 2B, monoclonal antibody 44 to the _MI-domain, which blocks with NIF binding to _M2 (56), inhibited its binding to the _MI-domain fusion protein to a level similar to that obtained with unlabeled NIF. As a control, monoclonal antibody IB4, which recognizes ₂ subunit (57), failed to affect NIF binding to the recombinant _MI-domain.

NIF Binding to "Triple Mutants" of the _MI-domain-- Mutant _MI-domains were expressed as GST fusion proteins in which the three segments, Pro¹⁴⁷-Arg¹⁵², Pro²⁰¹-Lys²¹⁷, or Asp²⁴⁸-Arg²⁶¹, implicated by homolog scanning mutagenesis, were swapped to the corresponding _LI-domain segments, and their binding of NIF was assessed. As shown in Fig. 3, the interaction of each of these mutants with ¹²⁵I-NIF was greatly diminished compared with the _MI-domain and was similar to that of the _LI-domain. Essentially no specific binding of NIF to these mutant I-domains was detected. To begin to identify the individual residues within these three segments that mediated NIF recognition, a series of 13 triple mutants were created. In each of these triple mutants, a set of three consecutive amino acids within the _MI-domain was changed to the corresponding _L residues. The specific substitutions made are identified in Fig. 4. If the _M and _L residues were the same, the amino acid was mutated to that present in the corresponding position in the _XI-domain; or if the residues were identical in all three I-domains, the amino acid was replaced with an Ala or Gly (see Fig. 4). After the DNA sequence of each mutant I-domain was confirmed, it was expressed in E. coli and purified on glutathione-Sepharose. When analyzed by SDS-PAGE, each mutant migrated as a single band of ~52 kDa (not shown), similar to the GST fusion proteins characterized in Fig. 1. ¹²⁵I-NIF binding to each triple mutant was then assessed. The results in Fig. 5A show the binding of the 13 triple mutants to a concentration of 170 pM ¹²⁵I-NIF, a concentration below its K_d for the _MI-domain. Of the 13 triple mutants, five, P147S/H148D/D149E, F150A/R151Q/R152K, G207L/R208L/T209A, G251T/Y252D/E253S, and P257D/E258A/A259G, showed a significant reduction in NIF binding. The K_d values of these five triple mutants for NIF were estimated from binding isotherms and are summarized in Table I. The values were reduced by at least 10-fold compared with the wild-type _MI-domain.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   NIF binding to homolog scanning mutants of the MI-domain. The MI-domain GST fusion proteins were as follows: wild-type MI-domain () and Pro147-Arg152 homolog scanning mutant (), Pro201-Lys217 homolog scanning mutant (), and Asp248-Arg261 homolog scanning mutant (). These were bound to glutathione-Sepharose and incubated with 125I-NIF, and binding was measured as in Fig. 2. Values are presented as the means ± S.E. of at least three experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Sequence alignments of the MI-, LI-, and XI-domains and the various mutants constructed. Amino acid residues 146-264 for the MI-domain are aligned with the corresponding sequences in the LI-domain and XI-domain (the numbering is based on the entire protein sequence of the M subunit including the signal peptide). The triple mutants are lined above. In cases where the amino acids in the MI- and LI-domains were identical, the mutation was made to the residue in the XI-domain, and when the residue was conserved in all three I domains, it was changed to an Ala or a Gly.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   NIF binding to MI-domain triple mutants (A) and single mutants (B). The GST fusion proteins were adsorbed into glutathione-Sepharose 4B, and 125I-NIF binding was measured as in Fig 2. Each data set is the mean ± S.E. of at least three independent experiments.

