(Received for publication, January 30, 1997, and in revised form, April 4, 1997)
From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Engagement of the
M
2 (CD11b/CD18, Mac-1) integrin on
neutrophils supports adhesion and induces various cellular responses. These responses can be blocked by a specific ligand of
M
2, neutrophil inhibitory factor (NIF).
The molecular basis of
M
2-NIF
interactions was studied. The single chain
M subunit,
expressed on the surface of human 293 cells, bound NIF with an affinity
equivalent to that of
M
2 heterodimer.
This observation, coupled with previous data showing that the
MI domain alone supported high affinity NIF binding,
indicated that the binding site for NIF is restricted to the I domain.
Guided by the crystal structure of the
MI domain, 16 segments corresponding to the entire outer hydrated surface of
MI domain were switched to their counterparts sequences
in
L, which does not bind NIF. Surface expression and
heterodimer formation were achieved for all mutants, and correct
folding was confirmed. Of the 16 switches, only 5 affected NIF binding
substantially, reducing affinity by 8-300-fold. These data confined
the NIF-binding site to a narrow region composed of
Pro147-Arg152,
Pro201-Lys217, and
Asp248-Arg261 of
M. Verifying
this localization, when these segments were introduced into the
XI-domain, the resulting chimeric receptor was converted
into a high affinity NIF-binding protein.
M
21
(Mac-1, CR3, CD11b/CD18) is a member of the
2 integrin
subfamily, which includes
L
2 (LFA-1,
CD11a/CD18),
X
2 (p150, 95, CD11c/CD18),
and
D
2. These integrins share a common
subunit of 95 kDa which is noncovalently linked to distinct but
homologous
subunits (1, 2). The physiological functions of
M
2 include roles in adhesion and
transmigration of leukocytes through endothelium (3), phagocytosis of
foreign materials (4), and activation of neutrophils and monocytes (5).
Excessive activation of
M
2 contributes to
sustained inflammation, reperfusion injury, and tissue damage (6). The
importance of the
2 integrin subfamily is underscored by
the severe phenotype of individuals with congenital deficiencies of
these integrins (7).
NIF (neutrophil inhibitory factor), a novel glycoprotein isolated from
canine hookworms, was originally identified as an inhibitor of a number
of neutrophil functions, such as adhesion to endothelial cells and
adhesion-dependent release of hydrogen peroxide (8). These
functional effects resulted from its specific binding to M
2 but not to other
2
integrins. Subsequently, we and others have reported that NIF
completely blocked
M
2-mediated binding of
C3bi (9), ICAM-1(10), and adhesion to protein-coated surfaces (11), and
partially blocked fibrinogen binding (12) to
M
2-bearing cells. As an antagonist, NIF
has been shown effective in attenuating the deleterious effects of
excessive neutrophil activation, such as tissue damage and
ischemia-reperfusion injury in animal models (13, 14).
The I domain, an inserted region of ~200 amino acids in
the M subunit (
MI-domain), contributes to
NIF binding to
M
2 (10, 12). I domains
with highly conserved amino acid sequences are also found in several
other integrin
-subunits as well as in other proteins, such as vWF,
and mediate a variety of protein-protein interactions, including ligand
binding to integrins (15). Using blocking mAbs which map to the I
domain or recombinant
MI domain itself, this region has
been implicated in the binding of ICAM-1, fibrinogen (16), and C3bi
(9), as well as NIF, to
M
2. Collectively, these data suggest that the
MI domain is an independent
structural unit, capable of interacting with many different proteins.
Nevertheless, the ligand binding functions of
M
2 are not solely a property of its I
domain. We (11) and Bajt et al. (17) have shown that mutations of Asp134, Ser136, or
Ser138 in
2 subunit abrogated the binding of
M
2 to C3bi and adhesion to protein-coated
surfaces, suggesting that, in addition to I domain, the
subunit
also is involved in the recognition of certain ligands by
M
2.
