Characteristics of Cation Binding to the I Domains of LFA-1 and MAC-1
THE LFA-1 I DOMAIN CONTAINS A Ca2+-BINDING SITE*

David W. Griggs, Christina M. Schmidt, and Christopher P. CarronDagger

From Discovery Pharmacology, Searle Research and Development, Monsanto Company, St. Louis, Missouri 63198

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The crystal structures of the I domains of integrins MAC-1 (alpha Mbeta 2; CD11b/CD18) and LFA-1 (alpha Lbeta 2; CD11a/CD18) show that a single conserved cation-binding site is present in each protein. Purified recombinant I domains have intrinsic ligand binding activity, and in several systems this interaction has been demonstrated to be cation-dependent. It has been proposed that the I domain cation-binding site represents a general metal ion-dependent adhesion motif utilized for binding protein ligands. Here we show that the purified recombinant I domain of LFA-1 (alpha LI) binds cations, but with significantly different characteristics compared with the I domain of MAC-1 (alpha MI). Both alpha LI and alpha MI bind 54Mn2+ in a conformation-dependent manner, and in general, cations with charge and size characteristics similar to Mn2+ most effectively inhibit 54Mn2+ binding. Surprisingly, however, physiological levels of Ca2+ (1-2 mM) inhibited 54Mn2+ binding to purified alpha LI, but not to alpha MI. Using 45Ca2+ and 54Mn2+ in direct binding studies, the dissociation constants (KD) for the interactions between these cations and alpha LI were estimated to be 5-6 × 10-5 and 1-2 × 10-5 M, respectively. Together with the available structural information, the data suggest differential affinities for Mn2+ and Ca2+ binding to the single conserved site within alpha LI. Antagonism of LFA-1, but not MAC-1, -mediated cell adhesion by Ca2+ may be related to the Ca2+ binding activity of the LFA-1 I domain.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

LFA-1 (alpha Lbeta 2) and MAC-1 (alpha Mbeta 2) are closely related leukocyte integrins that are essential for normal immune system functions (1, 2). Both integrins bind to several distinct ligands, but share in the ability to bind ICAM-1, a widely expressed cell surface protein (3-5). Recent studies suggest that in the beta 2 and other non-RGD binding integrins, additional and perhaps multiple subdomains within both the alpha  and beta  subunits may contribute to form the complete ligand-binding domain. One of these subdomains is the I domain (also known as the A domain), a region of approximately 200 amino acids found in a variety of proteins as well as the alpha  subunit ectodomain of all beta 2 integrins and VLA-1 (alpha 1beta 1), VLA-2 (alpha 2beta 1), and alpha Ebeta 7 (6, 7).

Several lines of evidence suggest that the I domain in the context of the complete integrin may play a significant and direct role in ligand binding. The activities of both integrin-neutralizing and integrin-activating monoclonal antibodies, and NIF, a hookworm-derived MAC-1 inhibitor have been mapped to the I domain region (8-14). Significantly, purified recombinant forms of the I domains derived from LFA-1, MAC-1, and VLA-2 have intrinsic ligand binding activity (12, 15-19) and in several systems it has been shown that this protein-protein interaction is cation-dependent (12, 19). Determination of the I domain crystal structures has provided a structural basis for conceptualizing the role of cations in alpha MI and alpha LI interaction with ligands (19-21). The structures show that alpha MI and alpha LI domains contain a single metal cation-binding site, and that residues involved in coordinating the metal ion in each protein are completely conserved (21). Lee and co-workers (19) proposed that this novel cation-binding site represents a general metal ion-dependent adhesion (MIDAS)1 motif for binding protein ligands. Interestingly, crystallization of alpha MI in the presence of different cations has been shown to result in significant changes in metal coordination and protein structure (19), suggesting that differential effects of cation binding on integrin function may be possible.

