Identification of the Binding Site for Fibrinogen Recognition Peptide gamma 383-395 within the alpha MI-Domain of Integrin alpha Mbeta 2*

Valentin P. YakubenkoDagger , Dmitry A. SolovjovDagger , Li Zhang§, Vivien C. YeeDagger , Edward F. PlowDagger , and Tatiana P. UgarovaDagger

From the Dagger  Joseph J. Jacobs Center for Thrombosis and Vascular Biology and the Department of Molecular Cardiology, Lerner Research Institute, Cleveland, Ohio 44195 and the § J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855

Received for publication, November 8, 2000, and in revised form, January 8, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The leukocyte integrin alpha Mbeta 2 (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha Mbeta 2 mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma 383TMKIIPFNRLTIG395, P2-C, within the gamma -module of the D-domain of fibrinogen, is a recognition site for alpha Mbeta 2 and alpha Xbeta 2. We have now identified the complementary sequences within the alpha MI-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha Mbeta 2, alpha Xbeta 2, and alpha Lbeta 2, i.e. the alpha MI- and alpha XI-domains bind P2-C, and the alpha LI-domain did not bind this ligand. The Lys245-Arg261 sequence, which forms a loop beta D-alpha 5 and an adjacent helix alpha 5 in the three-dimensional structure of the alpha MI-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha MI-domain segments 245KFG and Glu253-Arg261 were switched to the homologous alpha LI-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys245-Tyr252 and Glu253-Arg261 sequences directly bound the D fragment and P2-C derivative, gamma 384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys245-Arg261 segment, Phe246, Asp254, and Pro257, resulted in the loss of the binding function of the recombinant alpha MI-domains; and 4) grafting the alpha M(Lys245-Arg261) segment into the alpha LI-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha M(Lys245-Arg261) segment, a site of the major sequence and structure difference among alpha MI-, alpha XI-, and alpha LI-domains, is responsible for recognition of a small segment of fibrinogen, gamma Thr383-Gly395, by serving as ligand binding site.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrin alpha Mbeta 2 participates in the attachment of leukocytes to the endothelial lining of blood vessels and the subsequent transmigration of adherent cells during immune-inflammatory responses (1-3). The engagement of fibrinogen (Fg)1 by alpha Mbeta 2 on the surface of leukocytes and by intercellular adhesion molecule-1 (ICAM-1) on the endothelium may play a role in mediating the adhesion of leukocytes to the vessel wall (4, 5) and in facilitating their subsequent extravasation across the endothelial monolayer (6). In addition, the binding of deposited fibrinogen or fibrin to alpha Mbeta 2 may mediate leukocyte adhesion at extravascular sites of inflammation (7-9).

In previous studies, Altieri et al. (10, 11) demonstrated that a peptide (designated P1), corresponding to residues 190-202 of the gamma -chain of the D-domain of Fg, was recognized by alpha Mbeta 2. However, when residues key to the recognition of P1 by alpha Mbeta 2-bearing cells were mutated in the gamma -module, gamma 148-411, this recombinant fragment was as active as its wild-type counterpart in supporting alpha Mbeta 2-mediated adhesion (12). This observation led to the search for additional alpha Mbeta 2 recognition sites within the gamma -chain, and ultimately the P2 peptide, corresponding to gamma 377-395, was identified (12). Indeed, in comparative analyses, P2 was 10-15-fold more potent than P1 in inhibiting adhesion of the alpha Mbeta 2-expressing cells to the D fragment of Fg. Further analyses of the adhesion-promoting activity of overlapping peptides showed that its COOH-terminal part, gamma 383TMKIIPFNRLTIG395, designated P2-C, was the primary site of its biological activity (12). Recently, a second leukocyte integrin, alpha Xbeta 2, which is highly homologous to alpha Mbeta 2, was demonstrated to bind to the gamma -module and P2-C peptide (13), and soluble P2-C peptide efficiently blocked the alpha Xbeta 2-mediated adhesion.

Within the heterodimeric alpha Mbeta 2 receptor, the I-domain, a region of ~200 amino acid residues, inserted in the alpha M subunit, contributes broadly to the recognition of ligands by alpha Mbeta 2 (14) and specifically to the binding of Fg to this integrin (14, 15). In addition to Fg, this region also was implicated in the binding of ICAM-1(15), iC3b (16), and neutrophil inhibitory factor, NIF (17, 18). We have shown previously that P2 interacts with the recombinant alpha MI-domain and that NIF partially blocked this interaction (12). Previous studies suggested that overlapping but not identical sites are involved in the recognition of iC3b, NIF, and Fg (19). However, although the binding sites for iC3b and NIF in the alpha MI-domain were mapped extensively (20-23), the recognition site for Fg has not been studied. Recently, sequences key to the binding of NIF and iC3b to the alpha MI-domain were mapped using a homolog scanning mutagenesis strategy (22, 24). This approach is based upon the structural similarity of the I-domains of alpha M and alpha L and the differences in their ligand recognition; i.e. the crystal structures of the I-domains of alpha M and alpha L are very similar (25-28), but only the I-domain of alpha M binds NIF with high affinity (17, 18). Fg, together with NIF and iC3b, does not bind to alpha Lbeta 2, suggesting that differences in the structure of the alpha MI- and alpha LI-domains may be responsible for their distinction in ligand binding specificity. In this study, we have sought to localize the binding site for the P2-C sequence of Fg within the alpha MI-domain. The strategy developed was based on the differences in the binding of P2-C to the alpha MI-, alpha XI-, and alpha LI-domains and involved several independent approaches, including screening of mutant cells, synthetic peptides, site-directed mutagenesis, and the gain-in-function analyses. The binding site for P2-C was localized within the segment alpha M(Lys245-Arg261), a site of the major structure divergence between alpha MI-, alpha XI-, and alpha LI-domains. The grafting of this segment into the alpha LI-domain converted it to the P2-C-binding protein. Thus, a small amino acid sequence, P2-C, with a defined structure within a crystallized domain of fibrinogen (29) is shown to interact with a small segment that also has a defined structure within the alpha MI-domain.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins, Peptides, and Monoclonal Antibodies-- Human kidney 293 cells expressing wild-type and the mutant forms of the alpha Mbeta 2 receptor were described and characterized in detail previously (19, 22). These cells were grown as adherent monolayers in Dulbecco's modified Eagle's medium/F-12 medium (BioWhittaker, Walkersville, MD), supplemented with 10% fetal bovine serum, 25 mM HEPES, and antibiotics. Human Fg was purified from fresh human blood by differential ethanol precipitation (30) or obtained from Enzyme Research Laboratories (South Bend, IN). The D100 (Mr 100,000) fragment was prepared by digestion of human Fg with plasmin (Enzyme Research Laboratories, South Bend, IN) and purified as described (31). The D98 fragment (Mr 98,000) was produced by digestion of the D100 with plasmin. This fragment lacks 5-15 amino acid residues from the COOH terminus of the gamma -chain and will be described elsewhere.2 D98 was biotinylated with EZ-link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer's instructions. P1 (gamma 190-202), P2 (gamma 377-395), P2-C (gamma 383-395), H19 (gamma 340-357), H2O (gamma 350-374), L10 (gamma 402-411), and H12 (gamma 400-411) peptides were synthesized and purified as described (12). In addition, an analog of P2-C, P2-Ce, a peptide with the extended COOH-terminal end, gamma 384-402, was also synthesized to test for direct binding to alpha MI-domain peptides. The following peptides duplicating selected sequences within the alpha MI-domain were synthesized: 147PHDFRR152 (Pro147- Arg152), 201PITQLLGRTHTATGIRK217 (Pro201-Lys217), 245KFGDPLGY252 (Lys245-Tyr252), 253EDVIPEADR261 (Glu253-Arg261), 245KFGDPLGYEDVIPEADREG263 (Lys245-Gly263), and 223FITNGARKN232 (Phe223-Asn232). The numbers indicate the positions of the residues within the alpha M subunit (numbered according to Arnaout et al. (32)). NIF (a gift from Corvas International, San Diego) was labeled with EZ-link Sulfo-NHS-LC-Biotin according to the manufacturer's protocol. mAbs OKM1, 44a, and 904 were obtained from ATCC (Rockville, MD). mAb 4-2 (33) was a generous gift from Dr. B. Kudryk, the New York Blood Center, and mAb 4A5 (34) was obtained from Dr. G. Matsueda (Bristol-Meyers Squibb).