In this study, we have utilized NIF as a model
M
2 ligand and have sought to delineate
the molecular basis for its interaction with
M
2. Homolog-scanning
mutagenesis (18) in which sequences within the
MI
domain have been switched to the homologous sequences within the
LI domain has been used to map the NIF-binding site in
M
2. The NIF-binding site identified
through the loss-of-function mutations was confirmed by a
gain-in-function experiment, whereby the I domain of
X
was converted into a NIF-binding protein. Given the ability of NIF to
inhibit the binding of many ligands to
M
2 and the structural similarities among I domains, the molecular details
of NIF-
M
2 interactions may also apply to
other ligands of
M
2 and to other I domain
containing intergrins.
Human kidney 293 cells and the expression vector,
pCIS2M, were generous gifts from Dr. F. J. Castellino (Notre Dame, IN). The cDNA of CD11b and CD18 were obtained from Dr. B. Karan-Tamir (Amgen, Thousand Oaks, CA). NIF was a gift from Corvas Int., San Diego,
CA. TS1/18, OKM1, M1/70, IB4, 44a, 904, and LM2/1 were from ATCC (ATCC,
Rockville, MD). G418, Dulbecco's modified Eagle's medium/F-12, HBSS
(Hank's balanced solution), DH5 cell, and restriction enzymes were
from Life Technologies, Inc. (Grand Island, NY). All other reagents
were the highest grade available from Sigma unless otherwise noted.
Stretches of
7-12 amino acids within the MI domain were switched to
the corresponding sequences in
L. All such segment
switches were created by oligonucleotide-directed mutagenesis using
uracil-containing single stranded M13mp18 DNA (19). To facilitate the
mutagenesis, two unique restriction sites, ClaI at position
535 (Ile139), and NheI, at position 1141 (Ala342), flanking the I domain of
M, have
been introduced into
M previously (11). To switch a
segment of 7-11 amino acids from
M to
L, the mutagenic primer was designed to contain the corresponding mutations and 15 additional unchanged bases at each end. The length of
the primer was typically 54 bases. The site-directed mutagenesis was
performed according to our published procedure using T7 DNA polymerase
(19). The mutant was identified by DNA sequencing of 5 randomly picked
plaques. DNA sequencing of the entire I domain was conducted,
confirming the presence of the desired mutations and the correctness of
the rest of the I domain. The mutated I domain was transferred back to
the expression vector pCIS2M-
M using the unique
ClaI and NheI restriction sites. The cDNA of
M and
2 were inserted separately in the
pCIS2M expression vector employing XbaI and XhoI
sites; and expression of
M or
2 was under
the control of the human cytomegalovirus promoter and enhancer.
To generate the chimeric molecule,
M(I/
X)
2, where the
MI domain was replaced with
XI domain,
the fragment of the
XI-domain (Ile137 to
Val340) containing ClaI and NheI
restriction sites was prepared by reverse transcription and polymerase
chain reaction using total RNA from polymorphonuclear cells and the
following two primers: 5
-CCCAAGACAGGAGCAGGACATTG-3
(forward) and
5
-TGTGAAGCTAGCGCTGAAGCCCTC-3
(reverse). The reverse primer
contains a NheI site (GCTAGC), and a naturally occurring ClaI site exists in the
X cDNA at
nucleotide position 530.
To graft the NIF-binding site in the XI domain, five
segments of
MI domain (I142PHDFRR,
K200PITQLLG207, R208THTATGI,
D248PLGY, and E253DVIP) were substituted
sequentially for their counterparts (SSRNFAT, ASVHQLQG, FTYTATAI,
DSLDY, and KDVIP, respectively) in
XI domain using
M13mp19 vector and the following primers:
5
-GATGGCTCAGGCAGCATTATCCCCCTGACTTTCGCAGAATGATGAACTTCGTG-3
, 5
-CCCCTCAGCCTGTTGAAGCCTATCACACAGCTGCAAGGGTTT-3
,
5
-CCTATCACACAGCTGCTGGGACGCACACACACGGCCACCGCCATC-3
, and
5
-AAGAAAGAAGGCGACCCACTTGGCTACGAAGATGTCATCCCCATG-3
. The modified
XI domain was sequenced, confirming not only the
intended switches but also the correctness of the entire I domain. The
new chimera containing the grafted I domain,
M(I
/
X)
2, was created by
transferring the modified
XI-domain back into the
chimeric integrin
M(I/
X)
2 employing the ClaI and NheI sites identified
above.