Divalent cations have multiple effects on integrin-mediated cell adhesion including enhancement, suppression, and modification of ligand binding activity. Mg2+ and Mn2+ induce conformational alterations of several integrins, including LFA-1 and MAC-1, concomitant with activation of integrin-mediated adhesion to ligands (22-24). In contrast, Ca2+ has been shown to inhibit LFA-1, but not MAC-1, mediated adhesion to ligands (24-28). We speculated that the differential effects of Ca2+ and Mn2+/Mg2+ on LFA-1 and MAC-1 function might be related to differences in the divalent cation binding properties of their I domains. In the work described herein, we compared the cation binding properties of purified recombinant alpha L and alpha M I domains. Our results indicate that like alpha MI, alpha LI preferentially binds Mn2+ over most other cations. However, Mn2+ interaction with alpha LI was inhibitable to some degree using a variety of cations, and these studies revealed a pattern of binding selectivity that was clearly distinct from alpha MI. Interestingly, Ca2+ inhibited Mn2+ binding to alpha LI, but had little effect on Mn2+ binding to alpha MI, underscoring a fundamental difference in the cation-binding properties of these two I domains. Furthermore, experiments confirmed that alpha LI binds both Mn2+ and Ca2+ and the results are consistent with the notion that the alpha LI contains a single mixed-type Mn2+/Ca2+-binding site. It is possible that the activation state of LFA-1 is regulated in part by the interaction of the I domain with cations present in the extracellular environment. Ca2+ antagonism of LFA-1, but not MAC-1, mediated cell adhesion may be a consequence of the Ca2+ binding activity of the I domain of LFA-1 in that the Mn2+ and Ca2+ complexes of alpha LI may represent, respectively, high- and low-affinity ligand binding states.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Reagents-- Purified maltose-binding protein (MBP2) was obtained from New England Biolabs (Beverly, MA). Purified bovine serum albumin (BSA) was obtained from Pierce (Rockford, IL). Concentrations of proteins were determined using the Bio-Rad Protein Assay Reagent (Bio-Rad) with BSA as standard.

All buffer solutions were prepared in single-use plastic containers. Water was obtained by a Milli-RO/Milli-Q water system (Millipore Corporation, Bedford MA). Reagents used for the preparation of buffers were of the highest quality available. The concentration of Ca2+ and Mn2+ in the water and overlay buffer (described below) was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES) using a Jarell Ash Atomcomp 975 (Thermo Jarell Ash Corp., Franklin, MA) instrument. The lower limit for detection of Ca2+ and Mn2+ in water and overlay buffer by ICP-AES was 0.50 and 0.55 µM, respectively, and 2.5 and 0.91 µM, respectively. The measured Ca2+ and Mn2+ concentrations in water and buffer were below the limit of detection by ICP-AES.

Expression and Purification of I Domain Polypeptides-- alpha MI was expressed as a fusion protein with glutathione S-transferase (GST), purified from the soluble fraction of the cleared Escherichia coli lysate by affinity chromatography and then cleaved with thrombin to separate the I domain from the GST moiety. The alpha MI domain was amplified by polymerase chain reaction, using as template plasmid pMON24304 (obtained from B. Harding, Monsanto Co., St. Louis, MO) that contains a 2.45-kilobase cDNA fragment that includes the I domain region of CD11b. The primers used were 5'-GCGGATCCAACCTACGGCAGCAG-3' and 5'-GCGCGGCCGCGCAAAGATCTTCTCCCGAAG-3'. The fragment was digested with BamHI and NotI, cloned into pGEX 4T-1 (Pharmacia LKB, Uppsala, Sweden), and the accuracy of the DNA sequence was verified. The construct joins the glutathione S-transferase gene to the alpha MI coding sequence and adds five vector-derived amino acids (RPHRD) at the carboxyl terminus. The plasmid was transformed into E. coli strain DH5alpha , and expression and purification of the alpha MI polypeptide was carried out essentially by the procedure of Michishita et al. (29), in which glutathione-Sepharose 4B affinity chromatography is followed by thrombin digestion and gel filtration using a Superose 12 column (Pharmacia LKB, Uppsala, Sweden).

A similar strategy was employed for the expression of the I domain of LFA-1. A cDNA encoding the full-length alpha L (CD11a) protein was identified using standard hybridization techniques to screen a human inflamed colon library constructed in the lambda  ZAP II vector (Stratagene, San Diego, CA). The alpha LI coding region of the full-length alpha L (CD11a) template was amplified by polymerase chain reaction using the following oligonucleotides: 5'-GCGGATCCAATCTGCAGGGTCCCATGCTG-3' and 5'-GCGAATTCAGCTCCATGTTGAAGGAAGT-3'. The product was digested with BamHI and EcoRI, cloned into pGEX-2T (Pharmacia LKB, Uppsala, Sweden), and sequenced. This construct joins the glutathione S-transferase gene to the alpha LI-coding sequence and adds six vector-derived amino acids (NSIVTD) at the carboxyl terminus. alpha LI expressed in E. coli as a fusion protein with GST and grown under the conditions utilized for expression of alpha MI formed intracellular inclusion bodies and uncleavable fusion protein. However, modification of the conditions for expression of the GST-alpha LI fusion as described below yielded soluble and cleavable fusion protein. Transformed cells were grown in 3 liters of M9 minimal medium containing 50 µg/ml proline at room temperature to an optical density (A600) of 0.7. Expression was induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 24 h at room temperature. Cells were pelleted, stored at -70 °C overnight, and then lysed by sonication in 120 ml of column buffer (200 mM NaCl, 5 mM MgCl2, 20 mM Tris, pH 7.4). Triton X-100 (0.1%) was added and the lysate incubated for 30 min at 4 °C. The sample was then centrifuged, filtered (0.2 µM), and incubated with 3 ml of a 50% slurry of glutathione-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) for 30 min at room temperature. The resin was washed 4 times with column buffer, and the protein was eluted with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0. The sample was treated in column buffer with 1 unit of restriction grade thrombin (Novagen, Madison, WI) per mg of protein for 16 h at room temperature, and digestion was terminated with 1 mM phenylmethylsulfonyl fluoride. After dialysis of the sample against 2000 volumes of column buffer, free GST was removed by batch adsorption using the glutathione resin. The remaining protein was then characterized by protein sequencing, MALDI mass spectroscopy, and SDS-PAGE.