View this table: [in this window] [in a new window]   Table I Kd values (means ± S.D.) for binding of NIF to the wild-type and multiple mutants MI-domains

NIF Binding to Single Point Mutants of the _MI-domain-- Next, within the five triple mutants with defective NIF binding, each of the three amino acids was mutated individually to the corresponding residue in the _LI-domain (see Fig. 4). Thus, a total of 15 single mutants were created. After again confirming the DNA sequence of these single mutants, each was expressed as a GST fusion protein in E. coli and purified. The capacity of the 15 single mutants to bind NIF is summarized in Fig. 5B. Within each of the five triple mutants with reduced NIF recognition, only one of the three single mutants exhibited reduced ¹²⁵I-NIF binding. These five single mutants were D149E, R151Q, G207L, Y252D, and E258A. The K_d values of these single mutants for NIF were estimated and were increased by 10-16 times compared with the wild-type _MI-domain (see Table II).

View this table: [in this window] [in a new window]   Table II Kd values (means ± S.D.) for binding of NIF to the wild-type and single mutant MI-domains

Conformational Considerations-- Loss of NIF binding function in these single mutants could reflect direct involvement of the specific residues in NIF binding or conformational perturbation of the resulting _MI-domain due to substitutions at these positions. Two approaches were deployed to distinguish between these possibilities. First, we considered that alterations in the cation binding function of the MIDAS motif would provide a sensitive barometer of conformational alterations, since the three-dimensional fold of the _MI-domain is necessary to bring the five cation-coordinating residues into the appropriate spatially proximity. Tb³⁺, a fluorescent trivalent cation that replaces Ca²⁺ in most metal-binding proteins, including in I-domains (58), shows little luminescence when free but does so when it binds and can receive energy from an adjacent tryptophan residue (59, 60). An appropriate tryptophan is not present in the _MI-domain, but Phe²⁴⁶ is in close proximity to the MIDAS and resides on the hydrated surface in the _MI-domain crystal. Phe²⁴⁶ is not involved in NIF binding (see Fig. 5). Computer modeling using the known crystal structure of the _MI-domain as a template suggested that this residue could be mutated to tryptophan without perturbing conformation. The results shown in Fig. 6 indicate that the strategy of substituting Trp for Phe²⁴⁶ was effective. The Tb³⁺ luminescence of the _MI-domain with the Trp was >50 times greater than that of the wild-type _MI-domain with Phe at position 246. Therefore, this substitution was introduced into each of the five single point mutant _MI-domains with altered NIF binding function. Each of the five single mutants exhibited a similar emission spectrum (i.e. similar Tb³⁺ luminescent properties to the wild-type _MI-domain with the Trp substitution) (Fig. 6). The affinities of all five mutants estimated from concentration curves were also similar. The estimated K_d values of the mutants for Tb³⁺ and the wild-type _MI-domain are summarized in Table III. The values for the five mutants varied by less than 26% and were not significantly different. These data suggest that five point mutations did not perturb the conformation of the _MI-domain.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Terbium luminescence of mutant MI-domains. The five point mutants were introduced individually into the MI-domain also containing the F246W substitution. Tb3+ was added at 125 µM. The excitation wavelength was 285 nm, and the emission spectra are recorded at 545 nm. The spectra are as follows: wild-type MI-domain (), F246W (), D149E (), R151Q (), G207L (), Y252D (), and E258A ().

View this table: [in this window] [in a new window]   Table III Kd values (means ± S.D.) for binding of Tb3+ to wild-type and mutant MI-domains

As a second approach, we tested the prediction that introduction of these five point mutations into the _LI-domain should impart NIF binding function to the mutant protein. The chimeric I-domain was expressed as a GST fusion protein and purified, and its NIF binding properties were evaluated. This chimeric I-domain did bind NIF with an affinity substantially greater than the wild-type _LI-domain (see Fig. 7). Indeed, its affinity for NIF was similar to that of the wild type of the _MI-domain. The K_d of the chimeric I-domain for NIF was 7 nM compared with 5 nM for the wild-type _MI-domain. Thus, the insertion of these five amino acids was sufficient to impart high affinity NIF binding to the mutated _LI-domain.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   NIF binding to the chimeric LI-domain. 125I-NIF binding was measured as in Fig. 2 to wild-type MI-domain (), the LI-domain (), and the chimeric LI-domain () containing the five amino acids Asp149, Arg151, Gly207, Tyr252, and Glu258 from the MI-domain implicated in NIF recognition. Values are presented as the means ± S.E. of at least three separate experiments.