The expression vectors containing wild-type and mutated
M (pCIS2M-
M) and
2
(pCIS2M-
2) were purified using CsCl gradients and
transfected, together with pRSVneo (neomycin-resistant gene), into 293 cells according to our established procedures (19). G418 (600 µg/ml)-resistant colonies were pooled and
M
2-expressing cells were sorted by FACS
(FACStar, Becton-Dickinson, San Jose, CA), using
M-specific mAb, OKM1, which recognizes an epitope outside of the I domain (20).
Approximately 106 cells in HBSS
containing 1 mM Mg2+ were incubated with 5 µg
of mAb for 30 min at 4 °C. A subtype-matched mouse IgG served as a
control. After 3 washes with PBS (10 mM NaPO4, 150 mM NaCl, pH 7.4), cells were mixed with fluorescein
isothiocyanate goat anti-mouse IgG(H+L) F(ab)2 fragment
(1:20 dilution) (Zymed Laboratory, San Francisco, CA), kept at 4 °C
for another 30 min, washed with PBS, and then resuspended in 500 µl
of PBS. The FACS analysis was performed using FACScan, counting 10,000 events. For dual color FACS analysis, the cells (106) were
stained with OKM1 (5 µg) and biotinylated NIF (0.5 µg) for 30 min
at 4 °C, followed by three washes with PBS. These cells were then
mixed with fluorescein isothiocyanate-avidin conjugate and
phycoerythrin goat anti-mouse IgG(H+L) F(ab
)2 fragment
(1:20 dilution) (Zymed Laboratory), kept at 4 °C for another 30 min, washed with PBS, and then resuspended in 500 µl of PBS. FACS analyses was performed as described above. The same procedure was used for mAb
24 staining, except that 1 mM Mn2+ was
substituted for the 1 mM Mg2+ and incubations
were at 37 °C.
The procedures used for NIF binding,
and surface labeling and immunoprecipitation of cells have been
described (11). Briefly, different concentration of NIF (0-20 µg)
were incubated with 2 × 106
M
2-expressing cells at 4 °C for 30 min. The bound NIF was separated from the free NIF by centrifugation
through a 20% sucrose solution and counted with a
-counter. The NIF
titration data were fitted to a single site binding model using the
equation: [NIF]bound = Bmax*[NIF]/(Kd + [NIF]),
where Bmax is the maximal NIF binding and
Kd is the dissociation constant, using a program in
SigmaPlot (Jandel Co., San Rafael, CA).
When introduced into human kidney 293 cells in the
absence of transfected 2, the
M subunit
was expressed on the cell surface, albeit at low levels relative to the
heterodimer. Immunoprecipitation of surface-labeled cells with OKM1
yielded a band of 165 kDa on polyacrylamide gels in SDS under both
reducing and nonreducing conditions (Fig.
1A). No bands in the vicinity of 95 (
2) or 120 (
1) kDa were observed,
suggesting that the surface-expressed
M is not complexed
with its natural partner,
2, or with other typical
integrin
subunits. No bands were observed for mock transfection with either OKM1 or TS1/18 (Fig. 1B), verifying the
specificity of the immunoprecipitation. The presence of
M was confirmed by FACS analysis. The transfected cells
were positive with OKM1, LM2/1, 2LPM19c, 44a, and 904, mAbs to the
subunit, whereas mAbs to the
2 subunit, TS1/18, IB4, or
MHM23 were unreactive. Bilsland et al. (21) also detected
surface expression of the
M subunit in the absence of
2 in COS cells. The ability of the
M
subunit to interact with soluble 125I-NIF was assessed. As
shown in Fig. 1C, the
M-expressing cells bound NIF in a dose-dependent and saturable fashion. The
specificity of this binding was verified by addition of a 20-fold
excess of unlabeled NIF or 1 mM EDTA: in both cases,
125I-NIF binding was reduced by more than 99%. Scatchard
plots of the NIF binding isotherms were consistent with a single class of binding sites with respect to affinity and yielded a dissociation constant (Kd) of 2.1 nM. This value is
very similar to the Kd of 7 nM for NIF
binding to heterodimeric
M
2. To determine
the nature of the molecule on the cell surface that binds NIF, lysate
of surface-labeled
M-transfected cells were incubated
with biotinylated NIF. The NIF-receptor complex was captured with
avidin-agarose resin and analyzed on 7% SDS-PAGE. As shown in Fig.