The GST-alpha LI fusion protein expressed by the methods described above yielded relatively small quantities of soluble fusion protein. To generate larger amounts of protein needed for cation-binding studies, a soluble MBP (maltose-binding protein) fusion form of alpha LI was produced. MBP-alpha LI was generated by polymerase chain reaction-mediated amplification of the I domain-coding region of the cDNA encoding the full-length alpha L (CD11a) using the following oligonucleotides as primers: 5'- GCGAATTCAATCTGCAGGGTCCCATGCTG-3' and 5'-GCAAGCTTTCACAGCTCCATGTTGAAGGAAGT-3'. The product was digested with HindIII and EcoRI, cloned into pMAL-c2 (New England Biolabs), and the accuracy of the DNA sequence was verified. The resulting coding sequence joins the MBP to CD11a at amino acid Asn110 and extends to Leu324 corresponding ot the same residues contained in the free alpha LI protein described above. An overnight culture of E. coli strain DH5alpha containing the expression plasmid was subcultured 1:10 into 2 liters of LB broth with ampicillin and grown at 37 °C for 1 h. Expression was induced by the presence of 0.3 mM isopropyl-1-thio-beta -galactoside for 2 h. Cells were centrifuged and stored frozen at -80 °C overnight. After thawing, cells were resuspended in 60 ml of MBP column buffer (20 mM Tris pH7.4, 200 mM NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride), frozen in a dry ice/ethanol bath, thawed, and sonicated on ice for 30-s intervals until lysis was achieved. The sample was centrifuged, and the cleared lysate filtered (0.2 µM pore size) before applying to an amylose resin (New England Biolabs) column (70 ml bed volume) equilibrated with MBP column buffer. The column was washed extensively with MBP column buffer and the protein eluted into fractions with MBP column buffer containing 10 mM maltose. Protein identity and purity was confirmed by amino-terminal peptide sequencing and SDS-PAGE analysis, respectively.

Cation Binding Assays-- 54Mn2+ and 45Ca2+ binding to purified proteins was measured by procedures based on those previously described by Michishita et al. (29). Equimolar amounts of purified recombinant proteins were bound to nitrocellulose (PH79, 0.1 µM pore size, Schleicher and Schuell, Keene, NH) using the Mini-Fold II Slot Blot System (Schleicher and Schuell). Filters were washed twice for 10 min at room temperature with overlay buffer (10 mM imidazole, pH 6.8, 60 mM KCl) containing 10 mM EDTA, followed by four washes for 30 s with overlay buffer lacking EDTA. The filter was then incubated for 10 min in overlay buffer containing 0.5 µCi/ml 54MnCl2 (>40 Ci/g, NEN Life Science Products, Boston, MA) or 45CaCl2 (10-75 Ci/g, NEN Life Science Products) in the presence or absence of cold competitor cations diluted from freshly prepared stock solutions. Filters were washed twice in 50% ethanol and dried. Bound radioactive material was measured either by autoradiography or by PhosphorImage analysis using a Molecular Dynamics (Sunnyvale, CA) PhosphorImager system. The efficiency of protein binding to nitrocellulose was assessed by staining the filter with naphthol blue black as described previously (30), and scanning using an LKB Ultrascan XL densitometer (LKB, Uppsala, Sweden). Binding to filters was determined to be dose-dependent and was highly consistent (i.e. less than 10% variation) between identically loaded wells. In some experiments using cold competitor cations, IC50 values were derived from a two parameter logistic model using nonlinear regression analysis.