C3bi Binding-- The five segments implicated in NIF binding were previously shown to be involved in C3bi recognition using the same set of homolog scanning mutants (61). To determine whether the same residues involved in NIF binding were also involved in C3bi engagement, the binding of C3bi to the chimeric _LI-domain that bound NIF was assessed. As shown in Fig. 8, wild-type _LI-domain and the chimeric _LI-domain failed to bind C3bi, whereas C3bi showed a positive reaction with the wild-type _MI-domain in the assay used. We verified that C3bi bound to the _MI-domain with the appropriate specificity profile (i.e. the interaction was inhibited by NIF and by mAb CBL189 to C3bi). Thus, the five residues that implicated in NIF binding were not sufficient to impart C3bi binding to the _LI-domain. In support of this conclusion, we tested C3bi binding to each of the five single mutants, D149E, R151Q, G207L, Y252D, and E258A, with impaired NIF binding. These were immobilized onto microtiter wells, and their binding of biotinylated C3bi was assessed as in Fig. 8. The C3bi titration curves were essentially the same as that obtained with wild-type _MI-domain (data not shown). Half-maximal binding for all mutant and wild type _MI-domains was obtained at a 1:6 dilution of C3bi (see Fig. 8). Thus, none of the five _MI-domain residues involved in NIF binding was essential for C3bi recognition.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   C3bi binding to the chimeric LI-domain. Different concentrations of biotinylated C3bi-coated erythrocytes in Tris buffer, pH 7.6, containing 100 mM NaCl and 2 mM CaCl2, were added to the wells of microtiter plates coated with 50 µg/ml wild-type MI-domain (), wild-type LI-domain (), or the chimeric LI-domain () and postcoated with 2% BSA. Bound biotinylated C3bi was quantitated by adding avidin-alkaline phosphatase followed by p-nitrophenyl phosphate. Values are presented as the means ± S.E. of at least three separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we have sought to identify the residues within the _MI-domain that establish its high affinity and specificity for NIF, thereby serving as a model for the basis for ligand recognition by _M2. The approach of homolog scanning mutagenesis had previously identified five loops, two adjacent and three other discontinuous in amino acid sequence on the MIDAS face of the _MI-domain, as providing key contact sites for NIF recognition (50). The previous study had been performed in the context of the intact _M2 heterodimer, and the involvement of these segments has been confirmed in the present analysis by expressing these homolog scanning mutations in a different context (i.e. as I-domains). Whereas the _MI-domain, either as a GST or as a free recombinant protein, bound NIF with the same high affinity as intact _M2, the homolog scanning mutants showed negligible binding. It is also noteworthy that the _LI-domain failed to bind NIF. This result is consistent with previous reports for both the _LI-domain and _L2 (22, 37, 50), although other studies have provided evidence for an interaction of NIF (62) or NIF peptides with _L2 (63).

To further define the contact residues for NIF binding, a set of triple mutants was developed in which sets of three amino acid residues within the three implicated segments were changed to the corresponding _LI-domain residues (or to _x, Ala, or Gly residues if conserved in _L), and the capacity of the resulting mutants to bind NIF was assessed. Of the 13 triple mutants, five lost their ability to bind NIF with high affinity. The reduction in affinity of each of these triple mutants for NIF was at least 10-fold. Then, within these five triple mutants, the individual amino acids were mutated. The outcome of these data is the identification of residues Asp¹⁴⁹, Arg¹⁵¹, Gly²⁰⁷, Tyr²⁵², and Glu²⁵⁸ within the _MI-domain as being critical to NIF binding. Mutation at any one of these positions reduced affinity for NIF by at least 10-fold.

