J Biol Chem, Vol. 274, Issue 45, 32182-32191, November 5, 1999
Determinants of Ligand Binding Specificity of the
1
1 and 21
Integrins*
S. Kent
Dickeson
,
Nancy L.
Mathis,
Mariam
Rahman§,
Jeffrey
M.
Bergelson§¶, and
Samuel A.
Santoro
From the Department of Pathology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
§ Children's Hospital of Philadelphia,
Philadelphia, Pennsylvania, 19104
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ABSTRACT |
The 
1
1 and
21 integrins are cell surface collagen
receptors. Cells expressing the 11
integrin preferentially adhere to collagen IV, whereas cells expressing
the 21 integrin preferentially adhere to
collagen I. Recombinant 1 and 2 integrin
I domains exhibit the same collagen type preferences as the intact
integrins. In addition, the 2 integrin I domain binds
echovirus 1; the 1 I domain does not. To identify the
structural components of the I domains responsible for the varying
ligand specificities, we have engineered several
1/2 integrin I domain chimeras and
evaluated their virus and collagen binding activities. Initially, large secondary structural components of the 2 I domain were
replaced with corresponding regions of the 1 I domain.
Following analysis in echovirus 1 and collagen binding assays, chimeras
with successively smaller regions of 1 I were
constructed and analyzed. The chimeras were analyzed by ELISA with
several different 2 integrin monoclonal antibodies to
assess their proper folding. Three different regions of the
1 I domain, when present in the 2 I
domain, conferred enhanced collagen IV binding activity upon the
2 I domain. These include the 3 and 5 helices and
a portion of the 6 helix. Echovirus 1 binding was lost in a chimera
containing the C-6 loop; higher resolution mapping identified
Asn289 as playing a critical role in echovirus 1 binding.
Asn289 had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of
multiple determinants of ligand binding specificities within the
1 and 2 integrin I domains.
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INTRODUCTION |
The integrins constitute a large family of cell adhesion
molecules that are involved in both cell-cell adhesion as well as the adhesion of cells to the extracellular matrix (for review see Ref.
1). The integrins are involved in many important physiologic processes
including development and differentiation, cell migration, wound
healing, thrombosis and hemostasis, metastasis, and immune system
function. Structurally, integrins are heterodimeric glycoproteins composed of two noncovalently associated integral membrane subunits. The subunits range in size from 120 to 180 kDa, and the subunits range in size from 90 to 110 kDa. Many integrin subunits
associate with more than a single or subunit resulting in a
large number of different integrins.
The 1 integrin subunit associates with at least 11 different subunits. Members of the resulting 1
subfamily of integrins are cell surface receptors for specific
components of the extracellular matrix. The
11 integrin is a cell surface receptor
for several different collagens and laminin-1 (2).
11 integrin-dependent adhesion of cells to collagens or laminin requires the presence of
divalent cations (3). Similarly, the 21
integrin also serves as a cell surface receptor for collagens and
laminins (for review see Ref. 4), and the adhesion of cells via the
21 integrin to these ligands also depends
upon the presence of divalent cations (5). However, the substrate
specificity of the 21 integrin depends
upon the cell type on which it is expressed. The
21 integrin on platelets and fibroblasts
is a collagen receptor (5); on endothelial and epithelial cells it is a
receptor for both collagens and laminin-1 (6, 7). The
21 integrin also mediates echovirus 1 attachment and infection (8).
Near their amino termini, the 1 and the 2
integrin subunits share (along with approximately half of the integrin
subunits) an autonomously folding domain of approximately 220 amino
acids known as the I (inserted) domain (for review see Ref. 9). The subunit I domains are critical determinants for ligand recognition and
binding of both the 11 and the
21 integrins. Function blocking
antibodies directed against both of these integrins map to their subunit I domain, and several mutations in the I domains adversely
affect ligand binding activity (10, 11). Bacterially expressed I
domains from both of these integrins bind collagens and laminin-1 in a
manner that is saturable, blocked by function blocking antibodies
against the parent integrin, and, like the parent integrins, dependent
upon the presence of divalent cations (12-14).
The crystal structures of several different integrin I domains have
been solved, providing much insight into I domain structure. I domain
structures that have been solved include those of the M,
L, and 2 subunits (15-17). The
three-dimensional structures of these three I domains are very similar.
Each has a core that consists of a largely parallel sheet
structure, and the core is surrounded by several helices. All three
contain a single divalent cation-binding site in a crevice near the top
of the sheet. Unique to the 2 I domain, however, is
an additional short helix at the top of the domain, in close
proximity to the divalent cation-binding site (17).