Estimation of Ca2+ and Mn2+ Equilibrium Dissociation Constants-- The Ca2+ and Mn2+ binding parameters for MBP-alpha LI were examined using the methods described above with the following modifications. MBP-alpha LI was dialyzed exhaustively against 200 mM NaCl, 20 mM Tris, pH 7.5, and 5 mM MgCl2 and passed through a 0.2-µm filter prior to use. 45Ca2+ and 54Mn2+ were diluted with non-radioactive Ca2+ and Mn2+, respectively, to obtain total concentrations of these cations that ranged from 0.01 to 10 mM. MBP-alpha LI was bound to filters, washed with overlay buffer containing EDTA to remove bound metal, and then rinsed exhaustively with overlay buffer to remove residual EDTA. Filters were incubated with various concentrations of 45Ca2+ and 54Mn2+ for 10 min at room temperature, removed, and washed with 50% ethanol as described above. The filters were air dried and the amount of Ca2+ or Mn2+ bound determined by PhosphorImager analysis. A 45Ca2+ or 54Mn2+ standard curve was utilized to determine the specific activity of each labeled cation expressed as arbitrary units/pmole of cation.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of Recombinant Proteins-- The I domain coding regions were derived by polymerase chain reaction amplification using CD11a and CD11b cDNA clones as templates as described under "Experimental Procedures." The sequence of the MAC-1 I domain (alpha MI) was identical to that reported previously (31). However, the sequence of the LFA-1 I domain (alpha LI), as determined from two independent clones from the cDNA library, varied from that reported by Larson et al. (32) at a single nucleotide that results in substitution of tryptophan 189 by arginine. This substitution has also been identified by other researchers (21) and may represent a natural allelic variation. The position of this residue lies on the surface of the protein, far from the MIDAS motif, and thus is unlikely to affect the metal binding characteristics.

alpha MI was expressed as a fusion protein with GST, purified from the soluble fraction of the cleared E. coli lysate by affinity chromatography, and then cleaved with thrombin to separate the I domain from the GST moiety. In contrast, alpha LI expressed in E. coli as a fusion protein with GST, using conditions similar to those utilized for the expression of alpha MI, formed intracellular inclusion bodies. Modification of the expression conditions, as described under "Experimental Procedures," yielded small amounts of soluble GST-alpha LI that was cleavable with thrombin.

In order to generate larger amounts of soluble protein, alpha LI was expressed as a fusion protein with MBP. In this expression context, the protein was found almost exclusively in the soluble fraction and at high concentration. Unfortunately, cleavage of the MBP-alpha LI fusion protein with thrombin resulted in I domain aggregation and precipitation. Due to the apparent ability of the MBP moiety to enhance the solubility of the LFA-1 I domain and the high yields of recovered protein (e.g. up to 30 mg/liter of cell culture) the majority of experiments utilized alpha LI expressed as a fusion protein with MBP. However, to verify that structural differences imposed by the nature of the MBP-alpha LI fusion protein construct did not contribute to alpha LI cation binding properties, some experiments were conducted with alpha LI cleaved and purified from the GST-alpha LI fusion protein. For the sake of clarity, we subsequently use the term alpha LIf to refer to MBP-alpha LI fusion protein and the term alpha LIi to refer to the purified I domain of LFA-1 isolated from the thrombin cleavage of the GST-alpha LI fusion protein. The accuracy of protease cleavage of the fusion proteins for the release of alpha MI and alpha LIi was confirmed by identification of the correct amino terminus by amino acid peptide sequencing. The molecular masses of alpha MI and alpha LIi, determined by MALDI mass spectroscopy, were within 0.05 and 0.01%, respectively, of their expected values. Purity of each protein was greater than 95% as determined by SDS-PAGE analysis (data not shown).

The I Domains of LFA-1 and MAC-1 Bind Metal Cations-- To demonstrate that the LFA-1 and MAC-1 I domains contain qualitatively similar cation-binding sites, alpha MI and alpha LIf were immobilized on nitrocellulose filters and incubated with 54Mn2+ as described under "Experimental Procedures." The autoradiogram shown in Fig. 1A shows that both alpha LIf and alpha MI, but not BSA, bind 54Mn2+ and that unlabeled Mn2+ competitively inhibits binding in a dose-dependent fashion. Quantitative PhosphorImager analysis of the nitrocellulose filters confirmed that 54Mn2+ binding to control proteins BSA or MBP was less than 10% of 54Mn2+ binding to either of the purified I domains (Fig. 1, B and C). Moreover, boiling of alpha LIf prior to immobilization on nitrocellulose reduced 54Mn2+ binding to background levels. These results showed that the purified I domains of each integrin contain a conformationally sensitive Mn2+-binding site.


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Fig. 1.   Binding of 54Mn2+ to the purified I domains of LFA-1 (alpha LIf) and MAC-1 (alpha MI). Equimolar amounts of protein were immobilized on nitrocellulose, incubated with 54Mn2+ in the presence of the indicated concentrations of cold Mn2+ and then washed and analyzed either by autoradiography (A) or PhosphorImager analysis (B and C). The results shown in A and B were obtained from the same experiment. Boiling of alpha LIf for 5 min prior to immobilization inhibited 54Mn2+ binding (C). 54Mn2+ binding to alpha LIf, black-triangle; to alpha MI, bullet ; and BSA, black-square.

To assess possible qualitative variation in the cation binding activity of the I domain of each integrin, we screened various divalent and trivalent metal ions for the capacity to inhibit 54Mn2+ binding to alpha MI and alpha LIf. The results in Fig. 2 and Fig. 3A demonstrate that 500 µM unlabeled Mn2+ reduced 54Mn2+ binding to alpha MI and alpha LIf by approximately 90%. In these experiments, the concentration of 54Mn2+ was approximately 0.2 µM, and hence the observed residual binding is likely to be nonspecific. In agreement with the earlier data of Michishita et al. (29), Mg2+, Ni2+, Co2+, Zn2+, and Cd2+ all were excellent competitors of Mn2+ binding to alpha MI, as were two previously untested divalent cations, Fe2+ and Cu2+ (Fig. 2). Relative to these divalent cations, those with higher states of oxidation (Cr3+, Fe3+, and Au(III)) were less effective and reduced the binding of 54Mn2+ by only approximately 50%. The cations with the largest ionic radii of those tested, Ba2+, Sr2+, and Ca2+, although divalent, had no effect on Mn2+ binding to alpha MI. In contrast to the results obtained with alpha MI, all of the tested cations inhibited to some extent 54Mn2+ binding to alpha LIf (Fig. 2B). However, the small trivalent cations (Fe3+ and Cr3+) and large divalent cations (Sr2+, Ba2+, and Ca2+) were less effective inhibitors than the divalent cations with ionic radii more similar to Mn2+ (e.g. Cd2+, Co2+, Fe2+, Zn2+, and Cu2+). The smallest of the divalent cations tested, Ni2+ and Mg2+, exhibited intermediate inhibitory activity. Notably, the Au(III) ion, which has an ionic radius very close to that of Mn2+, was among the best competitors. Of particular interest was the observation that the physiologically relevant Ca2+ ion had no effect on 54Mn2+ binding to alpha MI, but did inhibit 54Mn2+ binding to alpha LIf by greater than 70%. Together the data suggest that ionic size may be a more important attribute than charge in determining cation binding specificity to alpha LI, although the specific coordination geometry preferred by the cations may also play a roll. The data further suggest that the LFA-1 I domain may be complexed with Ca2+ at physiological concentrations.


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Fig. 2.   Various metal cations inhibit 54Mn2+ binding to alpha MI (A) and alpha LIf (B). 54Mn2+ (0.2 µM) binding to alpha MI and alpha LIf was determined in the presence of excess cold competitor cation (500 µM) to examine the cation binding specificity. Binding is expressed as the percentage of binding observed in the absence of inhibitor. Bound 54Mn2+ was quantified by PhosphorImager analysis as described under "Experimental Procedures." The results represent the mean of values derived from three independent experiments.


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Fig. 3.   Ca2+ is an effective inhibitor of 54Mn2+ binding to alpha LI, but not alpha MI. Binding of 54Mn2+ to immobilized alpha LIf, alpha MI (panel A), and alpha LIi (panel B) was measured in the presence of varying concentrations of unlabeled Mn2+ and Ca2+. A, 54Mn2+ binding to alpha MI and inhibition by unlabeled Ca2+ (Delta ) and Mn2+ (black-triangle). 54Mn2+ binding to alpha LIf and inhibition by unlabeled Ca2+ (open circle ) and Mn2+ (bullet ). B, 54Mn2+ binding to alpha LIi and inhibition by unlabeled Ca2+ (open circle ) and Mn2+ (Delta ). Values shown are the mean of duplicate determinations and the error bars indicate the standard deviation. The data presented in each panel are the results of a single experiment that was representative of two separate experiments that yielded similar results.

The I Domain of LFA-1, but Not MAC-1, Contains a Binding Site for Calcium-- Ca2+ inhibits LFA-1, but not MAC-1, mediated cell adhesion (24-28, 33), an effect that conceivably may be related to differences in the divalent cation binding properties of the I domains of these integrins. Since the results presented above suggested that Ca2+ binds to alpha LI but not alpha MI, we examined in further detail the binding of this cation to these purified polypeptides. The effect of increasing concentrations of unlabeled Mn2+ and Ca2+ on 54Mn2+ binding to alpha MI and alpha LIf is presented in Fig. 3A. In agreement with the results of Michishita et al. (29), unlabeled Mn2+ and Ca2+ inhibited 54Mn2+ binding to alpha MI with estimated IC50 values (inhibitory concentration resulting in 50% control binding) of 1-2 µM and greater than 10,000 µM, respectively. In contrast to these results, unlabeled Mn2+ and Ca2+ were both potent inhibitors of 54Mn2+ binding to alpha LIf inhibiting 54Mn2+ binding with IC50 values of 10-20 and 50-100 µM, respectively. Furthermore, 1 mM Ca2+ reduced 54Mn2+ binding to alpha LIf to a level equivalent to that observed with the control proteins BSA and MBP (data not shown). These results showed clearly that Ca2+ inhibited 54Mn2+ binding to alpha LIf but not alpha MI, and indicate that the I domain of LFA-1, but not MAC-1, has a Ca2+-binding site.

In the experiments described above, the cation binding comparison was made between alpha LIf (i.e. the MBP-alpha LI fusion protein) and alpha MI, the I domain of alpha M prepared by cleavage of the GST-alpha MI fusion protein. To rule out the possibility that the differences in the cation binding activities of the two I domains was related to structural differences imposed by the nature of the protein constructs, 54Mn2+ binding to alpha LIi, the I domain of LFA-1 prepared by thrombin cleavage of the GST-alpha LI fusion protein, was determined in the presence of increasing concentrations of unlabeled Mn2+ and Ca2+. In agreement with the results shown in the previous experiment, Fig. 3B shows that 54Mn2+ binds to alpha LIi, and that both unlabeled Mn2+ and Ca2+ inhibit 54Mn2+ binding (IC50 = 39 ± 19 and 278 ± 108 µM, respectively). Furthermore, the results suggest that the cation binding activities of alpha LIi and alpha LIf are similar, and that the Ca2+-binding site is associated with the I domain peptide.

The hypothesis that the LFA-1, but not the MAC-1 I domain, has a Ca2+-binding site was further confirmed by direct binding studies utilizing 45Ca2+. In the first experiment, alpha LIf, MBP, and alpha MI were immobilized on nitrocellulose paper and then incubated with 0.2 µM 45Ca2+. Fig. 4A shows that 45Ca2+ binds directly to alpha LIf, but not to MPB or alpha MI, results consistent with those in Fig. 3, and that together confirm that the 45Ca2+ binding activity is contained within the LFA-1 I domain rather than the associated MBP moiety. To further confirm these results, the experiment was repeated utilizing alpha LIi, which lacks all MBP sequence. alpha LIi was immobilized on nitrocellulose and incubated with 45Ca2+ in the absence or presence (>1000-fold excess) of unlabeled Ca2+. The results shown in Fig. 4B demonstrate that 45Ca2+ binds to alpha LIi, but not to alpha MI, and that excess unlabeled Ca2+ completely inhibited 45Ca2+ binding to alpha LIi. Together, the results of these experiments strongly suggest that the LFA-1 I domain contains a Ca2+-binding site. Furthermore, these results suggest that Ca2+ binding to the I domain of LFA-1, but not MAC-1, may occur at cation concentrations found in plasma (34).


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Fig. 4.   Binding of 45Ca2+ to alpha LI, but not alpha MI. A, equimolar amounts of alpha LIf, MBP, and alpha MI were immobilized on nitrocellulose, incubated with 0.2 µM 45Ca2+, and the amount of bound cation was determined by PhosphorImager analysis as described under "Experimental Procedures." B, equimolar amounts of alpha LIi and alpha MI were immobilized on nitrocellulose and incubated with 0.2 µM 45Ca2+ in the presence (>1000-fold excess) or absence of unlabeled Ca2+. Each experiment was repeated twice with similar results. The values shown are the mean of duplicate determinations and the error bars indicate the standard deviation.

Determination of the Dissociation Constant (KD) for Mn2+ and Ca2+ Interaction with alpha LIf-- The affinity constant for Ca2+ and Mn2+ binding to alpha LIf were determined by hot saturation binding studies and analysis of the equilibrium binding data as described below. Initial experiments showed that Ca2+ and Mn2+ binding to alpha LIf immobilized on nitrocellulose was reversible, reached equilibrium in less that 1 min, and that bound 45Ca2+ and 54Mn2+ dissociated rapidly when filters were incubated in overlay buffer containing cold competitor cation (data not shown). Cation dissociation from alpha LIf was stopped completely by immersing and washing nitrocellulose filters in 50% ethanol as described above (data not shown). Quantitation of Ca2+ or Mn2+ bound to the filters was performed by autoradiography and PhosphorImaging, and the data was analyzed using the EBDA/LIGAND software (Biosoft, Milltown NJ). Ca2+ and Mn2+ binding to alpha LIf is shown in Fig. 5. Binding to alpha LI was dose dependent and saturable (Fig. 5, A and B, insets), and the Scatchard plot of the binding data was curvilinear. LIGAND resolved the binding isotherm into two components corresponding to high and low affinity binding sites. The estimate for the KD of the high affinity Ca2+- and Mn2+-binding sites was 5.6 ± 0.7 × 10-5 and 1.4 ± 0.2 × 10-5 M, respectively. The estimate of the KD values for the low affinity Ca2+- and Mn2+-binding sites was 1-5 × 10-3 M. The maximal binding capacity of the high affinity site for Mn2+ and Ca2+ was 0.4 and 0.8 mol of cation bound/mol of alpha LIf, respectively. The slope of the Hill plot of the binding data for both Ca2+ and Mn2+ was close to unity, indicating the apparent absence of cooperativity between the high and low affinity sites (data not shown).