To consider whether mutation at these individual positions altered NIF binding by perturbing the conformation of the _MI-domain, the effect of the substitutions on the affinity of the MIDAS motif for cation was assessed by Tb³⁺ luminescence spectroscopy. Since the five amino acids of the MIDAS are brought into the appropriate spatial distance to coordinate with the bound cation by the three-dimensional fold of the I-domain, changes in the affinity of the MIDAS for cation should be a sensitive indicator of conformational change within the _MI-domain. In a sense, this approach is similar to the use of the Ca²⁺ monoclonal antibody 24, which has been utilized previously (64, 65) to assess the conformational integrity of the _M2, but the specific reporter is different. In order to implement this approach, Phe²⁴⁶, which resides on the hydrated surface and in close proximity to the MIDAS based on the crystal structure of the _MI-domain, was mutated to a tryptophan to potentially enhance energy transfer to bound Tb³⁺. Substitution at this position did not alter NIF binding (see Fig. 5), was not predicted to alter the conformation of the _MI-domain based upon computer modeling, and did, indeed, enhance the efficiency of energy transfer to bound Tb³⁺ (see Fig. 6). Using this approach, all five point mutants were found to bind Tb³⁺ with the same affinity as the wild-type _MI-domain.

The involvement of the five residues identified through the loss-of-function resulting from their mutation was corroborated by a gain-in-function approach. When these five residues were inserted into the I-domain of _L, the chimera acquired NIF binding function. The affinity of the chimeric I-domain was 7 nM compared with 5 nM for the authentic _MI-domain. The slight difference in affinity may indicate that still other residues might contribute to NIF recognition. It is important not to overinterpret the result of this experiment to imply that these are the only residues involved in NIF binding. Residues that are conserved between the _MI- and _LI-domains may be necessary to mount a high affinity interaction of NIF with the _MI-domain but may not by themselves support an interaction of sufficient affinity to detect NIF binding to the _LI-domain. It is also noteworthy that two of the five residues implicated in NIF binding to the _MI-domain, Gly²⁰⁷ and Ala²⁵⁸, are conserved in the _XI-domain, which itself does not bind NIF with high affinity (22). This suggests that the three remaining residues may play a particularly prominent role in defining the specificity of the _MI-domain for NIF.

Based on the crystal structure (54), all of the five amino acids involved in NIF binding reside at the hydrated surface of the _MI-domain; three of the five lie on the top of the MIDAS face, and two lie at its edges (see Fig. 9). Arg¹⁵¹, Gly²⁰⁷, and Tyr²⁵² are in relatively close proximity to the cation bound in the MIDAS motif, whereas Asp¹⁴⁹ and Glu²⁵⁸ are more spatially distant. The capacity of NIF to contact several spatially distant residues over the _MI-domain may account for the high affinity of its interaction and its capacity to inhibit the binding of multiple ligands that interact with the MIDAS face. The involvement of the MIDAS face is consistent with the role of cation in NIF binding (37), which is again corroborated in this study. In a previous study, Rieu et al. (45) mutated residues in the surface loops and helices of the MIDAS face of the _MI-domain and implicated Gly¹⁴³, Asp¹⁴⁹, Glu¹⁷⁸, Glu¹⁷⁹, and Arg²⁰⁸ in NIF binding. Only one of these residues, Asp¹⁴⁹, coincides with our findings. The disparity regarding the role of Arg²⁰⁸ is particularly difficult to explain, since the same mutation, R208L, was introduced by both groups. The segment containing the two glutamic acids, Glu¹⁷⁸ and Glu¹⁷⁹, had been previously changed to Thr and Ser, respectively, in our original homolog scanning mutagenesis study without effect (50), as contrasted to the alanines by Rieu et al. (45). Gly¹⁴³ had not been altered in our original mutagenesis study but was absent in an _MI-domain that bound NIF with an affinity comparable with the intact receptor. One potential explanation for these differences in results relates to the activation states of the _MI-domains utilized. Relatively subtle differences in the length of _MI-domain can influence its activation state (66-68), which can, in turn, exert a profound influence on ligand recognition. Differences in activation state may also account for reports that integrin _L2 can recognize NIF (62, 69).