Although cells expressing either the 11
or the 21 integrins adhere to several
different collagens, they exhibit different relative affinities
depending on the identity of the substrate. For instance, cells
expressing the 11 integrin preferentially adhere to collagen IV, whereas cells expressing the
21 integrin preferentially bind to
collagen I (18). Likewise, the binding of purified recombinant
1 and 2 integrin I domains to collagen types I and IV reflects the same relative affinities to these ligands
as do the parent integrins (13). To identify the structural components
of these I domains responsible for conferring the observed substrate
specificities, we have engineered a series of
1/2 integrin I domain chimeras and
examined their ligand binding activities in collagen I and IV and
echovirus 1 binding assays. Initially, with the aid of the crystal
structure data mentioned above, relatively large structural components
of the 2 integrin I domain were replaced with the
corresponding region from the 1 integrin I domain. The chimeras were examined for both a gain of function (enhanced collagen IV binding activity) and a loss of function (echovirus 1 binding). Regions that satisfied one or both of these criteria were subjected to
subsequent rounds of chimera construction, purification, and analysis
to define further the critical structural and functional determinants.
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EXPERIMENTAL PROCEDURES |
Cloning and Mutagenesis of Integrin I Domain cDNAs--
The
cloning and expression of the 2 integrin I domain has
been previously described (12). Briefly, cDNA encoding the I domain
was amplified using the polymerase chain reaction
(PCR)1 with the full-length
2 integrin cDNA as the template. This cDNA encodes Ser124-Met349 of the published
2 integrin sequence (19). In addition, a cDNA
encoding a shorter I domain protein lacking the 35 amino-terminal amino
acids was also prepared. The shorter protein, referred to as 
I,
contains Trp159-Met349. This proteins lack the
DXSXS portion of the metal ion dependent adhesion
site (MIDAS) motif (15). The PCR primers were designed such that both
of the amplification products would contain a BglII restriction site at their 5' ends and a stop codon followed by an
XhoI restriction site at their 3' ends. The PCR products
were digested with BglII and XhoI, purified in
agarose gels, and cloned into BamHI- and
XhoI-digested glutathione S-transferase (GST) fusion protein expression vector pGEX-5X-1 (Amersham Pharmacia Biotech).
cDNA encoding the 1 integrin I domain was amplified
by PCR using the full-length human 1 cDNA (Dr.
Eugene E. Marcantonio, Columbia University) as the template. Analogous
to the 2 I domain construct, this cDNA encodes
Ser124-Met349 of the published
1 integrin sequence (20). As with the 2 integrin I domain, the PCR primers were designed to create a product that contained a BglII site at the 5' end and a stop codon
followed by a XhoI site at the 3' end. The PCR product was
digested, purified, and cloned into pGEX-5X-1 as described above.
To facilitate the construction of chimeric
1/2 integrin I domain cDNAs encoding
the 2 integrin I domain with an internal region replaced
with 1 I domain sequence, the cDNAs encoding both
the I domains were transferred into pBluescript KS
(Stratagene). A 661-bp EcoRI-XhoI fragment of
pGEX-5X-1/1 I, encoding the entire 1 I
domain with the exception of the first seven amino acids, was isolated
and ligated into pBluescript KS digested with
EcoRI and XhoI. For the 2 I
domain, a 1157-bp BalI-XhoI fragment of
pGEX-5X-1/2 I, encoding all of the 2 I domain and containing approximately two-thirds of the GST coding sequence, was isolated and ligated into pBluescript KS
digested with SmaI and XhoI.
Chimeric 1/2 integrin I domain cDNAs
were prepared by one of two methods, depending upon the length of the
internal portion of 2 to be replaced with
1 sequence. An example of the construction of one of
each type of chimera will be described. Chimera A, an example of one of
the chimeras with a longer replacement, consists of the
2 I domain with an internal region from the
1 I domain consisting of the 1 helix and the
1-B loop. First, pBluescript/2 I was
mutated using Kunkel's method (21) and oligo 1 as the mutagenic oligo
(Table I). Oligo 1 introduced an
Eco47III site into the 2 sequence at the
amino-terminal end of the 1 helix to create p4. Simultaneously,
pBluescript/1 I was mutated using oligo 2 as the
mutagenic oligo. This introduced an EcoRV site at the
amino-terminal end of the 1 helix (a position analogous to the
position of the Eco47III site in p4) to create p3.
Eco47III and EcoRV were chosen so that the proper
amino acids, serine and isoleucine, would be encoded by the 3' end of
the 2 fragment and the 5' end of the 1
fragment, respectively, upon restriction enzyme digestion. The 3482-bp
XhoI-Eco47III fragment of p4 and the 591-bp
EcoRV-XhoI fragment of p3 were isolated and
ligated to create p5, encoding amino acids 124-155 of 2
followed by amino acids 155-349 of 1. (Note that amino
acid 155 is present from both the 1 I and
2 I sequences. This is due to the fact that a one-amino
acid gap must be introduced into the 1 protein sequence between Pro135 and Val136 to align the two
protein sequences (see Fig. 1). Thus Ile155 of
1 corresponds to Ile156 of
2.)
p5 was mutated using oligo 3 to introduce a StuI site at the
carboxyl-terminal end of the B strand to create p6. Simultaneously, pBluescript/2 I was mutated using oligo 4 to introduce
an Eco47III site at the carboxyl-terminal end of the B
strand to create p7. StuI and Eco47III were
chosen so that the proper amino acids, glycine and leucine, would be
encoded at the 3' end of the 1 portion of p6 and the 5'
end of the 2 fragment, respectively, upon restriction
enzyme digestion. The 3565-bp XhoI-StuI fragment of p6 and the 505-bp Eco47III-XhoI fragment of p7
were isolated and ligated to create p8, encoding amino acids 124-155
of 2 followed by amino acids 155-182 of
1, which is in turn followed by amino acids 184-349 of
2. It is important to note that although amino acids
155-182 of 1 are encoded in this chimeric I domain,
only amino acids 160-178 differ in the two I domains. This is due to the fact that amino acids 155-159 of 1 are identical to
amino acids 156-160 of 2 and that amino acids 179-182
of 1 are identical to amino acids 180-183 of
2 (Figs. 1 and 3).
Finally, the 966-bp Bst BI-XhoI fragment of p8
and the 4669-bp XhoI-Bst BI fragment of
pGEX-5X-1/2 I were isolated and ligated to create the
expression plasmid pGEX-5X-1/Ch A, capable of directing the expression
of the GST-chimera A fusion protein.
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Fig. 1.
Comparison of the protein sequences of
the 1 and the
2 integrin I domains.
Numbers refer to the amino acid sequences of the mature
1 and 2 integrin subunits. The secondary
structure is represented by bars and arrows for
helices and strands, respectively. Shaded areas
represent identical residues. The residues that comprise the MIDAS
motif are boxed.
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Chimera B2, an example of one of the chimeras with a shorter internal
portion of the 1 integrin I domain, consists of the 2 I domain with an internal region from the
1 I domain consisting only of the 3 helix, amino
acids 205-210 (VLVAAK). Amino acids 206-211 (MIVATS) of the
2 I domain sequence were replaced with amino acids
205-210 (VLVAAK) of the 1 I domain sequence in a single
mutagenesis reaction using pBluescript/2 I and oligo 5 (Table I) to create pBluescript/Ch B2. The 966-bp Bst
BI-XhoI fragment of pBluescript/Ch B2 and the 4669-bp
XhoI-Bst BI fragment of
pGEX-5X-1/2 I were isolated and ligated to create
pGEX-5X-1/Ch B2. The sequences of the cDNAs used in this study,
including all of the chimeras, were determined using the BigDye
terminator cycle sequencing method (PE Applied Biosystems), and were
compared with the published 1 and 2
integrin sequences (19, 20).
Expression and Purification of GST-I Domain Fusion
Proteins--
Trial inductions were performed to determine whether the
selected clones could direct the expression of appropriately sized GST
fusion proteins. Escherichia coli DH5 containing each of the plasmid constructs was grown at 37 °C in 5 ml of 2× YT medium supplemented with 0.2% glucose and 100 µg/ml ampicillin. Uninduced samples were removed from each culture after 1 h.
Isopropylthiogalactoside was then added to a final concentration of 1 mM, and the cultures were returned to the incubator for
3 h to permit for accumulation of the expressed proteins. Cell
lysates from the uninduced and induced samples were analyzed by
SDS-polyacrylamide gel electrophoresis (22) followed by Coomassie Blue
staining. Constructs that directed the expression of recombinant
proteins of the expected size were used for large scale induction of
protein expression.
For the purification of the fusion proteins, the inductions were
performed as above except that the culture volume was increased to 500 ml and the expression was induced with 1 mM
isopropylthiogalactoside when the A550 reached
0.3-0.4. At the end of the induction period, the cells were recovered
by centrifugation at 2600 × g for 10 min, washed twice
with 10 ml of ice-cold phosphate-buffered saline (10 mM
Na2HPO4, 1.8 mM
KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4), and stored at 
70 °C until needed. The
GST-I domain fusion proteins were expressed, purified, and
characterized as recently described (12).
Collagen Binding Assays--
The wells of a 96-well microtiter
plate (Immulon 2, Dynatech) were coated overnight at 4 °C with 0.1 ml/well of 30 µg/ml collagen I from calf skin (Sigma) or collagen IV
from human placenta (Sigma) in 0.09% acetic acid. The wells were
washed twice with 0.15 ml of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and then blocked for 1 h at room
temperature with 0.15 ml of 300 µg/ml bovine serum albumin in TBS.
Purified recombinant I domain proteins were diluted to the desired
concentration in wash buffer (TBS containing 0.05% Tween-20, 30 µg/ml bovine serum albumin, and either 1 mM EDTA or 2 mM MgCl2). The wells were washed once with 0.15 ml of the appropriate wash buffer, and then 0.1 ml of each recombinant
protein was added and allowed to interact with the collagen substrate for 1.5 h at room temperature. Wells were then washed three times with 0.15 ml of the appropriate wash buffer and 0.1 ml of a 1:8,000 dilution of anti-GST antiserum (Amersham Pharmacia Biotech) in the
appropriate wash buffer was added for 1 h at room temperature. Following this incubation, the wells were again washed three times, and
then 0.1 ml of a 1:20,000 dilution of pig anti-goat IgG secondary antibody horseradish peroxidase conjugate (Roche Molecular
Biochemicals) in the appropriate wash buffer was added per well for
1 h at room temperature. The wells were again washed three times,
and 0.1 ml of tetramethylbenzidine dihydrochloride (Sigma) was prepared according to the manufacturer's directions was added per well. After
15 min of substrate conversion, reactions were stopped with 0.025 ml of
4 N H2SO4, and the plates were read
at 450 nm using a Molecular Devices Emax
microplate reader. Each protein was expressed and purified twice, and
both preparations were tested in collagen I and IV binding assays. All
data are presented as means of triplicate determinations.
Echovirus 1 Binding
Assays--
[35S]Methionine-labeled echovirus 1, prepared as described (23), was added to purified fusion proteins
immobilized on glutathione-Sepharose (10,000 cpm, 2 µg of
protein/aliquot) and then incubated for 1 h at room temperature
with rocking. Beads were washed to remove unbound virus and then
dissolved for liquid scintillation counting. Additional aliquots of
immobilized fusion proteins were boiled in Laemmli sample buffer (22)
and analyzed by SDS-polyacrylamide gel electrophoresis to confirm equal
protein loading.
ELISA--
I domain-containing proteins were diluted to 10 µg/ml in TBS containing 2 mM MgCl2 and used
to coat the wells of a 96-well microtiter plate (Immulon 2, Dynatech).
Coating was carried out overnight at 4 °C with 0.1 ml of
solution/well. The wells were washed twice with 0.15 ml of TBS
containing 2 mM MgCl2 and then blocked for
1 h at room temperature with 0.15 ml of TBS containing 300 µg/ml
bovine serum albumin and 2 mM MgCl2. Primary
antibodies used include the human 2 integrin I domain
monoclonal antibodies 6F1 (Dr. Barry S. Coller, Mt. Sinai Medical
Center), 12F1 (Dr. Virgil L. Woods, Jr., University of California at
San Diego), 5E8 (Dr. Richard B. Bankert, Roswell Park Cancer
Institute), P1E6 (Chemicon), and AK7 (Chemicon). The monoclonal
antibodies were diluted to 1 µg/ml in wash buffer (TBS containing
0.05% Tween-20, 30 µg/ml bovine serum albumin, and 2 mM
MgCl2). Following blocking the wells were washed once with
0.15 ml of wash buffer, and then 0.1 ml of primary antibody was added
and allowed to interact for 1 h at room temperature. The wells
were washed three times with 0.15 ml of wash buffer, then 0.1 ml of a
1:10,000 dilution of goat anti-mouse IgG secondary antibody horseradish
peroxidase conjugate (Roche Molecular Biochemicals) in the appropriate
wash buffer was added per well for 1 h at room temperature. The
wells were again washed three times, and 0.1 ml of tetramethylbenzidine dihydrochloride (Sigma) prepared according to the manufacturer's directions was added per well. After 10 min of substrate conversion, reactions were stopped with 0.025 ml of 4 N
H2SO4 and the plates read at 450 nm using a
Molecular Devices Emax microplate reader. Each
protein was expressed and purified twice, and both preparations were
analyzed in the ELISA assays. All data represent means of triplicate determinations.
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RESULTS |
Binding of the 11 and
21 integrins to a variety of
extracellular matrix molecules has been examined previously. The
11 integrin preferentially binds to
collagen IV, whereas the 21 integrin
preferentially binds collagen I (18). Analysis of the binding of the
1 and 2 integrin I domains to collagens I
and IV using surface plasmon resonance and solid phase binding assays indicate that the substrate preferences of the 1 and
2 I domains reflect those of the parent integrins (13).
We also have examined the binding of these I domains to collagens I and
IV in solid phase substrate binding assays. In agreement with the
above, we observed that the 1 I domain preferentially
bound collagen IV, and the 2 I domain preferentially
bound collagen I (Fig. 2A). The collagen type preferences of the 1 and
2 I domains were confirmed by more extensive studies
over a wide range of I domain concentrations (see Fig. 7,
left and middle panels).
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Fig. 2.
Binding of the
1 and
2 integrin I domains to ligands of
the
21
integrin. A, the binding of the 1 and
2 integrin I domains (50 nM) to collagen
types I and IV was measured in a solid phase binding assay. Binding was
determined in the presence of either 1 mM EDTA or 2 mM Mg2+. B, the binding of
[35S]methionine-labeled echovirus 1 to the
1 and 2 integrin I domains.
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The 21 integrin is also a cell surface
receptor for echovirus 1 (8). Binding of echovirus 1 to the integrin is
mediated by the 2 I domain (24). We tested the binding
of echovirus 1 to both the 1 and 2
integrin I domains. As expected, the virus bound to the
2 I domain. However, the virus failed to bind to the
1 I domain (Fig. 2B). To determine which
regions of the 1 I domain were responsible for enhanced
collagen IV binding activity and which regions of the 2
I domain were required for echovirus binding, we designed a series of
1/2 integrin I domain chimeras and
examined their collagen I and IV and echovirus 1 binding activities. As
a guide for designing the chimeras, we used the recently published crystal structure of the 2 integrin I domain (17).
Initially, relatively large portions of the 2 I domain
were replaced with the analogous regions of the 1 I
domain. Because loops and helices are exposed on the I domain surface,
the initial round of chimeras contained loops and helices from the
1 I domain. Fig. 3
(A
F) shows the initial round of six
1/2 I domain chimeras and indicates the
regions of the 2 I domain that were replaced with
1 I domain sequence.
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Fig. 3.
Structure of initial six
1/2
integrin I domain chimeras, ELISA, and ligand binding results.
Initially six 1/2 integrin I domains
(A-F) were designed, expressed, and purified. Each consists
of 2 sequence with an internal (shaded)
region from the 1 I domain. The structural components
from the 1 I domain are given above the
schematic representation of each construct. The numbers
given above the shaded areas refer to the 1 I
domain residues (inclusive) transferred to 2. Each
chimera was tested by ELISA using a panel of five 2
integrin I domain monoclonal antibodies, including 6F1, 12F1, 5E8,
P1E6, and AK7. Antibody reactivity was considered positive if a
majority of the antibodies reacted strongly (comparable with the
2 I domain). Collagen I and IV binding data are given as
percentages ± S.E. of control (2 I domain binding
to collagen I). Echovirus binding data are given as percentages ± S.E. of control (echovirus 1 binding to the 2 integrin I
domain).
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Each of the chimeras was tested in collagen I and IV binding assays and
the echovirus 1 binding assay. In addition, to assess the folded state
of the I domain chimeras, each was tested by ELISA using a panel of
five 2 integrin I domain monoclonal antibodies. Because
any given epitope could be lost because of replacement of the epitope
with protein sequence from the 1 I domain, the chimeras
were considered to be folded properly if a majority of the monoclonal
antibodies bound. The results of these analyses are shown in Fig. 3.
The collagen binding data are given as percentages ± S.E. of
control (binding of the 2 I domain to collagen I); the
echovirus binding data are also expressed as the percentages ± S.E. of control (binding of echovirus 1 to 2 I domain).
Of the six chimeras, A and C failed to bind to collagen I, collagen IV,
and echovirus 1. Furthermore, neither of these chimeras was effectively
recognized by any of the 2 I domain monoclonal
antibodies. Therefore, we assume chimeras A and C are incorrectly
folded, and no conclusions can be drawn from their lack of ligand
binding. The remaining four chimeras, B, D, E, and F, were recognized
by a majority of the antibodies and bound at least one of either collagen I, collagen IV, or echovirus at a level comparable with or
greater than that of the 2 I domain. Of these four
chimeras, B, D, and E showed enhanced collagen IV binding activity.
This represents a gain of function; these chimeras have become more 1 I domain-like with respect to their collagen binding
activity. Also, of the four chimeras showing positive antibody
reactivity, only chimera E lost the ability to bind echovirus 1. This
finding indicated an important role in echovirus 1 binding for the
region of the 2 I domain consisting of the C helix,
the C-6 loop, and the 6 helix.
To further localize regions of the 1 I domain
responsible for enhanced collagen IV binding activity and regions of
the 2 I domain necessary for echovirus 1 binding,
iterative rounds of chimera design, expression, purification, and
analysis were undertaken. The design of each round was based on the
results of the analysis of the previous round. Fig.
4 shows the resultant B series of chimeras. Because chimera B possessed enhanced collagen IV binding activity and contained both the C-3 loop and the 3 helix from the 1 I domain, chimeras B1 and B2 contained just the
C-3 loop (and one residue from the amino-terminal end of the 3
helix), or the remainder of the 3 helix from the 1 I
domain, respectively. The enhanced collagen IV binding activity of
chimera B segregated completely into chimera B2 containing only the
3 helix from the 1 I domain. Chimera B1 reverted to
the parental 2 I domain phenotype of preferential
binding to collagen I. An attempt to further map the region of the 3
helix necessary for the enhancement (Fig. 4, chimeras B2a and B2b)
resulted in reversion to the 2 I domain collagen binding
properties, indicating the requirement of the entire 3 helix from
the 1 I domain for the effect.
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Fig. 4.
Structure of B series of
1/2
integrin I domain chimeras and ligand binding results. Chimera B
contained the C-3 loop and the 3 helix from the
1 I domain. Chimeras B1 and B2 contained just the loop
and one residue from the amino-terminal end of the 3 helix, or the
remainder 3 helix, respectively. Chimeras B2a and B2b contained the
mutations MI  VL and TS AK within the 3 helix. Each of these
chimeras was tested in the collagen I and IV binding assays. The
binding data are given as percentages ± S.E. of control
(2 I domain binding to collagen I).
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Chimera D, containing the 5 helix from the 1 I
domain, as well as three amino-terminal flanking residues and one
carboxyl-terminal flanking residue, had also shown enhanced collagen IV
binding activity. To determine which region was required for the
effect, chimeras D1 and D2 were prepared. These chimeras contained the mutations GSMLKA NHRLKK (three residues from the D-5 loop and
the amino-terminal half of the 5 helix) and DQCNHD QDCEDE (carboxyl-terminal half of the 5 helix and one residue from the 5-E loop). The results of collagen I and IV binding assays using these chimera are shown in Fig. 5.
Chimera D1, containing the amino-terminal half of the 5 helix of the
1 I domain, bound to both collagens I and IV
considerably less effectively than did chimera D. Chimera D2,
containing the carboxyl-terminal half of the 5 helix of the
1 I domain, bound to collagen I as well as chimera D,
but again the binding to collagen IV was diminished with respect to
that of chimera D. Thus it appears that the entire 5 helix from the
1 I domain is required to observe the enhanced collagen
IV binding activity.
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Fig. 5.
Structure of D series of
1/2
integrin I domain chimeras and ligand binding results. Chimera D
contained the 5 helix as well as three amino-terminal flanking
residues and one carboxyl-terminal flanking residue from the
1 I domain. Chimeras D1 and D2 contained the
amino-terminal mutations (GSMLKA NHRLKK) and the carboxyl-terminal
mutations (DQCNHD QDCEDE) found in chimera D, respectively. Each of
these chimeras was tested in the collagen I and IV binding assays. The
binding data are given as percentages ± S.E. of control
(2 I domain binding to collagen I).
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Both enhanced collagen IV binding activity as well as complete loss of
echovirus binding activity was observed for chimera E, containing the
C helix, the C-6 loop, and the 6 helix. Chimeras E1, E2,
and E3 were prepared, containing only the C helix, the C-6
loop, and the 6 helix, respectively (Fig.
6). Chimera E3 reverted to the collagen
binding activity profile and the echovirus binding activity of the
2 I domain. Chimera E2 showed both enhanced collagen IV
binding activity as well as significantly diminished echovirus binding
activity. Chimera E1, which contained a two-residue mutation in the
C helix (YL SY), bound to both collagens I and IV. However, as
opposed to the binding of both the 1 and
2 I domains as well as chimera E, the binding of chimera E1 to both collagens I and IV was independent of divalent cations. The
binding of chimera E1 to collagen IV was not as pronounced as the
binding of chimeras E and E2 to collagen IV. Chimera E1a, which
contained a single amino acid replacement (Y S) within the C
helix, also bound both collagens I and IV in a divalent cation-independent manner. However, chimera E1R, in which the residues
YL in the C helix of the 2 I domain were reversed to LY, showed enhanced collagen IV binding activity and bound collagens I
and IV in a divalent cation-dependent manner.
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Fig. 6.
Structure of E series of
1/2
integrin I domain chimeras and ligand binding results. Chimera E
contained the C helix, the C-6 loop, and the 6 helix from
the 1 I domain. Chimeras E1, E2, and E3 contained just
the C helix, the C-6 loop, or the 6 helix from the
1 I domain, respectively. Chimera E1a contained a point
mutation (Y S) within the C helix; chimera E1R contained a
reversal of two residues in the C helix of the 2 I
domain (YL LY). Chimeras E2a, E2b, and E2c contain mutations in the
C-6 loop (E2a and E2b) or at the amino-terminal end of the 6
helix (E2c). These mutations are NA GN, D S, and KN EK,
respectively. Chimeras E2a1 and E2a2 contain mutations in the C-6
loop, N G and A N, respectively. Chimera E2c1 contains a
mutation in the amino-terminal end of the 6 helix, K E. Each of
these chimeras were tested in the collagen I and IV binding assays. The
collagen binding data are given as percentages ± S.E. of control
(2 I domain binding to collagen I). Data marked with a
lowercase letter a reflect divalent cation-independent
binding to collagen. Many of these chimeras were also tested for
echovirus 1 binding. Echovirus binding data are given as
percentages ± S.E. of control (echovirus 1 binding to the
2 integrin I domain).
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To localize the regions of E2 required for enhanced collagen IV binding
activity and loss of echovirus binding, E2a, E2b, and E2c were prepared
(Fig. 6). The amino acid replacements in these chimeras are NA GN,
D S, and KN EK, respectively. The enhanced collagen IV binding
activity segregated to chimera E2c, containing residues EK from the
1 I domain at the amino-terminal end of the 6 helix.
Chimera E2c1, containing only the point mutation K E at the
amino-terminal end of the 6 helix, reverted to the collagen binding
properties of the 2 I domain, indicating the importance
of K294 for the enhanced collagen IV binding activity of chimera E2c.
The loss of echovirus binding activity segregated completely to E2a, in
which NA in the C-6 loop had been replaced. Chimeras E2a1 and
E2a2, containing point mutations N G and A N in the C-6
loop, were prepared to determine which of the two residues from the
1 I domain present in E2a was responsible for the loss
of echovirus 1 binding activity. This analysis revealed that
Asn289 of the 2 I domain, within the
C-6 loop, is required for echovirus binding.
Because chimeras E2 and E2a containing amino acids 288-294 and
288-289 of the 1 I domain had severely diminished
echovirus 1 binding activity (E2) or exhibited in a complete loss (E2a) of echovirus 1 binding activity, reciprocal I domain chimeras were
constructed in which amino acids 288-294 and 288-289 of the 1 I domain were replaced with the corresponding residues
from the 2 I domain to determine whether either of these
replacements could confer echovirus 1 binding activity upon the
1 I domain. Neither of these two chimeras, E2' or E2a',
gained echovirus 1 binding activity (data not shown). However, these
two chimeras also failed to bind collagens I and IV in a divalent
cation-dependent manner and therefore may represent
incorrectly folded species.
To gain a more complete understanding of the reciprocal collagen type
specificities of the 1 I domain and the 2
I domain, as well as the cation-independent collagen binding activity
of chimera E1, the binding of each of these proteins to collagens I and
IV was measured over a wide range of I domain concentrations and in the
presence of 1 mM EDTA or 2 mM Mg2+
(Fig. 7). The preferential binding of the
1 I domain to collagen IV and of the 2 I
domain to collagen I was evident over the entire range of I domain
concentrations. In addition, the divalent cation-independent binding of
chimera E1 to collagens I and IV was confirmed over the entire range of
protein concentrations tested.
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Fig. 7.
Binding of integrin I domain proteins to
collagens I and IV. The binding of the 1 I domain,
the 2 I domain, and chimeric I domain E1 to collagens I
and IV was measured in a solid phase binding assay over a range of I
domain concentrations from 1.6 to 400 nM. Binding was
determined in the presence of either 1 mM EDTA or 2 mM Mg2+.
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Chimera F, containing the 7 helix from the 1 I
domain, was recognized by a majority of the 2 integrin I
domain monoclonal antibodies and echovirus 1 but showed significantly
impaired collagen (both I and IV) binding activity. Three more
chimeras, F1, F2, and F3, were prepared to ascertain which region of
the 7 helix from the 1 I domain was necessary for the
loss of collagen binding activity. Chimeras F1, F2, and F3 contained
the mutations A L, LEKAG VTIVK, and Q R within the 7
helix, respectively. Chimera F1 reverted to the collagen binding
activity profile of the 2 I domain (Fig.
8). Chimera F2 showed somewhat diminished collagen I binding activity (in comparison to the 2 I
domain) and severely impaired collagen IV binding activity. Of the
chimeras F1-F3, F3 showed the most impaired collagen I binding
activity, only marginally higher than chimera F. Thus it appears that
the loss of collagen binding activity seen in chimera F is a result of
a combination of effects from the F2 region (LEKAG) of the 7 helix
as well as the Q R mutation near the carboxyl terminus of the 7
helix.
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Fig. 8.
Structure of F series of
1/2
integrin I domain chimeras, and ligand binding results. Chimera F
contained the 7 helix from the 1 I domain. Chimeras
F1, F2, and F3 contained residues from the amino-terminal third (A L), the middle third (LEKAG VTIVK), and the carboxyl-terminal third
(Q R) of the 7 helix of the 1 I domain,
respectively. Each of these chimeras were tested in the collagen I and
IV binding assays. The binding data are given as percentages ± S.E. of control (2 I domain binding to collagen
I).
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As mentioned earlier, five 2 integrin I domain
monoclonal antibodies recognizing complex conformational epitopes were
used as probes in ELISAs to assess the folded state of chimeric I
domain molecules. The chimeras were considered to be properly folded if
a majority of the antibodies were able to bind. All of the chimeras
presented in this study were deemed properly folded with the exception
of chimeras A and C in the first round and E2' and E2a'.
New epitope mapping information for three of the five 2
integrin I domain monoclonal antibodies was obtained in this study from
the analysis of their binding to the 1/2
integrin chimeras (Table II). These
antibodies were 6F1, 12F1, and 5E8. These antibodies had previously
been mapped to the same relatively large region (residues 173-259) of
the I domain using human/bovine chimeric 2 I domain
proteins (11). In our experiments, 6F1 binding was abrogated by
mutation of the sequence TS (residues 210 and 211) at the
carboxyl-terminal end of the 3 helix (chimera B2b). The binding of
12F1 required the sequence TYKTK (residues 199-203) within the
C-3 loop (chimera B1). P1E6 recognition required DQCNHD (residues
268-273), the carboxyl-terminal half of the 5 helix (chimera D2).
With the exception of the P1E6-binding site, these regions are all
within the range of amino acids previously specified as required for
binding using the human/bovine chimeras. Amino acids 268-273, required
for P1E6 binding in this study, are close in the primary structure to
the region previously shown to be important for P1E6 recognition (11);
it appears that P1E6 binding may require amino acids within both
regions.
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Table II
Results of ELISA analysis of recombinant I domain proteins
The data are given as percentages ± S.E. of the absorbance
obtained with the 2 I domain.
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DISCUSSION |
Although purified 11 and
21 integrins each bind both collagen I
and collagen IV, the 11 integrin exhibits
a greater affinity for collagen IV than for collagen I, whereas the
21 integrin preferentially binds collagen
I (18). BIAcore and solid phase binding studies using isolated subunit I domains from these two integrins demonstrate that the
collagen type specificities of the I domains are those of the parent
integrins (13). The 21 integrin has been
shown to be a cell surface receptor for echovirus 1 (8). The
2 I domain was shown to contain the echovirus 1-binding
site (24). Murine 2 I domain fails to bind the virus (24) and human/murine 2 I domain chimeras have been used
to identify some regions of the human 2 I domain
required for echovirus binding (25).
To determine which regions of the 1 integrin I domain
are responsible for enhanced collagen IV binding activity, and to
identify additional regions of the 2 integrin I domain
required for echovirus 1 binding, we undertook a systematic study
involving the preparation and analysis of human
1/2 integrin I domain chimeras.
Initially, a series of six chimeras were constructed that contained
primarily 2 sequence, each with a single internal region
that had been replaced with the analogous residues from the
1 I domain. The design of the initial round of chimeras
was undertaken with the aid of the recently published crystal structure
of the 2 integrin I domain (17). For the most part,
chimeras in the initial round (Fig. 3, A-F) contained a
combination of secondary structural elements from the 1
I domain, for instance a helix and a loop, as in chimera A. Helices and
loops were chosen, as opposed to strands, because of their surface
exposure. Two of the chimeras (D and F) contained only a helix and
chimera E contained two helices and a loop. Following the
identification of chimeras with enhanced collagen IV binding activity
or impaired echovirus binding activity, additional chimeras were
constructed that had progressively smaller regions of replacement by
1 sequence. The pattern of chimera design, expression,
purification, and analysis was repeated until the chimeras reverted to
the 2 phenotype with regard to ligand binding or until
the effect was isolated to a single amino acid.
The only chimera in the first round to lose echovirus 1 binding
activity was chimera E. This chimera contained the C helix, the
C-6 loop and the 6 helix from the 1 I domain.
Through iterative rounds of expression and analysis, the loss or severe impairment of echovirus binding was traced through chimera E2 (C-6 loop) and chimera E2a (residues NA in the loop) and finally to chimera E2a1 (Asn289). The fact that the loss of
echovirus 1 binding can be traced through these four chimeras in the E
series provides compelling evidence for the involvement of this region
and Asn289 in the interaction of echovirus 1 with the
2 I domain.
A similar approach has been previously used to map the echovirus
1-binding site on the 2 I domain (25). However, chimeras in the earlier study were human/murine 2 integrin I
domain chimeras. Murine 2 integrin I domain fails to
support echovirus 1 binding (24). Two regions of the I domain were
shown to be required for maximal echovirus 1 binding activity. These
regions consisted of amino acids 199-201 in the C-3 loop and
amino acids 212, 214, and 216 in the 3-4 loop. In a reciprocal
experiment, replacement of the analogous residues within the mouse
2 I domain with human residues 199-216 conferred
echovirus 1 binding upon the mouse 2 I domain. This
provided unambiguous evidence for the role of this region of the
2 I domain in echovirus binding. A similar attempt was
undertaken in this study involving the C-6 loop. Two chimeras
were prepared in which portions of the 1 I domain were
replaced with the analogous regions of the 2 I domain.
These chimeras were analogous to E2 and E2a and thus are referred to as
E2' and E2a'. Although neither of these two chimeras bound echovirus,
they also failed to bind collagens I and IV, indicating that they may
be incorrectly folded. We conclude that Asn289 is necessary
for echovirus 1 binding; it may not be sufficient.
The mouse/human 2 I domain chimera study did not
implicate Asn289 or the region around the C-6 loop in
echovirus binding. A comparison of the mouse and human 2
I domain sequences (25) provides a possible explanation for this. Not
only is Asn289 conserved in these two I domains, but the
entire region containing the C helix, the C-6 loop, and the
6 helix as well as some flanking residues is completely conserved
between the human and mouse 2 I domain structures. Thus
it would have been impossible to ascertain the importance of this
region for echovirus 1 binding using mouse/human chimeras.
Fig. 9 shows the residues involved in
echovirus binding by the 2 integrin I domain in
green. Residues implicated in the previous study (25)
(residues 199-201, 212, 214, and 216) and in this study
(Asn289) are shown. It has been hypothesized that the
2 integrin I domain may bind to echovirus 1 within
depressions that exist on the viral surface at both the 5- and 2-fold
symmetry axes (25, 26). The topography of these depressions may explain
how residues that are quite distant from each other on the I domain,
such as Asn289, and residues 199-201, could be involved in
virus binding by the I domain.
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Fig. 9.
Location of residues affecting the ligand
binding specificity of the 2
integrin I domain. Shown are two representations of the
2 integrin I domain structure (17). In the space-filling
model (left) and the main-chain schematic (right)
residues affecting the collagen type specificity of the I domain are
shown in purple, and residues required for echovirus 1 binding to the I domain are shown in green. The replacement
of any of three groups of residues including 206-211 (3 helix),
260-273 (5 helix with some flanking residues), and 294-295 in the
6 helix with the analogous residues from the 1 I
domain results in chimeras with enhanced collagen IV binding activity,
an 1 I domain-like quality. Tyr285, in the
C helix, when mutated to serine resulted in enhanced collagen IV
binding activity and divalent cation-independent binding to both
collagens I and IV. Residues 199-201 and 212-216 have previously been
shown to be required for echovirus 1 binding using human/mouse
2 I domain chimeras (25). Based on this study,
Asn289 is also required for echovirus 1 binding. In the
figure on the right, the secondary structural elements are
labeled |
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TOP
ABSTRACT
INTRODUCTION