Expression of Recombinant alpha MI-, alpha XI-, and alpha LI-Domains and Site-directed Mutagenesis-- The I-domains were expressed as fusion proteins with glutathione S-transferase (GST) and purified from soluble fractions of Escherichia coli lysates by affinity chromatography. The coding regions for the alpha MI-domain (residues Asp132-Ala318) and alpha LI-domain (Gly153-Ile333) were amplified by polymerase chain reactions using as template plasmids pCIS2M-alpha M (19) and pCIS2M-alpha L, which contain the cDNA fragments coding for the full-length of alpha M and alpha L, respectively. The primers used for the alpha MI-domain were 5'-TGTCCTGGATCCGATAGTGACATTGCCTTCTTGA (forward) and 5'-TGAGTACCCGCGGCCGCCGCAAAGATCTTCTCCC (reverse). The primers used for the alpha LI-domain were 5'-CAGGAAGGATCCAAGGGCAACGTAGACCTGGTATT (forward) and 5'-GTCCTGTTTGCGGCCGCCCTCAATGACATAGA (reverse). The underlined nucleotides are BamHI and NotI recognition sequences that were introduced in the primers. The fragments were digested with BamHI and NotI and cloned in the expression vector pGEX-4T-1 (Amersham Pharmacia Biotech). The accuracy of the DNA sequence was verified by sequencing. The plasmid was transformed in E. coli strain BL-21(DE3)pLysS competent cells, and expression was induced by adding 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3-5 h at 37 °C.

To express the alpha XI-domain, the following primers were used to amplify a cDNA fragment encoding the alpha XI-domain (residues Glu148-Ala335) from a randomly primed cDNA library of U937 monocytoid cell line: 5'-AGGCTACCGGGATCCAGACAGGAGTGCCCAAGA (forward) and 5'-CATCTCCCAATTTGGCGGCCGCACTGCTTGTGGTC (reverse). The product was digested with BamHI and NotI and cloned into pGEX-4T-1. The plasmid was transformed in E. coli BL-21(DE3)pLysS cells, and the correctness of the alpha XI-domain insertion was confirmed by sequencing. The alpha XI-domain was expressed and purified from the cell lysates as a fusion protein with GST under the conditions used for the alpha MI- and alpha LI-domains.

Site-directed mutagenesis of the alpha MI-domain was performed by using QuickChangeTM mutagenesis kit (Stratagene, San Diego). The pGEX-4T-1 construct containing DNA encoding the alpha MI-domain was modified by site-directed mutagenesis using two mutagenic primers containing the desired mutation. The mutations introduced in the alpha MI-domain and the primers used are listed in Table I. The oligonucleotide primers, each complementary to opposite strands of the vector, were extended during temperature cycling by using PfuTurboTM DNA polymerase. Following temperature cycling, the product was treated with DpnI endonuclease to digest the parental DNA template. The nicked vector DNA incorporating the desired mutations was then transformed into the Epicurian Coli® XL1-Blue supercompetent cells, and cDNA from individual bacterial clones was analyzed by sequencing. The E. coli BL-21(DE3)pLysS host cells were then transformed with the mutant plasmids, and the mutant alpha MI-domains were prepared as described above for the recombinant wild-type alpha MI-domain. The immunoreactivity of wild-type and mutant recombinant I-domains was analyzed by enzyme-linked immunosorbent assay with mAbs 44a and 904 using our standard protocol (35).


                              
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Table I
Mutations in the Lys245-Arg261 sequence of the recombinant alpha MI-domain and nucleotide sequences used for their constructing

Generation of the alpha L(alpha M (Lys245-Arg261))I-Domain Chimera-- The segment corresponding to the sequence Ala267-Asp278 within the alpha LI was exchanged to the homologous segment of the alpha MI-domain Lys245-Arg261. The segment switch was created by oligonucleotide-directed mutagenesis using polymerase chain reaction. The construction of the chimera was based on the observation that oligonucleotide sequence corresponding to alpha M(Lys245-Arg261) contains a unique restriction site for Eco81I (SauI), whereas the second site for this enzyme is present between 4760 and 4761 of the pGEX-4T-1 sequence. To switch the Lys245-Arg261 from alpha M to alpha L, the mutagenic primers were designed to contain the Eco81I site within the alpha M(Lys245-Arg261) segment and additional unchanged alpha L bases: 5'-CATCCCTGAGGCAGACAGAATCATCCGCTACATCATCG (forward), 5'-GCTGCCTC AGGGGATCACATCTTCATAACCCAATGGATCTCCAAACTTCTCCCCATCCGTGATGATGATAAG (reverse) (the alpha M sequences are in bold, and the restriction site for Eco81I is underlined). The pGEX-4T-1 vector containing DNA encoding the alpha LI-domain was modified by polymerase chain reaction using PfuTurbo DNA polymerase with the following cycling parameters: 95 °C for 30 s, 55 °C for 1 min, 68 °C for 11.5 min. Following temperature cycling, the product was treated with DpnI to digest the parental DNA template. The linear product was purified and digested with Eco81I to produce the two cDNA fragments with cohesive ends. These fragments were ligated and transformed into Epicurian Coli XL1-Blue supercompetent cells. The accuracy of the DNA sequence and the correctness of the I-domain direction were verified by sequencing. The E. coli BL-21(DE3)pLysS cells were transformed with the mutated plasmid, and the chimeric molecule was prepared following the procedure described above for the wild-type and mutant I-domains.

Flow Cytometry-- FACS analyses were performed to assess the expression of alpha Mbeta 2 on the surface of the cells transfected with wild-type and mutant forms of the receptor. The cells were harvested, and 106 cells were incubated with alpha M-specific mAbs OKM1 or 44a at 15 µg/200 µl of cell suspension for 30 min at 4 °C. The cells were then washed and incubated with fluorescein isothiocyanate-goat anti-mouse IgG (at a 1:1,000 dilution) for additional 30 min at 4 °C. Finally, the cells were washed and analyzed in a FACS Star (Beckton Dickinson, Mountain View, CA). Populations of cells expressing a similar amount of the receptor were selected by FACS. A clonal population of each mutant was isolated by limiting dilution. After propagation in culture, the amount of the alpha Mbeta 2 was again evaluated by FACS analysis with mAb OKM1.

Adhesion Assays-- The wells of tissue culture plates (Costar, Cambridge, MA) were coated with 6.1, 12.5, 25, 50, and 100 µg/ml P2-C peptide or 1, 5, and 25 µg/ml of D100 fragment for 3 h at 37 °C. The amount of peptides immobilized onto the wells was measured by utilizing radiolabeled peptides (12). The coated wells were postcoated with 0.5% polyvinylpyrrolidone for 1 h at 22 °C. The 293 cells expressing wild-type or mutated forms of alpha Mbeta 2 were harvested with cell-dissociating buffer (Life Technologies, Inc.) for 1 min at 22 °C and washed twice in HBSS/HEPES solution containing 10 mg/ml BSA, and resuspended at 5 × 105/ml in HBSS (without phenol red)/HEPES supplemented with 1 mM Ca2+ and 1 mM Mg2+ and 10 mg/ml BSA. 100-µl aliquots of the cells were added to each well and incubated on the adhesive substrates for 25 min at 37 °C in a 5% CO2 humidified atmosphere. The nonadherent cells were removed by three washes with colorless HBSS. The adherent cells were frozen overnight at -20 °C, then thawed and lysed by the addition of a buffer containing the CyQuant dye (Molecular Probes, Eugene, OR). The fluorescence was measured in a Cytofluorimeter (PerSeptive Biosystems, Framington, MA), and the number of adherent cells was determined from a reference standard curve prepared according to the manufacturer's protocol. Additionally, several experiments were performed using our established protocol with 51Cr-labeled cells (12), and the results were found to be identical with those obtained using the CyQuant reagent (see "Results").

Solid Phase Binding Assays-- To test the interaction of the wild-type alpha M, alpha X, alpha L, mutant alpha M, and chimeric I-domains, 96-well plates (Immulon 4BX, Dynex Technologies Inc., Chantilly, VA) were coated with P2-C or P2-Ce at 50 µg/ml overnight at 4 °C and postcoated with 3% BSA or 0.5% polyvinyl alcohol for 2 h. The GST-I-domains in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.05% Tween 20, and 5% glycerol were added to the wells and incubated for 3 h at 22 °C. After washing, bound I-domains were detected with an anti-GST mAb (Upstate Biotechnology, Lake Placid, NY) at a 1:5,000 dilution. After washing, goat anti-mouse IgG conjugated to alkaline phosphatase was added for 1 h, and the binding of the I-domains was measured by reaction with p-nitrophenyl phosphate. As a control, the binding of GST to immobilized P2-C was typically ~5-10% that of wild-type alpha MI-domain, and background binding to BSA or polyvinyl alcohol was subtracted.

To examine the interaction of the D fragment with the I-domain peptides, 96-well plates (Immulon 2 HB, Dynex Technologies Inc.) were coated with peptides Pro147-Arg152, Pro201-Lys217, Lys245-Tyr252, and Glu253-Arg261 at 100 µM (0.1 ml/well) overnight at 4 °C and postcoated with 3% BSA for 1 h at 22 °C. 10 µg/ml biotinylated D98 in 50 mM Tris-HCl buffer, pH 7.5, and 0.05% Tween 20 were added to the wells and incubated for 2.5 h at 37 °C. In parallel, the same amount of the D98 was mixed with different concentrations of P1, P2 peptides, or NIF and added to the wells with immobilized alpha MI-domain peptides. After washing, streptavidin conjugated to alkaline phosphatase (Pierce) was added and incubated for 45 min at 37 °C. D98 binding was detected by reaction with p-nitrophenyl phosphate, measuring the absorbance at 405 nm.

To demonstrate the direct binding of the P2-C region (gamma 383-395) to the alpha MI-domain peptides, experiments were performed as follows. Different concentrations of the P2-Ce peptide, gamma 384-402, in 50 mM Tris-HCl and 0.05% Tween 20 were added to wells with immobilized alpha MI-domain peptides and incubated for 3 h at 37 °C. After washing, the binding of the gamma 384-402 was detected with mAb 4-2. The mAb 4-2 recognizes an epitope within this peptide, although it reacts poorly with authentic P2-C.3 The binding of this mAb to gamma 384-402 was measured by reaction with goat anti-mouse IgG, conjugated to alkaline-phosphatase (Pierce) and using p-nitrophenyl phosphate for detection.

Statistical Analyses-- Values of adherent cells bound to substrates are given as means ± S.E. and are based on two to six independent experiments with each alpha Mbeta 2 cell line, performing triplicates at each experimental point.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of Wild-type Recombinant alpha MI-, alpha XI-, and alpha LI-Domains to the P2-C Peptide of Fg-- In previous studies, we have demonstrated that the P2-C peptide binds directly to the recombinant alpha MI-domain (12). alpha Xbeta 2 also recognizes P2-C (13); but the role of the alpha XI-domain in this interaction was not evaluated, and the recognition of P2-C by the alpha LI-domain had not been tested. Therefore, the three I-domains were expressed as GST fusion proteins and tested for their ability to interact with the immobilized P2-C peptide. As shown in Fig. 1, the recombinant alpha XI-domain exhibited a dose-dependent and saturable binding to the P2-C peptide similar to that of the recombinant alpha MI-domain. In contrast, the recombinant alpha LI-domain did not interact with P2-C even at the highest concentration of the alpha LI-domain added (200 µg/ml maximal testable concentration). The binding characteristics of the isolated I-domains parallel the binding properties of the corresponding intact receptors on cell surfaces, i.e. the alpha Mbeta 2- and alpha Xbeta 2-expressing cells adhere to Fg and P2-C, whereas the alpha Lbeta 2-bearing cells do not (see Figs. 2 and 3). Thus, these data confirm that binding of P2-C to two highly homologous integrins, alpha Mbeta 2 and alpha Xbeta 2, is mediated by alpha MI- and alpha XI-domains, respectively, and further suggest that the lack of binding of P2-C to alpha Lbeta 2 may be the result of sequence and/or structural differences.



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Fig. 1.   Binding of the recombinant wild-type alpha MI- (), alpha XI- (open circle ), and alpha LI- (black-down-triangle ) domains to the P2-C peptide of Fg. Different concentrations of recombinant I-domain-GST proteins in Tris buffer, pH 7.4, containing 100 mM NaCl, 1 mM Mg2+, 1 mM Ca2+, 0.05% Tween 20, and 5% glycerol were added to the microtiter plates coated with 50 µg/ml P2-C peptide and postcoated with 0.5% polyvinyl alcohol and incubated for 3 h at 22 °C. After washing, anti-GST mAb (1:5,000) was added to the wells and incubated for an additional 1.5 h. The binding of the I-domains then was detected with a secondary goat anti-mouse IgG conjugated to alkaline phosphatase with subsequent development of the reaction with p-nitrophenyl phosphate.

Binding of P2-C to Mutant Cell Lines-- As the first step to define the binding site for P2-C within the alpha MI-domain, mutant cell lines, each expressing a mutant alpha Mbeta 2 in which a short alpha MI-domain sequence was replaced for the corresponding region of the alpha LI-domain, were tested for their adhesion to immobilized P2-C or D100 fragment. These mutant cell lines have been used previously to examine the binding of NIF and iC3b to alpha Mbeta 2 (22, 24). For such experiments to be readily interpretable, cell lines expressing high and similar levels of wild-type and mutant receptors were selected by cell sorting using mAbs OKM1 and 44a followed by cloning by limiting dilution. The expression of the receptors as assessed by the mean fluorescence intensity in FACS analyses differed by less than 2-fold from the internal wild-type control.

The adhesion of each mutant was measured with increasing concentrations of immobilized P2-C and D100 fragment to determine the maximal level of adhesion. In all cases where a cell line bearing a mutant receptor did adhere to either substrate, the adhesion was dependent upon the concentration of the immobilized ligand and reached a plateau. This maximal adhesion was compared with that of the cells expressing wild-type alpha Mbeta 2 receptor and mock-transfected cells in the same experiment to allow normalization of results. The cell lines exhibiting adhesion similar to or greater than the wild-type alpha Mbeta 2 cells were identified as "positive" mutants. The pattern of cell adhesion to P2-C obtained for wild-type alpha Mbeta 2 and a representative mutant, alpha M(K231NAF), is shown in Fig. 2. Adhesion of alpha M(K231NAF) to P2-C was dose-dependent and saturable with ~70% of added cells adherent to P2-C at the plateau. Adhesion of wild-type and mutant cells to a control peptide, H19, was tested and was found to be less than 5% of added cells. Also, as the essential control, the alpha Lbeta 2-expressing cells adhered poorly to either adhesive substrates, P2-C and D100, (~10-15% of added cells) consistent with lack of interaction of alpha Lbeta 2 with Fg. All negative mutants did not adhere to P2-C; adhesion of these cells was similar to that of mock-transfected cells.



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Fig. 2.   Adhesion of the alpha M(K231NAF) mutant and the cells expressing wild-type alpha Mbeta 2 and alpha Lbeta 2 to different adhesive substrates. 0.1-ml aliquots of 5 × 104 cells expressing alpha M(K231NAF) (), wild-type alpha Mbeta 2 (black-down-triangle ), and alpha Lbeta 2 (black-square) in HBSS/HEPES, supplemented with 1 mM Ca2+ and 1 mM Mg2+, were incubated in the wells of 48-well plates coated with different concentrations of P2-C. Adhesion of alpha M(K231NAF)-expressing cells to H19 is shown for comparison (dashed line). After 25 min at 37 °C in a humidified atmosphere containing 5% CO2, the nonadherent cells were removed by three washes with HBSS, and the amount of adherent cells was determined using the fluorescent dye CyQuant as described under "Experimental Procedures." Data are expressed as a percentage of added cells and are the mean ± S.E. of four individual experiments. The actual amounts of P2-C and H19 immobilized onto the plastic wells were determined as described (12) and are shown on the abscissa.

The results of adhesion of 16 mutants to P2-C and the D100 fragment are summarized in Fig. 3, A and B. The data are expressed as the percent adhesion of the wild-type alpha Mbeta 2-expressing cells to each substrate. Substitutions for the following regions of alpha MI-domain abrogated adhesion: alpha M(Pro147-Arg152), alpha M(Pro201-Gly207), alpha M(Arg208-Lys217), alpha M(K245FG), and alpha M(Glu253-Arg261). These cell lines form a group of negative mutants. The lack of adhesion of these mutants was not the result of decreased surface expression of the receptor because there was no correlation between adhesion and expression. Specifically, although surface expression of alpha M(R208-K217) was 2.0 lower than that of the wild-type alpha Mbeta 2 cells, adhesion was decreased 6-fold. On the other hand, surface expression of the alpha M(K245FG) was 1.2-fold higher than that of the cells expressing the wild-type alpha Mbeta 2 cells, but adhesion was abrogated completely. Surface expression of two other negative mutants, alpha M(P147-R152) and alpha M(E253-R261), was very similar to that of the wild-type receptor.



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Fig. 3.   Adhesion of the wild-type and the alpha MI-domain mutants to P2-C (panel A) and D100 (panel B). 0.1-ml aliquots of 5 × 104 cells were added to the wells coated with 5, 25, 50, and 100 pmol of each peptide and 1, 5, and 25 µg/ml D100 fragment. Adhesion was performed as described in Fig. 2. Adhesion of each mutant reached the maximal level at 25 pmol of P2-C and at 5 µg/ml of the D100 fragment. The results are presented as a percentage of maximal adhesion attained with the wild-type expressing cells (WT) at 25 pmol of the P2-C and 5 µg/ml D100. Dashed lines on each graph are drawn at the maximal level of adhesion achieved with the wild-type cells, and dotted lines show the maximal level of adhesion with mock-transfected cells (Mock). Adhesion of the cells expressing alpha Lbeta 2 and alpha Xbeta 2 is also shown. Data for each mutant are from two to six adhesion assays, performed in triplicate at each experimental point.

The following mutants supported the same or higher levels of adhesion to P2-C compared with the cells expressing the wild-type alpha Mbeta 2 receptor (positive mutants): alpha M(M153-T159), alpha M(E162-L170), alpha M(E178-T185), alpha M(Q190-S197), alpha M(K231NAF234), deletion mutant alpha M(D248PLGY252), alpha M(R281-I287), and alpha M(Q309-E314). Adhesion of dE262G and alpha M(F297-T307) was partially affected; the maximal level of adhesion to P2-C reached 64 ± 5% and 83 ± 7, respectively, of wild-type alpha Mbeta 2 cells. These two receptors were classified as "intermediate" mutants. alpha M(D273-K279) was the only mutant to exhibit differential recognition of P2-C and the D fragment; it supported adhesion to P2-C effectively (65%) but mediated adhesion to D100 poorly (15%).

Interaction of P2-C and D Fragment with the alpha MI-DomainPeptides-- In subsequent analyses, we focused on the five negative mutants. Peptides corresponding to the wild-type alpha MI-domain sequences were synthesized and tested for their ability to interact with the D fragment and P2-C. Because the sequences within two of the negative mutants, alpha M(Pro201-Gly207) and alpha M(Arg208-Lys217), were contiguous, one linear peptide spanning Pro201-Lys217 was prepared. Also, the peptide, Lys245-Tyr252, containing the sequence of the negative mutant alpha M(K245FG) was extended at its COOH terminus to include the D248PLGY252 sequence. Thus, four peptides were synthesized: Pro147-Arg152, Pro201-Lys217, Lys245-Tyr252, and Glu253-Arg261. In these experiments, we used a derivative of the D100 fragment, D98, which has a higher affinity for alpha Mbeta 2.2 D98 contains the entire P2-C but differs from D100 in the length of the constituent gamma -chain, which terminates at gamma 397/405 in D98 compared with the intact gamma 411 in D100. These alpha MI-domain peptides were immobilized onto microtiter plates, and the binding of biotinylated D98 fragment to them was assessed. As shown in Fig. 4A, two peptides, Lys245-Tyr252 and Glu253-Arg261, bound D98 effectively. The immobilized Pro201-Lys217 peptide exhibited low binding of D98, and Pro147-Arg152 and control peptide Phe223-Asn232 did not bind the fragment.



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Fig. 4.   Binding of the D98 fragment to the peptides from within the alpha MI-domain. Panel A, 10 µg/ml biotinylated D98 fragment in 50 mM Tris-HCl, pH 7.5, and 0.05% Tween 20 was added to the wells coated with 0.1 ml/well 100 µM each Pro147-Arg152, Pro201-Lys217, Lys245-Tyr252, Glu253-Arg261, and Phe223-Phe232 and incubated for 2.5 h at 37 °C. After washing, the bound D98 fragment was detected using streptavidin conjugated to alkaline phosphatase and p-nitrophenyl phosphate for disclosure. Panel B, 10 µg/ml biotinylated D98 in 50 mM Tris-HCl buffer, pH 7.5, with 0.05% Tween 20 was mixed with different concentrations of P2 (gamma 377-395) (open circle ), P1 (gamma 190-202) (), H19 (gamma 340-354) (black-down-triangle ), and H2O (gamma 350-374) (down-triangle) and added to the wells coated with 100 µM Glu253-Arg261 peptide for 2 h at 37 °C. The binding of the D98 was determined as above. Data are expressed as a percentage of the D98 binding in the absence of peptides. Shown in panels A and B are the representative experiments of three to five independent determinations.

The interaction of the D98 fragment with Lys245-Tyr252 and Glu253-Arg261 was characterized further. The binding of D98 to immobilized Lys245-Tyr252 and Glu253-Arg261 was effectively inhibited by P2-C and P2 (gamma 377-395), which contains the P2-C sequence (Fig. 4B; inhibition of D98 binding to Glu253-Arg261 by P2 is shown). In addition, P1 (gamma 190-202) inhibited the binding of D98 to Lys245-Tyr252 and Glu253-Arg261 (Fig. 4B; the effect of P1 on the binding to Glu253-Arg261 is shown). Two control peptides duplicating fibrinogen sequences gamma 340-357 (H19) and gamma 350-374 (H2O) did not affect the interaction. P1 inhibited the interaction as efficiently as P2, consistent with ability of this peptide to compete with P2 for binding to alpha Mbeta 2 (12). The interaction of the D98 with immobilized alpha MI-domain peptides was cation-independent; in fact, the higher level of binding was observed in the absence than in the presence of 1 mM Ca2+ or Mg2+ (Table II). Interestingly, NIF did not inhibit binding of the D98 fragment to the immobilized Lys245-Tyr252 and Glu253-Arg261 peptides at concentrations as high as 100 µg/ml (Table II), although it efficiently inhibited adhesion of the alpha Mbeta 2-expressing cells to the D fragment and P2-C peptide at a concentration as low as 0.1 µg/ml (not shown). These data suggest that NIF does not interact with the I-domain peptides, which are capable of binding P2-C and the D98 fragment. Indeed, when direct binding of biotinylated NIF to the immobilized alpha MI-domain peptides was tested, NIF did not bind to Lys245-Tyr252 and Glu253-Arg261 peptides (not shown).


                              
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Table II
Effect of cations and NIF on the binding of D98 fragment and the P2-C derivative, gamma 384-402, to immobilized Glu253-Arg261
10 µg/ml biotinylated D98 fragment in 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM Ca2+ 1 mM Mg2+, 0.05% Tween 20 or in 50 mM Tris-HCl, pH 7.5, 0.05% Tween 20 without cations was added to the wells coated with 100 µM Glu253-Arg261 and incubated for 2.5 h at 37 °C. After washing, bound D98 was detected with streptavidin conjugated to alkaline phosphatase and nitrophenyl phosphate. The effect of 100 µg/ml NIF on the binding of the D98 fragment in the absence of cations was tested in parallel. The inhibitory effect of 10 µg/ml each P2-C and P1 (both tested without cations) is shown for comparison. The binding of the gamma 384-402 was detected by using the mAb 4-2 as described in Fig. 5. The data shown are the mean values (±S.D.) of the absorbance at 405 nm of a representative experiment done in triplicate.

In addition, we were able to detect direct binding of P2-C to the immobilized alpha MI-domain peptides. Using a derivative peptide P2-Ce, gamma 384-402, which contains an epitope for the reporting mAb 4-2, we demonstrated that P2-Ce efficiently bound to immobilized Lys245-Tyr252 and Glu253-Arg261 in a dose-dependent and saturable manner (Fig. 5). The binding of P2-Ce to Pro201-Lys217 was low, whereas Pro147-Arg152 and control peptide Phe223-Asn232 did not bind P2-Ce. Similar to the interaction of the whole D98 fragment, the binding of P2-Ce to Lys245-Tyr252 and Glu253-Arg261 was cation-independent (Table II). To exclude further the possibility that the binding of P2-Ce to immobilized Lys245-Tyr252 and Glu253-Arg261 was nonspecific, we tested two control fibrinogen peptides, gamma 402-411 (L10) and gamma 400-411 (H12). These peptides duplicate sequences that reside in close proximity to P2-Ce (gamma 384-402) and interact with platelet integrin alpha IIbbeta 3. These peptides contain an epitope at gamma 405-411 recognized by the mAb 4A5 (34). L10 and H12 did not bind to the immobilized alpha MI-domain peptides as judged by the lack of the mAb 4A5 immunoreactivity, providing further evidence for the specificity of the interaction between P2-Ce and two alpha MI-domain peptides, Lys245-Tyr252 and Glu253-Arg261. Thus, the D98 and P2-C-derivative bound strongly to the same peptides from within the alpha MI-domain, Lys245-Tyr252 and Glu253-Arg261, and the interaction of both ligands with these peptides followed the same pattern. This is consistent with the binding of the D98 fragment being mediated by P2-C.



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Fig. 5.   Binding of the P2-Ce peptide to the alpha MI-domain peptides. Different concentrations of P2-Ce (gamma 384-402) in 50 mM Tris-HCl, pH 7.5, with 100 mM NaCl and 0.05% Tween 20 were added to the well coated with 100 µM solutions of Pro147-Arg152 (diamond ), Pro201-Lys217 (black-triangle), Lys245-Tyr252 (down-triangle), Glu253-Arg261 (), and Phe223-Asn232 (black-square) peptides and incubated for 3 h at 37 °C. After washing, the mAb 4-2 at 1:1,000 dilution was added for 1 h at 37 °C. To detect the binding of the mAb 4-2, the second goat anti-mouse IgG was added, and its binding was determined by reaction with p-nitrophenyl phosphate, measuring absorbance at 405 nm.

Grafting of the alpha M(Lys245-Arg261) Segment into the alpha LI-Domain Converts It to a P2-C-binding Protein-- We have sought to provide direct evidence that the two identified alpha MI-domain segments, Lys245-Tyr252 and Glu253-Arg261, contain a binding site for P2-C. Guided by the crystal structure of the alpha MI- and alpha LI-domains, the whole contiguous Lys245-Arg261 segment was switched from the alpha MI-domain into the counterpart region of the alpha LI-domain. The appropriate DNA sequence of the entire mutated I-domain was confirmed, and the chimeric I-domain was expressed as a GST fusion protein. The functional consequence of this switch is shown in Fig. 6A. The P2-C peptide was immobilized onto microtiter plates, and the binding of the chimeric alpha L(alpha M(Lys245-Arg261))I-domain molecule and the parental alpha LI-domain was tested. Whereas the parental alpha LI-domain does not bind to P2-C, the grafting of the alpha M segment imparted the P2-C binding function to the chimeric I-domain. Although the affinity of the interaction could not be assessed accurately from the experimental format used, the concentration of the immobilized P2-C required to obtain 50% of the chimeric I-domain binding was similar to that for wild-type alpha MI-domain, i.e. ~12 µg/ml for the chimera compared with ~20 µg/ml for the wild-type alpha MI-domain. The interaction of the chimeric molecule with P2-C was blocked by soluble P2-C, thus confirming specificity (Fig. 6B).



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Fig. 6.   Binding of wild-type alpha LI-domain and alpha L(alpha M (Lys245-Arg261)) chimeric I-domain to P2-C. Panel A, different concentrations of wild-type alpha LI- () and alpha L(alpha M(Lys245-Arg261))I- (open circle ) domains as fusions with GST in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM Mg2+, 1 mM Ca2+, 0.05% Tween 20, and 5% glycerol were added to the wells coated with 50 µg/ml P2-C and incubated for 3 h at 22 °C. After washing, bound I-domains were detected by anti-GST mAb (1:5,000 dilution). After washing, goat anti-mouse IgG conjugated to alkaline phosphatase was added for 1 h, and the binding of the I-domains was measured by reaction with the p-nitrophenyl phosphate. Background binding to BSA or polyvinyl alcohol was subtracted. Panel B, inhibition of the chimeric I-domain binding by P2-C. 50 µg/ml chimera was mixed with different concentrations of P2-C () or H19 () and then added to the wells coated with P2-C. The binding of the I-domain was determined as in panel A. Results are presented as a percentage of maximal chimera binding in the absence of P2-C.

Identification of Residues within the alpha M(Lys245-Arg261) Segment Critical for P2-C Binding-- The residues within the alpha M(Lys245-Arg261) segment, responsible for the P2-C binding, were identified by site-directed mutagenesis. The selection of residues for mutational analyses was based on the following considerations: 1) the side chains of residues that participate in direct docking of P2-C should be exposed on the hydrated surface of the alpha MI-domain; 2) given the similarities in the binding function between alpha M and alpha X I-domains, it is likely that the residues that interact with P2-C should be identical or conserved between two I-domains; 3) because the switch of the 248DPLGY252 segment did not affect adhesion of mutant cell line, this sequence was not included in mutational analyses. Nine residues, Lys245, Phe246, Gly247, Glu253, Asp254, Pro257, Glu258, Asp260, and Arg261 are exposed on the surface of the alpha MI-domain in the Lys245-Arg261 stretch, and eight residues are identical in the alpha M(Lys245-Arg261) and alpha X(Lys262-Ala277) sequences (see Fig. 7A). Thus, only 5 of the 17 residues in Lys245-Arg261 segment meet both criteria, being exposed and identical between alpha M and alpha X: Lys245, Gly247, Asp254, Pro257, and Asp260 (Fig. 7A). To examine the contribution of these residues in the ligand binding function, single or multiple point mutations to alanine were introduced into the alpha MI-domain (Fig. 7A), and the capability of mutant proteins to interact with the immobilized P2-C was tested (Fig. 7B). The binding of mutants containing single or double mutations of Asp254 and Pro257 (mutants 3, 4, 5, and 6) was significantly impaired compared with wild-type alpha MI-domain. In contrast, I-domains with substitutions of Lys245, Gly247, and Asp260 bound similarly to the wild-type alpha MI-domain. Because the switch of 245KFG impaired adhesion of the alpha Mbeta 2-expressing cells (see Fig. 3, A and B), Phe246 might be critically involved in P2-C recognition. Accordingly, Phe246 was mutated to Arg because previous data had indicated that substitution of Phe246 to the positively charged residue abrogated binding of the alpha MI-domain to iC3b, whereas mutation to Ala was without effect (23). As shown in Fig. 7B, the binding of the alpha MI-domain with mutation F246R was reduced significantly. The reduced binding of the three mutants, F246R, D254A, and P257A, to P2-C was apparently not caused by perturbation in conformation based upon the following observations: 1) The reactivity of the mutant I-domains with anti-alpha MI-domain mAbs 44a and 904 was similar to that the wild-type alpha MI-domain (not shown), consistent with what was reported previously for other mutations in this region (20). 2) Inspection of the three-dimensional structure of the alpha MI-domain suggested that introduction of these substitutions would not produce conformational changes because side chains of the three mutated residues do not interact with neighboring residues and thus do not contribute into stabilization the overall structure; instead, alanine substitution of Pro257 should improve the slightly imperfect alpha 5 helix. 3) Typical proteins can tolerate major mutations within a single loop and remain properly folded (36). Thus, these data suggest that the three residues, Phe246, Asp254, and Pro257, are directly involved in the P2-C binding.



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Fig. 7.   Binding of the recombinant wild-type I-domains and alpha MI-domain mutants to P2-C. Panel A, alignment of the alpha MI-, alpha XI-, and alpha LI-domain sequences. The alpha M(Lys245-Arg261) sequence was aligned with human alpha X(Lys262-Ala277) and alpha L(Ala269-Asp280) sequences using the NCBI Blast program. The solvent-exposed residues in alpha M(Lys245-Arg261) are in bold, and the residues identical between alpha M and alpha X are underlined. The residues mutated in the alpha MI-domain are illustrated in the lower part of panel A. Panel B, different concentrations of wild-type (WT) alpha MI-, alpha XI-, and alpha LI-domains and mutant alpha MI-domains as fusions with GST were added to microtiter wells coated with 50 µg/ml P2-C and incubated for 3 h at 22 °C. The binding of the recombinant I-domains was detected as in Fig. 6. The binding of each mutant reached the maximal level at 100 µg/ml of the added I-domain, and results are presented as a percentage of the binding attained with wild-type alpha MI-domain. The dashed line indicates the maximal level of binding achieved with wild-type alpha MI-domain, and the dotted line is drawn at the level of binding attained with the recombinant alpha LI-domain.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have identified key elements of the binding site for a small amino acid sequence of Fg, gamma 383-395 (P2-C), within the alpha MI-domain of alpha Mbeta 2. The strategy to define the ligand binding site was based on the difference in the P2-C binding properties of the alpha MI-, alpha XI-, and alpha LI-domains and entailed four complementary approaches. In the first approach, a series of homolog-scanning mutants, used previously to map the binding regions for NIF, iC3b, and Candida albicans (22, 24, 37), were screened for adhesion to P2-C and D100 fragment of Fg. In these mutants, 16 segments from the alpha MI-domain were replaced with the corresponding segments from the homologous alpha LI-domain, which does not bind Fg. Because all of these swapped segments are located at the hydrated surface of the alpha MI-domain, they should identify candidate sequences for interaction with the ligand. Five mutants lacked the ability to support adhesion to P2-C and D100: alpha M(P147-R152), alpha M(P201-G207), alpha M(R208-K217), alpha M(K245FG), and alpha M(E253-R261). Therefore, these alpha MI-domain segments may be critical for binding of these ligands. Alteration of three other regions, dE262G, Asp273-Lys279, and Phe297-Thr307, resulted in the partial loss of adhesive function. These segments may play an accessory role in ligand binding. Thus, the initial insight provided by these mutant receptors indicated that the P2-C binding interface within the alpha MI-domain was composed of several nonlinear sequences. Based on the crystal structure of the alpha MI-domain (25), the segments critical for recognition encompass a portion of helix 1, the loop between helix 3 and helix 4, the small 245KFG segment in the loop between helix 5 and beta -strand D, and the entire helix 5 (Fig. 8, left). These sequences form an almost continuous stretch on the upper face of the alpha MI-domain (colored in different shades of green in Fig. 8, left).



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Fig. 8.   Binding site for P2-C in the alpha MI-domain. Left, ribbon model of the alpha MI-domain based upon its crystal structure (25), code 1JLM. The 245KFG in the beta D-alpha 5 loop and Glu253-Arg261 in the helix alpha 5 are bright green. The segments Pro147-Arg152 and Pro201-Lys217 are light green. The numbers indicate the positions of segments. The side chains of residue mutations that were found to affect the P2-C binding, Phe246, Asp254, and Pro257, are shown in purple. beta -Strands and helix assignments are shown. Right, ribbon model of the alpha LI-domain, based upon its crystal structure, code 1LFA (27), shown for comparison. The region homologous to alpha M(Lys245-Arg261) is light blue. The models were drawn using the computer program Molscript, Bobscript and Raster (44-46).

The second approach entailed the use of synthetic peptides duplicating the sequences of the critical segments in the alpha M I-domain. These analyses showed that two of four critical segments, alpha M(245KFG) and alpha M(Glu253-Arg261), may contain amino acid residues that participate directly in binding the P2-C sequence of Fg because the peptides that duplicated Lys245-Tyr252 and Glu253-Arg261, bound P2-C. Although the peptide Pro147-Arg152 did not interact with the Fg derivatives, and Pro201-Lys217 interacted weakly, the negative results do not exclude a role for these peptides in binding function; the immobilized peptide may simply not assume the appropriate conformation for recognition by the ligand.

The Lys245-Tyr252 and Glu253-Arg261 sequences are contiguous, and the entire alpha M(Lys245-Arg261) sequence might serve as the primary binding site for P2-C. Of note, this segment is the most divergent between the alpha MI- and alpha LI-domains in terms of sequence homology and folding (Fig. 8, right). In fact, alpha L lacks most of helix 5, which is formed by residues Tyr252-Arg261 of alpha M (27, 28). In addition, the loop beta D-alpha 5 is longer in alpha M than in alpha L and assumes a different conformation. Therefore, this difference between alpha M and alpha L could account for inability of alpha L to bind Fg. To obtain direct evidence that the beta D-alpha 5 loop-alpha 5 helix in the alpha MI-domain constitutes the functional binding site for the Fg ligand, the third approach entailed grafting of the entire Lys245-Arg261 segment into the corresponding region of the alpha LI-domain. As demonstrated in Fig. 6, this manipulation imparted P2-C binding capacity to the chimeric molecule, and the binding affinity of the chimeric receptor for P2-C was very similar to that of wild-type alpha MI-domain. Thus, the role of the beta D-alpha 5 loop-alpha 5 helix in P2-C binding, which initially was inferred from the loss-in-function experiments, was verified by the gain-in-function approach.

Finally, the implementation of the fourth method, site-directed mutagenesis, served the dual purpose. First, it provided the independent confirmation that the Lys245-Arg261 segment is important for P2-C binding because mutations of the three residues resulted in the significant loss of P2-C binding. Second, it implicated Phe246, Asp254, and Pro257 as contact residues. Taken together, the four approaches substantiated independently the role of the beta D-alpha 5 loop-alpha 5 helix in the P2-C binding and provided evidence that three residues participate in ligand docking.

It is unclear why switches of the alpha M(Pro147-Arg152) and alpha M(Pro201-Lys217) resulted in the loss of function given that two other unsubstituted segments, alpha M(245KFG) and alpha M(Glu253- Arg 261) could potentially support adhesion. The effect of the switches of alpha M(Pro147-Arg 152) and alpha M(Pro201-Lys217) on P2-C recognition could conceivably arise from changes of conformation of the ligand binding region residing in the alpha M(Lys245-Arg261). In this regard, a subtle perturbation of the structure of alpha M(P201-G207) and alpha M(P147-R152) mutants was suggested previously by an altered reactivity with the conformation-dependent mAb 24 (22). In addition to the MIDAS motif (25), which is known to be required for the normal binding function (21, 38), single point mutations in the regions outside MIDAS also may abrogate ligand binding by altering conformation (21, 38, 39). For example, alanine substitution of Asp248 or Tyr252, the residues that are not exposed on the surface of the alpha MI-domain, eliminated the binding of mutants to iC3b (21), suggesting that a structural alteration might have been involved in the loss-of-binding function. Thus, even relatively small perturbations in the folding of the I-domain can lead to gross alterations in binding affinities.

Although alpha M(Lys245-Arg261) resides in close proximity to the cation binding MIDAS motif, none of the residues in this sequence is directly involved in coordination of the divalent cation (25). Our results indicate that the binding of Fg derivatives, the D fragment and P2-C, to immobilized peptides duplicating Lys245-Arg261 region was cation-independent. This finding is consistent with previous data showing that EDTA or mutation of Asp242, a residue which coordinates to the bound metal, only partially impairs the binding of Fg to the alpha MI-domain although it abolished the binding of other ligands, including NIF and iC3b to the recombinant fragment (25). Another report also suggests that ligands can bind to I-domains independent of cations. Peptides duplicating beta D-alpha 5 loop in the alpha MI-domain or immediately preceding it bound to iC3b in a cation-independent manner (16). Thus, at least in the case of P2-C, its binding to the alpha MI-domain does not occur through direct interaction with the metal ion as was proposed (25). This conclusion is further supported by the fact that P2-C sequence in Fg does not contain a candidate acidic residue to provide a missing coordination to the metal. At the same time, P2-C does contain an arginine residue, Arg391, which could displace cation, a model suggested from the crystal structure of the alpha 1I-domain (40).

The sequence alpha M(Lys245-Tyr252), which overlaps with the identified Fg-binding region alpha M(Lys245-Arg 261), was implicated previously in the binding of iC3b. Deletion of Phe246-Tyr252 abolished rosetting of iC3b-coated erythrocytes with alpha Mbeta 2 (21). In addition, mutation of Lys245 to Ala (24) and Phe246 to Lys (24) also significantly reduced iC3b binding. However, although the binding site for iC3b overlaps with the Fg binding site, the contribution of this region in recognition of two ligands appears to be distinct. For example, deletion of 248DPLGY did not affect adhesion to P2-C or D100 fragment in our experiments, but it reduced to some extent iC3b binding (19).

The overlapping nature of the NIF and Fg binding sites within the alpha MI-domain was suggested previously based on the ability of NIF to inhibit interaction of the alpha Mbeta 2-bearing cells with Fg (18, 19). The same segments which were identified as critical for Fg binding, alpha M(Pro147-Arg152), alpha M(Pro201-Gly207), alpha M(Arg208-Lys217), and alpha M(Glu253-Arg261), also have been shown to participate in NIF binding (22). The significant differences in the binding of alpha MI-domain mutants to these two ligands were: 1) switch of 245KFG, which completely abrogated adhesion to Fg peptides, was not critical for NIF binding; and 2) deletion of 248DPLGY, which affected NIF binding, was not detrimental for adhesion to Fg derivatives. The binding site for NIF was verified previously by grafting the identified segments into the alpha XI-domain because these swaps imparted NIF binding capacity to the chimeric receptor (22). However, because the identified segments were grafted simultaneously, it is uncertain whether all of these sequences contain NIF contact sites or if some may provide structural elements necessary to maintain a permissive conformation for NIF binding. That NIF did not inhibit the binding of the D fragment or P2-C to immobilized Lys245-Tyr252 and Glu253-Arg261 suggests that these segments of the alpha MI-domain may not contain contact sites for NIF. Therefore, one possibility is that the primary binding site for NIF may reside in the two other identified segments, alpha M(Pro147-Arg152) and alpha M(Pro201-Lys217). Indeed, switching alpha M(Pro147-Arg152), alpha M(Pro201-Gly207), and alpha M(Arg208-Lys217) decreased affinity for NIF 33-, 305- and 206-fold, respectively (22). At the same time, affinity of the alpha M(E253-R261) swapping mutant or alpha M(D248PLGY252) deletion mutant was reduced 8- and 13-fold, respectively (22). In addition, Rieu et al. (20) demonstrated the importance of Gly143, Asp149, and Arg208, which reside close or within alpha M(Pro147-Arg152) and alpha M(Pro201-Lys217), for NIF binding. The extension of this hypothesis is that NIF and P2-C do not compete for the same binding site on the alpha MI-domain but rather that NIF blocks adhesion to Fg by steric or allosteric effects. Also, the epitope for the mAb CBRM1/5 was recently mapped to a region that includes Pro147, His148, Arg151 within alpha M(Pro147-Arg152) and Lys200, Thr203, and Leu206 within the alpha M(Pro201-Arg208) segment (41). This mAb blocks the alpha Mbeta 2-mediated adhesion to several ligands including Fg (42). Furthermore, the epitope for the second inhibitory mAb, 7E3, which recognizes the active I-domain conformation and interferes with binding of Fg to stimulated leukocytes resides in the alpha M sequence Gly127-Phe150 (43). Therefore, this region in the alpha MI-domain may contain a unique regulatory element that controls ligand recognition to other portions of the alpha MI-domain.

In summary, we have determined the binding site for the P2-C sequence of Fg, gamma 383-395, within the alpha MI-domain of alpha Mbeta 2. The binding site was localized in the 17-mer linear sequence, Lys245-Arg261, thus mapping the site to a very narrow region on the MIDAS face of the alpha MI-domain. The interaction between P2-C and the alpha MI-domain Lys245-Arg261 may represent a minimal functional recognition unit and, thus, defines complementary binding sites within the alpha MI-domain and ligand. The alpha M(Lys245-Arg261) sequence is a region of the major difference between the I-domains of two related integrins, alpha Mbeta 2 and alpha Lbeta 2, in terms of sequence and structure. Thus, the regions of structural divergence may provide the potential mechanism whereby integrins specify ligand recognition.


    ACKNOWLEDGEMENTS

We thank Dr. C. Forsyth for valuable technical advice regarding the mutant cells propagation, Dr. B. Kudryk for providing mAb 4-2, Dr. G. Matsueda for mAb 4A5, and Corvas International Inc. for NIF. We thank Jane Rein for secretarial assistance. FACS analyses were performed at the Institutional core facility established with a gift from the W. M. Keck Foundation.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL-63199 (to T. P. U.) and HL-54924 (to E. F. P).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.

To whom correspondence should be addressed: Dept. of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, NB50, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8209; Fax: 216-445-8204; E-mail: ugarovt@ccf.org.

Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010174200

2 T. P. Ugarova, V. P. Yakubenko, B. Kudzyk, E. F. Plow, and V. C. Yee, manuscript in preparation.

3 V. P. Yakubenko and T. P. Ugarova, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: Fg, human fibrinogen; ICAM-1, intercellular adhesion molecule-1; I-domain, region of ~200 residues "inserted" in the alpha -subunit of alpha Mbeta 2, alpha Xbeta 2, and alpha Lbeta 2; NIF, neutrophil inhibitory factor; mAb, monoclonal antibody; GST, glutathione S-transferase; FACS, fluorescence activated cell sorting; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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