View larger version (60K): [in this window] [in a new window]   Fig. 9.   Location of the amino acid residues implication in NIF binding to the MI-domain. The structure of the I-domain is modeled according to the crystal coordinates of the MI-domain (54) using the Insight II program. The residues implicated in NIF binding (Asp149, Arg151, Gly207, Tyr252, and Glu258) are highlighted in yellow. The divalent cation bound in the MIDAS motif is shown in white.

To date, many lines of evidence have emphasized that many ligands share overlapping binding sites within _M2, including the fact that NIF blocks the interaction of many ligands (C3bi, ICAM-1, fibrinogen, and several immobilized substrates) with the receptor (22, 37, 70). However, while the binding sites for these ligands may be overlapping, they need not be identical (46, 61). Indeed, the _MI-domain residues involved in NIF binding were not critical for C3bi binding (Fig. 8), although all segments originally implicated in NIF binding were also involved in C3bi binding (61). The nonidentity of these ligand binding sites is consistent with our previous data showing that mutation of Lys²⁴⁵ to Ala significantly reduced C3bi but had no effect on NIF binding to _M2 (61). Thus, a model can now be proposed in which many of the same loops and helices of the _MI-domain, on or near its MIDAS face, may engage ligands, but different amino acid residues within these structures may contact the ligands. Accordingly, the _MI-domain may present a mosaic of different amino acids that are capable of contacting different ligands. Direct support for this mosaic model is seen within the Lys²⁴⁵-Arg²⁶¹ segment of the _MI-domain, which has been implicated in both NIF and fibrinogen recognition (71). Key contact amino acids in this loop for fibrinogen binding are Phe²⁴⁶, Asp²⁵⁴, and Pro²⁵⁷, whereas Tyr²⁵² and Glu²⁵⁸ are involved in NIF binding. This same _MI-domain segment is also involved in C3bi recognition by _M2 (61). Although it is not known which residues in this segment serve as contact sites for C3bi, the residues involved in NIF binding, Tyr²⁵² and Glu²⁵⁸, do not appear to be critical. Pro¹⁴⁷-Arg¹⁵² and Pro²⁰¹-Lys²¹⁷ are also involved in recognition of C3bi (61) and the fungal pathogen Candida albicans (although this latter ligand also interacts with several distinct regions of the _MI-domain (70)). Whether different residues within these shared loops will be involved in contacting these ligands remains to be assessed. The systematic approach delineated in this study involving loss of function, gain in function, and conformational assessments provides a means for assessing whether this mosaic model provides a molecular basis for the capacity of _M2 and other integrins to recognize multiple and in many cases unrelated ligands.

	ACKNOWLEDGEMENT

We thank Dr. T. A. Haas (The Cleveland Clinic Foundation) for help in molecular modeling.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant HL 66197.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by an American Heart Association postdoctoral fellowship from the Northeast Ohio Affiliate.

^§ To whom correspondence should be addressed: Joseph J. Jacobs Center for Thrombosis and Vascular Biology and Dept. of Molecular Cardiology/NB50, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8200; Fax: 216-445-8204; E-mail: plowe@ccf.org.

Published, JBC Papers in Press, March 5, 2002, DOI 10.1074/jbc.M110242200

	ABBREVIATIONS

The abbreviations used are: NIF, neutrophil inhibitory factor; I-domain, region of ~200 amino acid residues "inserted" in the -subunit of _M2 and some other proteins, such as von Willebrand factor; GST, glutathione S-transferase; MIDAS, metal ion-dependent adhesion site; ICAM-1, intracellular adhesion molecule-1; BSA, bovine serum albumin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES