1 identification and characterization of two cation binding sites in

1

Identification and Characterization of Two Cation Binding Sites

in the Integrin β3 Subunit

Aleksandra Cierniewska-Cieslak1, Czeslaw S. Cierniewski

1,2, Kamila Blecka

2,

Malgorzata Papierak2, Lidia Michalec

2, Li Zhang

3, Thomas A. Haas

4 and Edward F

Plow4*

Center for Microbiology and Virology1, Polish Academy of Sciences, Lodz; Department

of Biophysics2, Medical University in Lodz, Poland; American Red Cross Holland

Laboratory3, Rockville, MD; Joseph J. Jacobs Center for Thrombosis and Vascular

Biology and Department of Molecular Cardiology4. Cleveland Clinic Foundation,

Cleveland, OH

*To whom correspondence should be addressed:

Mail Code NB50, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH

44195

TEL: 216-445-8200; FAX: 216-445-8204; E-mail: [email protected]

Running Title: Cation and Ligand Binding to the Integrin β3 subunit

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 16, 2002 as Manuscript M112388200 by guest on M

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Cation and Ligand Binding to the Integrin β3 subunit

2

SUMMARY

The mid-segment of the β3 subunit has been implicated in the ligand and cation

binding functions of the β3 integrins. This region may contain a metal ion dependent

adhesion site (MIDAS) and fold into an I-domain like structure. Two recombinant

fragments, β3(95-373) and β3(95-301), were expressed and found to bind fibrinogen.

While 0.1 mM Ca2+

supported ligand binding to both recombinant fragments, 1.0 mM

Ca2+

suppressed binding to the longer but not the shorter fragment. These properties

suggest that β3(95-373) contains both the ligand competent (LC) and inhibitory (I) cation

binding sites and β3(95-301) lacks the I site. In equilibrium dialysis experiments, β3(95-

373) contained two divalent cation binding sites, one reactive with either Mg2+

or Ca2+

and one Ca2+

-specific while β3(95-301) lacked the Ca2+

-specific site. Mutant forms of

β3(95-373) suggested that the LC site is a MIDAS motif involving D119, S121, S123,

D217 and/or E220 as coordination sites, and the I site was dependent upon residues

within β3(301-323). In a molecular model of β3(95-373), a second Ca2+

could be docked

onto a flexible loop in close proximity to the MIDAS. These results indicate that the

ligand competent and Ca2+

-specific inhibitory cation binding sites are distinct and reside

in β3(95-373).

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αIIbβ3 is a typical member of the integrin family of cell adhesion receptors (1), being

composed of an α (αIIb) and a β (β3) subunit, which associate to form a noncovalent

heterodimer. This integrin is the most abundant membrane protein on the platelet surface

and serves as a receptor for multiple adhesive proteins including fibrinogen (Fg).1 Two

sets of peptides – HHLGGAKQAGDV, corresponding to the sequence at the C-terminus

of the Fg γ-chain (2), and RGD(X), corresponding to a sequence present in many protein

ligands of αIIbβ3 and recognized by many other integrins as well - define the recognition

specificity of αIIbβ3 for its macromolecular ligands [reviewed in (3)]. αVβ3, which shares

the same β3 subunit as αIIbβ3, is broadly distributed and binds many but not all of the

same ligands as αIIbβ3, including Fg, von Willebrand factor and fibronectin [reviewed in

(4,5)]. This integrin also exhibits a RGD recognition specificity but shows a much

weaker recognition of Fg γ-chain peptides (6). Numerous studies have suggested that

binding of macromolecular ligands to αIIbβ3, as well as αVβ3, involves multiple contacts

in each subunit (7-13). Essential residues for ligand binding to αIIbβ3 reside in two major

regions: a mid-segment of β3, β3(95~400), and the amino-terminal aspect of αIIb, αIIb(1-

334) (11), which contains seven structural repeats (14). The mid-segment of the β3

subunit is highly conserved among integrin β subunits and exhibits some structural and

functional features of an I-domain. I-domains are present in 9 integrin α subunits and

play major roles in the ligand binding functions of their integrin heterodimers (15-20).

The relationship between the conserved β mid-segments and α I-domains was proposed

based upon similarities in their hydropathy profiles, secondary structural predictions and

mutational analyses (18,20-22). A central feature of I-domains is a metal ion-dependent


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adhesion site, a MIDAS motif (15,18,19,23). In MIDAS motifs, three of the five cation

coordination sites are provided by a DxSxS sequence and two other coordination sites are

provided by oxygenated residues distant in primary sequence. Mutations of the cation

coordinating residues in a MIDAS motif often cause loss of ligand binding functions of

an integrin, and ligand binding sites map in close proximity to MIDAS motifs (18,24,25).

The β3 mid-segment does contain a D(119)xSxS sequence, and these residues also have

been implicated in the ligand binding functions of αIIbβ3 (24,26-28). While there is broad

consensus that the mid-segment of integrin β subunit contains a functional MIDAS, other

structural algorithms have predicted a protein fold for this region that is quite distinct

from that of I-domains (29).

The presence of divalent cations, such as Ca2+

and Mg2+

, is essential to the

integrity of the heterodimeric structure of αIIbβ3 [reviewed in (30)], its conformational

state (31,32) and for the ligand binding functions of this as well as all integrins (33).

αIIbβ3 has approximately five Ca2+

-binding sites, falling into at least two distinct affinity

classes as estimated by equilibrium dialysis (34) and Tb3+

luminescence spectroscopy

(35). To account for the influential role of cations in integrin function, it has been

proposed that ligand and cation may share a common binding pocket on the integrin (36).

Dissection of the ligand binding reaction into association and dissociation steps and

subsequent (37) surface plasmon resonance experiments (13,38) suggested that the

integrin β3 subunits contain two functionally distinct classes of ion binding sites. One

class must be occupied for ligand to bind, the ligand competent (LC) sites, and the second

class is specific for Ca2+

and has an inhibitory effect on ligand binding, the inhibitory (I)

site(s).


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Taking into consideration the significant role of the mid-segment of the β3 subunit

in the function of αIIbβ3 specifically and integrins in general, in this study we have

attempted to map the location of ligand and cation binding sites within this region. Two

series of mutants were produced: the first group was obtained by swapping individual

homologous segments from the β2 subunit, which does not form high affinity RGD-

binding integrins, for the corresponding segment in β3(95-373). The second group

contained mutants in which single amino acid residues known to coordinate cations in

authentic I-domains, such as in the αL I-domain, were substituted by a site-directed

mutagenesis. The results provide direct experimental support for the presence of not one

but of two cation binding sites in β3(95-373). The first of these sites displays the

characteristics of MIDAS motif and is critical for ligand binding while the second of

these sites exhibits the properties and specificity of the inhibitory Ca2+

-binding site.

EXPERIMENTAL PROCEDURES

Preparation of ααααIIbββββ3. αIIbβ3 was isolated from outdated platelets by RGD

affinity chromatography (39,40). Briefly, platelets were lysed in buffer containing 10

mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM leupeptin, 10 mM N-

ethylmaleimide, 1 mM phenylmethane sulfonyl fluoride, and 50 mM octyl-glucoside, pH

7.3, centrifuged for 1 hr at 30000 x g, and applied onto a GRGDSPK-Sepharose column

(12 cm x 2.5 cm). The affinity matrix was equilibrated with the lysis buffer. Detergent

extracts of platelet proteins were recycled over the affinity matrix at a flow rate of 0.5

ml/min at 4°C. Unbound protein was removed with 10 column volumes of column

buffer, identical to lysis buffer except that the octyl-glucoside concentration was lowered


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to 25 mM. Protein remaining bound onto RGD-affinity matrix was eluted with buffer

containing RGDF (1 mg/ml). Fractions were analyzed by electrophoresis on 7%

acrylamide gels in SDS under nonreducing conditions (41). Prior to ligand binding

experiments, samples of αIIbβ3 were dialyzed against 0.02 HEPES buffer, pH 7.3,

containing 0.15 M NaCl, 25mM octyl-glucoside and selected divalent cations.

Recombinant ββββ3 fragments. Two recombinant β3 fragments, β3(95-301) and β3(95-373),

were produced. Each contains the V107-S292 segment, which corresponds to the putative

I-domain in the β3 subunit based on its homology to the I domains of αL and αM.

Complementary DNAs encoding these fragments were generated by PCR using full-

length human β3 cDNA as a template. To express β3(95-301), a PCR fragment

containing BamH1 and XhoI restriction sites was generated using the following primers:

5'CTCCGCCTGggatccGATGATTCGAAG3' (upper primer) and

5'CTCATTctcgagTCAGGTGGCATTGAAGGA3' (lower primer). To express β3(95-

301), 5'CTCAATctcgagTCATTTCTGGGATAGCTTCTCAG3' replaced the lower

primer. The PCR products were digested with BamH1 and Xho1 restriction enzymes and

inserted into pRSETa (Invitrogen) for expression in Escherichia coli (DH5α; GIBCO-

BRL) as a His-tag fusion protein containing 12 residues (MRGSHHHHHHGS) at their

N-termini. To purify the recombinant β3 fragments, inclusion bodies were prepared,

dissolved in 6 M urea and separated by chromatography on Chelating Sepharose

(Pharmacia), loaded with Ni ions according to the manufacturer instructions.

Recombinant β3(95-301) or β3(95-373) fragments were eluted with 0.02 M Tris-HCl, pH

7.9, containing 1.0 M imidazole, 0.5 M NaCl and 6 M urea. Refolding of β3(95-301) and

β3(95-373) was performed by dialysis of dilute protein solutions (100-200 µg/ml) against


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1000 ml of the elution buffer at 4°C. Then, the urea concentration was reduced by a

continuous, slow drip of 1000 ml of 20 mM Tris buffer, pH 8.0, into the dialysis solution.

Afterwards, 1000 ml of the dialysis solution was removed and the next 1000 ml of 20

mM Tris, pH 8.0, was added by a continuous drip, and this procedure was repeated five

times at 4°C under constant stirring. A final dialysis was then performed against. 0.01 M

Tris-HCl, pH 8.0, containing 0.14 M NaCl, NaN3 (1 mg/ml). Then, the recombinant

fragments were concentrated to ~2 mg/ml by ultrafiltration. Each fragment migrated as a

single band on SDS-PAGE of either 31 kDa for β3(95-373) or 21 kDa for the β3(95-301).

Site-directed Mutagenesis. All mutations were created using QuikChange site

directed mutagenesis kits from Stratagene (La Jolla, CA) performed according to the

manufacturer’s instructions. Two sets of mutant the β3 fragments were produced. In the

first set, individual segments from β3(95-373) were replaced with the homologous

segments from the integrin β2 subunit. Nine mutants designated β3(F100-Y110),

β3(L128-Q141), β3(K159-P170), β3(C177-C184), β3(L194-E206), β3(Q210-D217),

β3(N279-S291), β3(Q301-E312), and β3(T311-S322) were expressed using the mutagenic

primers. All were expressed as fusion proteins with His-tags and were purified on

Chelating Sepharose as described above for the wild-type fragments. In the second set

of mutants, the cation coordinating residues of the putative MIDAS motif in the β3

subunit were changed to alanines. The specific mutations and the primers used were:

D119A (5'ggacatctactacttgatggcactgtcttactccatg3' and

5'catggagtaagacagtgccatcaagtagtagatgtcc3'); S121A

(5'cttgatggacctggcatactccatgaaggatgatctg3' and cagatcatccttcatggagtatgccaggtccatcaag3'),

S123A (5'ggacctgtcttacgcaatgaaggatgatctgtgg3' and


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5'ccacagatcatccttcattgcgtaagacagctac3'), D217A (5'gtcacggaaccgagcagccccagagggtggc3'

and 5'gccaccctctggggctgctcggttccgtgac3'), and E220A

(5'ccgagatgccccagcaggtggctttgatgccatc3' and 5'gatggcatcaaagccacctgctggggcatctcgg3').

All mutations were confirmed by dideoxy sequencing and were subsequently subcloned

into the pRSETa vector for expression.

Fibrinogen binding assays. Human Fg was purified and characterized as

described previously (42). The binding of Fg to immobilized αIIbβ3, β3(95-301), β3(95-

373) or its mutants was performed as described for intact receptor (35). Briefly,

microtiter wells (Corning Costar Corp., Cambridge, MA) were coated by incubation

overnight at 4°C with 200 µl of purified αIIbβ3 or β3 fragments dissolved at a

concentration of 5 µg/ml in 10 mM Tris buffer, pH 7.4, containing 0.15 M NaCl. The

plates were then washed and post-coated with 4% bovine serum albumin overnight at

4°C. In binding experiments, 20 µl of 125

I-fibrinogen with a specific radioactivity of ~0.2

µCi/µg was added to each well and mixed with 100 µl aliquots of buffer or inhibitors.

The plates were incubated for 3 hr at 37°C. After this and between each preceding step,

the wells were washed extensively with Tris buffer. Binding of Fg was determined by

direct γ counting of the plastic wells. Nonspecific binding was defined as the residual

binding observed in the presence of either 10 µM nonlabeled Fg or 2 mM EDTA (see

Results). This value was subtracted from the total binding to obtain specific binding

values. The amounts of recombinant β3 fragments and mutants bound to the plastic wells

were verified to be similar by ELISA using a rabbit antiserum raised to β3(95-373).

Complex formation between Fg and β3(95-373) or β3(95-301) was also assessed

in a fluid phase analysis. Fg (1 mg/ml) and the recombinant fragments of the β3 subunit


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(0.2 mg/ml) were mixed in a total volume of 4 ml 50 mM Tris buffer, pH 7.5, containing

100 mM NaCl, 5 mM CaCl2 and 0.5 M glucose to yield a molar ratio of Fg to the

recombinant β3 fragment of 1:3. After 4 hr at room temperature, the mixtures were

concentrated by centrifugation in 100K Centricon tubes (Millipore, Bedford, MA) and

washed three times, each time followed by concentration step in the Centricon tubes. The

retentates were analyzed by electrophoresis on either non-denaturing 7.5 % acrylamide

gels (43) or on 12 % acrylamide gels containing SDS and 2-mercaptoethanol (41).

Cation-binding assays. Binding of Ca2+

to purified αIIbβ3, β3(95-301), β3(95-

373) or its mutants was determined by equilibrium dialysis using a 16-mirocell dialyzer

(Sialomed Inc., Columbia, MD). Regulation of the free Ca2+

by EGTA was used to

obtain the Ca2+

concentrations needed to construct binding isotherms. The buffer used

was 50 mM Tris, 0.14 M NaCl, pH 7.3, containing 50 mM octyl glucoside (the

equilibrium buffer). Before use, glassware, plasticware, and dialysis tubing were treated

as described by Gulino et al. (44). Total Ca2+

concentrations were determined by

spectrofluorimetry, using Fura-2 and Ca2+

standard solutions from Molecular Probes, Inc

(Sunnyvale, CA). 45

Ca2+

was measured in a scintillation counter (Beckman LS6000LL).

Below 10 µM, the required free Ca2+

concentration was adjusted by addition of 7 mM

EGTA, as calculated according to Fabiato (45) to account for influence of pH,

temperature and ionic strength on the equilibrium constants used. Demineralization of

the analyzed proteins was done by dialysis for 1 hr at 22°C against the equilibrium buffer

containing enough EGTA to reduce the free Ca2+

to below 1 nM as described (34).

Equilibrium dialysis experiments were then performed as follows. Various

amounts of 10 mM CaCl2 were added in the equilibrium buffer containing 45

Ca2+

(3


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µCi/ml) to achieve free Ca2+

concentration between 0.01 and 1000 µM. Free Ca2+

concentrations were calculated from the contaminating Ca2+

, determined

spectrofluorimetrically, plus the amount of added Ca2+

. 45

CaCl2 (0.48 µCi) was injected

into one half cell, and the proteins into the other one. After dialysis for 24 hr at 21°C,

aliquots were removed for 45

Ca and protein determinations, SDS-PAGE and ligand

binding studies. αIIbβ3 and the recombinant β3(95-373) mutants were used at the

concentration of 5-10 µM and 30-40 µM, respectively. To test the effects of Mg2+

on

Ca2+

binding, the equilibrium dialysis was performed in the presence of 45

CaCl2 (500

µM), sufficient to saturate cation binding sites in the β3(95-373) and its mutants, and

serial dilutions of Mg2+

, ranging from 0 to 100 mM, were added.

Circular Dichroism (CD). The CD spectra of β3(95-301), β3(95-373) and various

mutants were measured in the 200 to 260 nm range using a CD6 JOVIN YVON

spectropolarimeter at protein concentrations of 80 µM using 0.1 mm pathlength cells.

The secondary structural content of the fragments was estimated by published protocols

(46-48).

Radioiodination. Na125

I (specific activity 15-17 µCi 125

I per mg iodine) from

Amersham Pharmacia Biotech (Piscataway, NJ) was used for radioiodination. Fg was

labeled using IODO-GEN (Pierce, Rockville, IL). The iodinated protein was separated

from free Na125

I by gel filtration on a Bio-Gel P2 column (Bio-Rad, Hercules, CA). The

specific radioactivity of the 125

I-Fg ranged from 0.5 to 1.0 Ci/g. Usually, >90% of the

radioactivity incorporated into Fg was precipitated by 10% trichloroacetic acid or by a

monospecific antiserum. Aliquots of the radioiodinated protein were stored at -20°C for

no longer than two weeks before use.


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Molecular modeling. Molecular dynamic simulations and model building were

carried out using the InsightII and Homology (Molecular Simulations, Inc., San Diego,

CA) Programs on a Silicon Graphics Indigo workstation as previously described (49).

The I-domain structures of αL (CD11a), 1zon (50), αM (CD11b), 1jlm (51), and α2, 1aox

(52), were structurally aligned based on homology, and the sequence of β2(75-397) was

then aligned to these known structures as previously described (49). The sequence of

β3(82-405) was then aligned to the α2 sequence by sequence homology and by secondary

structural predictions (Homology and Jpred). Structures for β3 segments were then

assigned from 1zon, 1jlm and 1aox, and gaps in structure were filled in by the generation

and selection of loops. The sides of each loop were then relaxed to remove/reduce

atomic overlap and torsions. The charges on the amino acids were adjusted to pH 7.2

using the Biopolymer Program. A minimized-energy conformation for β3 (82-405) was

then obtained by gradually minimizing the protein structure. A calcium ion was then

docked at the MIDAS site and another at the putative I site using the Docking Program.

The resulting minimized structure was then subjected to a series of molecular dynamic

simulations and energy minimizations using a time step of 1 fs, and a Van der Waals and

an electrostatic cut off of 12. The resulting structure was then minimized to a maximum

derivative of less than 0.05 kcal⋅(mol⋅Å)-1

using steepest descent and conjugate gradient

minimizations.

Antibodies. Monoclonal antibody (mAb) P37 was obtained from Dr. Rodriguez-

Gonzalez and reacts with β3(101-109) (53), and mAb 25E11 was originally described by

Burns et al. (54) and was purchased from Chemicon ( Temecula, CA) as mAb 1957. A


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polyclonal antibody was raised to β3(95-373) by immunizing rabbits with the purified

recombinant fragment.