1B, only one band of 165 kDa was present. In contrast, two
bands (165 and 95 kDa) were observed for the
M
2-expressing cells. The specificity of
this assay was demonstrated by the absence of any band for the mock
transfectant. These data indicate that, unlike other
M
2 ligands (C3bi and adhesion (11)), all
major contributing elements of the binding site for NIF reside in the
subunit of
M
2. Consistent with this
conclusion, we also found that: 1) mutations in the
2
subunit, which abrogated binding of other ligands, did not affect NIF
binding of
M
2 complex (11); and 2)
replacement of the
M with the
XI domain
in the context of the
M
2 heterodimer
completely abrogated NIF binding (see below, Fig. 5).
The I Domain Peptide N232AFKILVVITDGEK Is Not Required for NIF Binding
Rieu et al. (10) had previously used
synthetic peptides and implicated two candidate M
sequences in NIF binding: the A7 peptide,
N232AFKILVVITDGEK, was the most potent inhibitor of NIF
binding to the
MI domain (10). To test the role of this
sequence in NIF binding, we created two switch mutants in which the
amino- and carboxyl-terminal portions of this peptide were changed
individually to the corresponding sequences in
L
(
L
2 does not bind NIF (8):
M
(K231NAFKILVVITDGEK245FG)
to
L (PDATKVLIIITDGEATD). Thus,
the first mutant,
M(K231NAF), changed the
amino-terminal non-conserved KNAF to PDAT, and the second mutant,
M(K245FG), changed to non-conserved KFG to
ATD. The hydrophobic cluster in the center of the peptide sequence is
well conserved and was not altered. The mutant
M vectors
were transfected together with
2 into 293 cells. Both
mutants were expressed well on the cell surface as judged by FACS
analysis, and surface labeling and immunoprecipitation with OKM1 (data
not shown). When stained with LM2/1 mAb, the mean fluorescence for the
M(K231NAF)
2 mutant was 392.0, compared with 398.4 for wild-type. A similar result was obtained for
the
M(K245FG)
2 mutant. When
stained with OKM1, the mean fluorescence was 377.2 for the mutant and
380.1 for the wild-type. Both mutants also bound NIF with high affinity
(Fig. 2, and Table II), indicating that this peptide is
not centrally involved in NIF binding when placed in the context of the
intact receptor.
|
To systematically define the
NIF-binding site in M, a homolog-scanning mutagenesis
(18) strategy was implemented. Accordingly, guided by the crystal
structure (15), the entire hydrated surface of the
MI
domain was replaced with sequences of the
LI domain in
segments of 7-11 amino acids. To apply this approach to the I domain
of
M (~200 amino acids), 16 segments were switched. The primers used are listed in Table I. The efficiency
of mutagenesis was typically 60%, with the 7-amino acid segment
switches having substantially higher efficiency (>90%) than the
longer 10-amino acid segment switches (~30%). The appropriate DNA
sequence of the entire I domain (from Ile139 to
Ala332) was confirmed for each mutant before transferring
back into the
M subunit cDNA.
|
The functional consequences of these segment switches were initially
investigated by transient expression in 293 cells. Forty-eight hours
after transfection of M, together with
2
subunit, the cells were detached from tissue culture dish and double
stained with OKM1 and biotinylated NIF. A representative dual-color
FACS analysis is shown in Fig. 3. With wild-type
M
2 transfectant, 1.83% of the cells were
positive with both OKM1 and NIF, whereas less than 0.02% of mock
transfected cells were positive. Two distinct patterns were observed
for the mutants. One, represented by
M(E178-T185) in Fig. 3, in which
a similar percentage (1.12%) of the cells were positive for both
markers, i.e. exhibiting a pattern similar to the wild-type
M
2 cells. The second pattern is represent
by
M(
D248-Y252), in which a
substantial percentage of cells were positive with OKM1 (22.5%) but
negative with NIF (<0.05%), indicating a loss in NIF-binding
function. The following mutants belonged to the first category:
M(M153-T159),
M(E162-L170),
M(E178-T185),
M(Q190-S197),
M(K231NAF),
M(K245FG),
M(
E262G),
M(D273-K279),
M(R281-I287),
M(F297-T307), and
M(Q309-E314). Belonging to the
second category were:
M(P147-R152),
M(P201-G207),
M(R208-K217),
M(
D248-Y252), and
M(E253-R261).