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Fig. 5.   Determination of the dissociation constant (KD) for Mn2+ and Ca2+ interaction with alpha LIf. 54Mn2+ (A) or 45Ca2+ (B) binding to alpha LIf was determined by PhosphorImager analysis. The binding data was replotted (inset) by the method of Scatchard (43) and analyzed using the program LIGAND as described under "Experimental Procedures." The values reported are the results of a single experiment; this experiment was repeated three times with similar results.

We questioned whether the low affinity metal-binding site might represent Ca2+ and Mn2+ binding to the MBP component of the fusion protein. Therefore, the affinity of each of these cations for purified MBP, lacking any I domain sequences, was determined as described above. Scatchard analysis of the binding data showed a single low affinity site for Ca2+ and Mn2+ in MBP with a KD corresponding to the low affinity site identified in alpha LIf (data not shown). We conclude that the low affinity metal-binding site in alpha LIf is due to Ca2+ and Mn2+ binding to the MBP component, whereas the higher affinity metal-binding site is associated with the I domain of the LFA-1 integrin.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The results of this study clearly show that the purified I domains of LFA-1 and MAC-1 bind cations with distinct selectivity. Binding of 54Mn2+ to alpha LI was inhibitable by a variety of divalent and trivalent cations with a range of ionic radii. In contrast, binding to alpha MI appeared to be more specific for smaller divalent cations. Notably, Ca2+ inhibited 54Mn2+ binding to alpha LI at concentrations well below those found in physiological environments (34), whereas Ca2+ failed to inhibit 54Mn2+ binding to alpha MI even when present at approximately 105 M excess. These results demonstrate a fundamental biochemical difference between these two closely related integrins, a finding with potential functional significance in the modulation of cell-ligand interactions.

In initial experiments, the comparison of the cation binding activities of the I domains of alpha L and alpha M were made between the intact MBP-alpha LI fusion protein (alpha LIf) and alpha MI, the I domain of alpha M that was cleaved from the GST-alpha MI fusion protein. It seemed possible that the cation binding characteristics of alpha LIf were unique to the MBP-alpha LI fusion protein and not reflective of the inherent cation binding activity of the LFA-1 I domain. To test this notion, we purified the isolated I domain of LFA-1 (alpha LIi) and then compared the cation binding characteristics of both forms of the LFA-1 I domain. The results of these experiments showed that: 1) 54Mn2+ binds to both alpha LIi and alpha LIf in a saturable manner; 2) binding is inhibitable with both unlabeled Ca2+ and Mn2+; and 3) both alpha LIi and alpha LIf, but not alpha MI, bind 45Ca2+. Furthermore, in these experiments, the concentration of 54Mn2+ was approximately 0.2 µM, a Mn2+ concentration approximately 5000-fold below the KD of the MBP low affinity cation-binding site in alpha LIf. Under these conditions 54Mn2+ binding is restricted to the LFA-1 I domain component of alpha LIf, a conclusion supported by the observation that Ca2+ binds to immobilized alpha LIi and alpha LIf, but not to MBP, a consequence of the relatively disparate cation binding affinities of the I domain and MBP components of alpha LIf. Collectively, the results suggest that alpha LIi and alpha LIf have similar cation binding characteristics and appear to be interchangeable in these experiments.

The binding data suggest that Ca2+ and Mn2+ bind to a single site within the I domain of LFA-1. 45Ca2+ and 54Mn2+ binding was inhibited by either unlabeled cation, demonstrating that these two cations bind to the LFA-1 I domain in a mutually competitive manner. Ca2+ and Mn2+ bind to the I domain of LFA-1 with similar affinity and stoichiometry with 1 µmol of cation bound/mol of I domain indicative of a Ca2+/Mn2+ mixed type binding site. We speculate that the Ca2+/Mn2+-binding site in alpha LI may represent an equivalent Ca2+/Mg2+-binding site since, as we show here, Mg2+ is an effective inhibitor of Mn2+ binding to alpha LI. Collectively, these results are consistent with the available structural information showing that the I domains of LFA-1 and MAC-1 contain a single Mn2+/Mg2+-binding site (19, 21, 29). Comparable structural data describing an I domain Ca2+-binding site has not been presented; the information herein is the first demonstration that the I domain of LFA-1 binds Ca2+ as well as Mn2+ and that both cations bind to a single site in a competitive manner. It should be noted that these metal binding studies were conducted using solid-phase binding techniques and it is possible that the physical interaction of the proteins with the nitrocellulose membrane could affect the metal binding characteristics. Consequently, confirmation of the reported binding constants using solution phase equilibrium binding dialyisis would be useful.

