From Discovery Pharmacology, Searle Research and Development, Monsanto Company, St. Louis, Missouri 63198
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ABSTRACT |
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The crystal structures of the I domains of
integrins MAC-1 (M
2; CD11b/CD18)
and LFA-1 (
L
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
(
LI) binds cations, but with significantly different
characteristics compared with the I domain of MAC-1
(
MI). Both
LI and
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
LI, but not to
MI. Using
45Ca2+ and 54Mn2+ in
direct binding studies, the dissociation constants
(KD) for the interactions between these cations and
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
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.
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INTRODUCTION |
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LFA-1 (L
2) and MAC-1
(
M
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
2 and other non-RGD
binding integrins, additional and perhaps multiple subdomains within
both the
and
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
subunit ectodomain of all
2 integrins and VLA-1
(
1
1), VLA-2 (
2
1), and
E
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 MI and
LI interaction with ligands
(19-21). The structures show that
MI and
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
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 L and
M I domains. Our results indicate that like
MI,
LI preferentially binds
Mn2+ over most other cations. However, Mn2+
interaction with
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
MI.
Interestingly, Ca2+ inhibited Mn2+ binding to
LI, but had little effect on Mn2+ binding to
MI, underscoring a fundamental difference in the cation-binding properties of these two I domains. Furthermore, experiments confirmed that
LI binds both
Mn2+ and Ca2+ and the results are consistent
with the notion that the
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
LI may represent,
respectively, high- and low-affinity ligand binding states.
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EXPERIMENTAL PROCEDURES |
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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--
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
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
MI coding sequence
and adds five vector-derived amino acids (RPHRD) at the carboxyl
terminus. The plasmid was transformed into E. coli strain
DH5
, and expression and purification of the
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).
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-LI were examined using the
methods described above with the following modifications. MBP-
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-
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.
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RESULTS |
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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
(MI) was identical to that reported previously (31).
However, the sequence of the LFA-1 I domain (
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.
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, MI and
LIf were immobilized on nitrocellulose
filters and incubated with 54Mn2+ as described
under "Experimental Procedures." The autoradiogram shown in Fig.
1A shows that both
LIf and
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
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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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 LI but not
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
MI and
LIf is presented in Fig. 3A. In
agreement with the results of Michishita et al. (29),
unlabeled Mn2+ and Ca2+ inhibited
54Mn2+ binding to
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
LIf inhibiting
54Mn2+ binding with IC50 values of
10-20 and 50-100 µM, respectively. Furthermore, 1 mM
Ca2+ reduced 54Mn2+ binding to
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
LIf but not
MI, and indicate
that the I domain of LFA-1, but not MAC-1, has a
Ca2+-binding site.
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Determination of the Dissociation Constant (KD) for
Mn2+ and Ca2+ Interaction with
LIf--
The affinity constant for
Ca2+ and Mn2+ binding to
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
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
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
LIf is shown in
Fig. 5. Binding to
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
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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DISCUSSION |
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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 LI was inhibitable by
a variety of divalent and trivalent cations with a range of ionic
radii. In contrast, binding to
MI appeared to be more
specific for smaller divalent cations. Notably, Ca2+
inhibited 54Mn2+ binding to
LI
at concentrations well below those found in physiological environments
(34), whereas Ca2+ failed to inhibit
54Mn2+ binding to
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 L and
M were made
between the intact MBP-
LI fusion protein
(
LIf) and
MI, the I domain of
M that was cleaved from the GST-
MI fusion
protein. It seemed possible that the cation binding characteristics of
LIf were unique to the MBP-
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 (
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
LIi and
LIf in a
saturable manner; 2) binding is inhibitable with both unlabeled
Ca2+ and Mn2+; and 3) both
LIi and
LIf, but
not
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
LIf. Under these conditions
54Mn2+ binding is restricted to the LFA-1 I
domain component of
LIf, a conclusion
supported by the observation that Ca2+ binds to immobilized
LIi and
LIf, but
not to MBP, a consequence of the relatively disparate cation binding
affinities of the I domain and MBP components of
LIf. Collectively, the results suggest that
LIi and
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 LI may
represent an equivalent Ca2+/Mg2+-binding site
since, as we show here, Mg2+ is an effective inhibitor of
Mn2+ binding to
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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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: 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.
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REFERENCES |
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