The NIF-binding Site Is Composed of Segments P147-R152, P201-G207, R208-K217, D248-T252, and E253-R261
To quantitate the binding
affinities of each mutant receptor for NIF, stable cell lines were
established. All 16 M
2 mutants were cell
surface expressed as heterodimers: immunoprecipitations of
125I-surface-labeled cells with OKM1 yielded
subunits
of 165 kDa and
subunits of 95 kDa on 7% SDS-PAGE (a representative
gel is shown in Fig. 4A), similar to the
patterns obtained with that of recombinant wild-type and
naturally-occurring
M
2 (11). Identical
patterns were obtained when the immunoprecipitations were performed
with TS1/18, a mAb to the
2 subunit (data not shown).
The specificity of this assay was verified by the absence of any band
when 125I-surface-labeled mock transfected cells were
immunoprecipitated with either OKM1 or TS1/18 (Fig. 1B), or
when wild-type
M
2-expressing cells were
immunoprecipitated with IV.3, an irrelevant mAb (11). NIF binding was
measured as a function of ligand concentrations, and representative
binding isotherms are presented in Fig. 4B. With all
mutants, dose-dependent and saturable binding of
125I-NIF was observed, which could be inhibited by
unlabeled NIF and EDTA. The binding isotherms could be fitted to a
one-site model as described under "Experimental Procedures." The
Kd values calculated from these binding data are
summarized in Table II. The Kd of 7 nM for wild-type
M
2 was
nearly identical to the Kd of 5 nM
reported for naturally-occurring
M
2 on
neutrophils (8). The expression levels, reflected by the maximal NIF
binding, were similar for all mutants, differing by less than 4-fold.
The following mutants had similar Kd values
(<4-fold different than wild-type
M
2):
M(M153-T159),
M(E162-L170),
M(E178-T185),
M(Q190-S197),
M(K231NAF),
M(K245FG),
M(
E262G),
M(D273-K279),
M(R281-I287),
M(F297-T307), and
M(Q309-E314). The most dramatic
increases in Kd values were found for five mutants:
M(P147-R152) (33-fold),
M(P201-G207) (305-fold),
M(R208-K217) (206-fold),
M(
D248-Y252) (13-fold), and
M(E253-R261) (8-fold).
The Defective Mutants Possess Correct Conformations
Given the
similarities in the three-dimensional structures of MI
and
LI domains (15, 22), it is unlikely that the defects in NIF binding of the five identified mutants arise from incorrect folding of their
MI domain. This assertion was supported
by a series of additional experiments. First, FACS analyses were
performed for all 16 mutants with a panel of mAbs against
M
2, including OKM1, LM2/1, M1/70, TS1/18,
and MHM23. The former three are
M-specific, and the
latter two are
2-specific. In all cases, the mAbs
reacted well with wild-type and all five mutant receptors. Second, the reactivity of the five mutants with a
conformation-dependent antibody, mAb 24, which has been
used to probe the cation-dependent conformational integrity
of
MI domain (23, 24), was assessed. FACS analyses were
performed on the five mutants, together with the wild-type receptor. As
a control, FACS analyses were performed in parallel with OKM1, a
conformation-independent mAb. The ratios of the mean fluorescence
intensities of the various cell lines with mAb 24 and OKM1 are shown in
Fig. 4C. All of the mutants defective in NIF binding
exhibited the capacity to bind mAb 24. Mutant
M(P201-G207) showed a slightly
attenuated reactivity with mAb 24, suggesting a subtle perturbation of
its structure. Nevertheless, binding of mAb 24 to this and the other
mutants remained cation-dependent; addition of 1 mM EDTA completely abrogated mAb 24 binding. Thus, all
mutants retained the cation-dependent conformation reported by mAb 24.
When the I domain of M (Ile139
to Ala342) was replaced with that of
X
(Ile137 to Val340), the most closely related I
domain to
MI domain in terms of primary structure but
which still does not bind NIF (8), the expressed heterodimeric
receptor,
M(I/
X)
2, had no
NIF binding capacity (Fig. 5), albeit expressed well on
293 cell surface. When stained with mAb OKM1, the mean fluorescence of
the
M(I/
X)
2 chimera was
281.2, compared with 279.0 for wild-type
M
2 receptor. This chimeric receptor
continued to recognize the
X
2 mAb, clone 3.9, and to rosette with C3bi (data not shown), indicating the retention of functional integrity. To provide direct evidence that the
identified segments (Pro147-Arg152,
Pro201-Gly207,
Arg208-Lys217,
Asp248-Thr252, and
Glu253-Arg261) constitute a functional
NIF-binding site, we sought to impart NIF binding function to the
chimeric receptor,
M(I/
X)
2, by grafting the
five segments from
M into their counterparts' sequences in the
XI domain. The chimeric molecule,
M(I
/
X)
2, containing the
reconstructed
XI domain, was expressed in 293 cells, and NIF binding was assessed using different concentrations of
125I-NIF (Fig. 5). While the chimeric receptor,
M(I/
X)
2, containing the
wild-type
XI domain, did not bind NIF, upon grafting the five
M segments into
XI domain, full NIF
binding function was imparted to the modified receptor. The
reconstructed I domain has a Kd of 2 nM
for NIF binding, essentially identical to that of the original
wild-type
MI domain (7 nM). This interaction was blocked completely by EDTA (1 mM) or unlabeled NIF (10 µg), verifying that NIF binding to the reconstructed
XI domain remained cation-dependent and
specific.
In this study, we have sought to map the binding site for NIF, a
model ligand for the I domain of M
2.
First, we have demonstrated that the binding surface for NIF is located
exclusively in the I domain of the receptor. This conclusion is
supported by the observations that
M alone (Fig. 1C) or
the expressed I domain (10, 12) support NIF binding with an affinity
similar to that of the intact
M
2
heterodimer; that mutations in
2 subunit, known to
abolish the binding of several other ligands to
M
2 (11, 17), have no effect on NIF
binding; and that a swap of the
MI domain for the
XI domain in the context of the intact
M
2 receptor completely abolishes the NIF
binding activity of the resulting chimeric receptor (Fig. 5). These
observations also distinguish NIF binding from C3bi binding and
adhesion to protein substrates mediated by
M
2, interactions which require both
subunits (11). Second, we have scanned the entire hydrated surface of I
domain by substituting 16
L segments of 7-10 amino
acids for their corresponding sequences in the I domain of
M
2. The approach of homolog-scanning
mutagenesis had been previously employed to identify receptor and
antibody epitopes in human growth hormone (18). Since the homologous
segments of
M and
L adopted similar structures (15, 22), and the structurally important residues involved
in cation coordination (Asp140, Ser142,
Ser144, Thr209, and Asp242) are
preserved, the structural integrity and the essential cation-binding site was maintained. Therefore, any functional changes resulting from
the switches should reflect sequences that have a direct role in NIF
binding. This strategy does not exclude a role for residues conserved
among the two homologous proteins in NIF binding. Nevertheless, our
study does identify segments that are required for a high affinity
interaction with this ligand. Of these 16 segment-switch mutants, only
five (P147-R152,
P201-G207, R208-K217,
D248-Y252, and
E253-R261) lost their ability to bind NIF with
high affinity, suggesting that the NIF-binding site is composed of
these five short segments, two of which are contiguous (Fig.
4B, Table II). The loss of NIF binding function in these
five mutants is unlikely to arise from incorrect conformational folding
since: 1) these, as well as all 16 mutants, were expressed on the cell
surface and heterodimeric complexes were formed (Fig. 4A);
and 2) they all reacted with a panel of
M
2 mAbs, including the
conformation-dependent antibody, mAb 24 (Fig.
4C). The reactivity of this mAb has been shown to depend on
an intact conformation of the I domain (23). For mutant P201-G207, an attenuated reactivity with mAb 24 was observed, although its binding to mAb 24 remained
cation-dependent. It is possible that this mutant has a
slightly altered conformation; however, when this segment was grafted
together with the other four segments into
XI domain,
cation-dependent NIF binding activity was observed.
Recently, the crystal structures of both the MI and
LI domains have been solved (15, 22). These two I
domains adopt very similar folds (
/
Rossmann folds), and both
contain a unique metal-binding site (MIDAS motif) on their surface. The
similarity in three-dimensional structures of
MI and
LI domains is in agreement with their highly homologous
primary sequences and suggest that these regions have a common
evolutionary origin. Based on the
MI domain structure
(15) and the data from homolog-scanning, the binding interface for NIF
is located in a very narrow region on the I domain surface (see Fig.
6). This region entails the beginning portion of helix
1, the loop between helix 3 and helix 4, the small segment leading to
helix 5, and the entire helix 5. The binding site for Mg2+
is located at the edge of this binding surface. As a result, coordination to Mg2+ ion by NIF may provide additional
energy for
M
2-NIF interactions. Such a
ligation mechanism has been suggested for other integrin-ligand interactions (25); is consistent with the availability of additional coordination sites for a cation bound within a MIDAS motif (15); and
may explain the cation dependence of NIF binding to
M
2. In support of our findings, while our
study was in progress, Rieu et al. (26) recently identified
several specific residues which are important for
NIF-
M
2 interaction. Their study
implicated residues Gly143 and Asp149, which
reside in the Pro147-Arg152 segment, and
Arg208 which resides in the
Arg208-Lys217 segment. However, a discrepancy
exists regarding the roles of Glu178 and
Glu179; Rieu et al. (26) found that mutation of
these two residues disrupted NIF binding, whereas we found that the
Glu178-Tyr185 segment which contains these
residues, was nonessential. As one possible explanation, in the study
of Rieu et al., Glu178-Glu179 both
were mutated to Ala, whereas we changed them to Thr and Ser,
corresponding to their homologous sequences in
L; the
former substitutions may indirectly disrupt the conformation of the
spatially adjacent loop between helix 3 and 4 (see Fig. 6) which are
directly involved in NIF binding.
Since NIF blocks the interactions of M
2
with many of its ligands, such as C3bi, ICAM-1, fibrinogen, and many
immobilized substrates, the binding sites for these ligands are
overlapping. Nevertheless, we have previously shown that the contact
regions for these ligands are not identical (11). Thus, it is expected that the NIF-binding site identified here will constitute at least part
of the binding surface for other
M
2
ligands. Indeed, both we (11) and Bajt's group (23) have shown that
the Asp248-Tyr252 region is required for
rosetting of EC3bi with
M
2 receptor. The
binding interface on the
LI domain for ICAM-1 has been
partially identified (25): key amino acids involved in this interaction are Met140, Glu146, Thr243, and
Ser245. These residues reside within the regions
corresponding to Pro147-Arg152 and
Asp248-Tyr252 of
M, which we
have implicated in NIF binding. The importance of the regions
corresponding to Pro201-Gly207,
Arg208-Lys217, and
Glu253-Arg261 in
M were not
tested for their involvement in ICAM-1 binding to
L
2.
As demonstrated in Fig. 5, grafting of the
Pro147-Arg152,
Pro201-Gly207,
Arg208-Lys217,
Asp248-Tyr252, and
Glu253-Arg261 segments into XI
domain imparted NIF binding capacity to the chimeric receptor. The
affinity of this chimeric receptor for NIF, 2 nM, was
essentially the same as that for the
MI domain, suggesting that the NIF contact site is dependent upon the swapped regions. Thus, the loss-in-function identified by the homolog-scanning mutagenesis is supported by this gain-in-function experiment and is not
due to disruption of conformation or loss of cation binding functions
of the resultant
MI domain mutants. The successful implantation of the homolog-scanning mutagenesis strategy marks the
first time that a ligand-binding domain within any integrin molecule
has been systemically probed over its entire surface. With these
analyses serving as proof of principle, the homolog-scanning strategy
should allow mapping of the ligand binding site for other
M
2 ligands and other I domain-containing
integrins. Experiments to test this hypothesis are in progress.
We thank Corvas International Inc. for NIF,
Dr. N. Hogg of Imperial Cancer for mAb 24, and Dr. B. Karan-Tamir of
Amgen for the cDNA for M and
2. We
are very grateful to Xiaohua (Sue) Wu for excellent technical
support.