Considerable evidence supports the idea that cation-binding to integrin I domain MIDAS motifs plays a role in modulating the interaction of intact integrin with ligand. Mg2+ and Mn2+ induce conformational changes in integrins and both cations stimulate LFA-1-mediated cell adhesion to ICAM-1 (22-24,26,27). In contrast, Ca2+ inhibits Mn2+-induced activation of LFA-1, and Mg2+ stimulation of LFA-1 mediated cell adhesion to ICAM-1 requires prior chelation of Ca2+ by EGTA treatment (24, 26, 27). That the effects of divalent cations on integrin-mediated cell adhesion may be related to cation binding and cation-induced changes in I domain conformation is suggested by several observations. First, the recombinant forms of the I domains derived from a variety of integrins have intrinsic ligand binding activity. Second, ligand binding activity is cation-dependent and supported by Mn2+ and Mg2+, results that reflect the activity of the these cations on the behavior of the intact integrin (12, 16-19). Third, activation-specific conformational changes (neoepitopes) have been mapped to the I domain of MAC-1 (17, 35). Finally the structural data reported by Lee et al. (20) showed alpha MI crystals grown in Mg2+ display large differences in conformation and dramatic alteration of the surface of the protein implicated in ligand binding compared with crystals grown in Mn2+. These investigators proposed that the Mg2+ and Mn2+ structures represent conformations of the I domain that exist in the active and inactive states of the integrin, respectively (20). In addition, it was speculated that Ca2+ binding to the integrin I domain might stabilize the inactive form of the integrin (20). Together, these observations suggest that cation binding to the I domain modifies integrin interaction with ligand and raises the question whether cation-I domain complexes might exert activating or inactivating ligand-binding effects that are dependent upon the cation type.

Ca2+, Mg2+, and Mn2+ are present in the extracellular environment and all are available to compete for binding to the I domain. The normal concentration of Ca2+ and Mg2+ in serum is approximately 1 mM (34), and it has been estimated that the concentration of Mn2+ may range from 1 to 50 µM depending upon the particular tissue environment (36). Assuming that the cation binding properties of the isolated recombinant I domain and the native I domain within the context of the holoprotein are similar, the high concentration of cation relative to the Kd for cation binding to the I domain predicts that the metal-binding site in the I domain of LFA-1 will be occupied by cation. Furthermore, based on the evidence discussed above supporting the idea that cation-binding to integrin I domain MIDAS motifs play a role in modifying the integrin interaction with ligand, it is conceivable that I domain of low affinity LFA-1 on circulating leukocytes may be complexed with calcium, and that activation to the high affinity form may be accompanied by the replacement of Ca2+ with Mg2+ or Mn2+.

It seems possible that integrin activation could be driven in some environments by dynamic alteration in the ratio of cation concentration, for example, at sites of vascular or tissue injury or bone resorption (22, 36-39). However, a more likely scenario is that cation binding to the I domain of LFA-1 is only one of multiple factors that together coordinate and regulate LFA-1 activation. Additional components of the activation process likely include conformational changes conferred by inside-out signaling mechanisms and ligand binding (40-42). Consequently, structural changes in the integrin ectodomain mediated by inside-out signaling processes, ligand binding, and the association of Mg2+ or Mn2+ cation binding with the I domain, may combine to produce the activated form of LFA-1.

    ACKNOWLEDGEMENTS

We thank Elizabeth Harding for the gift of CD11b cDNA, Pat Sullivan for providing the inflamed colon cDNA library for CD11a screening, Khai Huynh for assistance with protein purification, and Chaur-Sun Ling for ICP-AES analysis.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Searle Research and Development, Monsanto Co. AA3C, 700 Chesterfield Village Pkwy., St. Louis, MO 63198. Tel.: 314-737-6847; Fax: 314-737-7310; E-mail: CPCARR{at}monsanto.com.

The abbreviations used are: MIDAS, metal ion-dependent adhesion; MBP, maltose-binding protein; BSA, bovine serum albumin; ICP-AES, inductively coupled plasma-atomic emission spectrometry; GST, glutathione S-transferasePAGE, polyacrylamide gel electrophoresis.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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