Statistical analysis. All the final data are presented as the means of the averaged

replicates ± S.D. The normal distribution of data was confirmed using the Shapiro-Wilk's

test. The analysis of variance and Tukey's test for multiple comparisons (55) were

employed to assess the significance of differences (p values) among groups.

RESULTS

Ligand binding to the recombinant fragments of the integrin ββββ3 mid-segment.

Two recombinant fragments of the β3 subunit, β3(95-301) and β3(95-373), each

containing the putative I-domain, β3 (107-272) and its MIDAS, were expressed in

Escherichia coli as His-tag fusion proteins, purified on nickel chelating columns in 6M

urea, and refolded by sequential dialysis to remove the denaturant. The final products

were soluble in aqueous buffers and were homogenous as assessed by SDS-PAGE.

When immobilized on microtiter plates, each of the recombinant β3 fragments reacted

with mAb 937 to β3(101-109)(53) and mAb 25E11 to β3(237-248)(54) in an ELISA

format. Circular dichroism was performed on the renatured recombinant fragments. The

spectra were very similar and used to assess their secondary structure. The helical

content was estimated to be 17.3 ± 2.5% for β3(95-301) and 16.5 ± 3.1% for β3(95-373).

These values are consistent with those predicted for these fragments based upon

secondary structural algorithms [Chou-Fasman (10.8%) and jpeg (24.5%) in the Insight II

Molecular Simulations Program] for β3(95-373). Both fragments also contained a β sheet


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content that was destroyed upon addition of 6 M urea [29.2 ± 1.0% for β3(95-301) in 20

mM Tris, pH 7.3 and <0.1 ± 3.1% in 6M urea].

Initially, the capacity of the two recombinant β3 fragments to form complexes

with Fg was assessed with all components in solution (Fig. 1). In these experiments, Fg

was mixed with the recombinant fragments at the ratio of 1:3 for 4 hr at room

temperature, and then the bound and free β3 fragments were separated by centrifugation

using filters with 100,000 molecular weight cut-offs. The formation of complexes was

evaluated by electrophoresis performed in polyacrylamide gels under non-denaturing or

denaturing (SDS) conditions. When complexes of the β3(95-301) or β3(95-373) with Fg

were separated on non-denaturing gels, no free recombinant β3 fragments were detected

in the gels (Fig. 1A). However, both β3(95-301) and β3(95-373) could be released from

their complexes with Fg by SDS (Fig. 1B). Thus, both the β3(95-301) and β3(95-373)

were capable of interacting with Fg.

In the next set of analyses, the two β3 fragments were immobilized onto microtiter

wells, and their binding of 125

I-Fg was evaluated. As assessed by an ELISA using a

rabbit antiserum raised to β3(95-373), similar amounts of the two recombinant fragments

bound to the microtiter wells. Both recombinant β3 fragments displayed the capacity to

bind 125

I-Fg (see Fig. 2). The interactions were specific since they were inhibited by

nonlabeled Fg or EDTA. Thus, by two approaches, the fluid phase (Fig. 1) and the solid

phase analyses (Fig. 2), the recombinant fragments were capable of binding Fg.

Although the binding of 125

I-Fg to both β3 fragments was divalent ion dependent, each

interaction was differentially influenced by Ca2+

. Whereas changes in the Ca2+

concentration from 0.1 mM to 1 mM had little effect on Fg binding to the shorter β3(95-


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301), the increase in Ca2+

concentration inhibited the binding of Fg to the β3(95-373) by

~ 85 %. These data suggest that the cation binding properties of β3(95-301) and β3(95-

373) may be different. With both recombinant β3 fragments, the total 125

I-Fg binding was

inhibited well by excess nonlabeled Fg (~85%) but less well by EDTA (~60%). A

divalent ion independent interaction of Fg with recombinant β3 fragments has been

previously reported (56). Since the interaction of Fg with the intact β3 integrins requires

divalent ions, we have focused our subsequent studies on this component of the

interaction.

Cation binding to the recombinant fragments of the integrin ββββ3 mid-segment.

The binding of Ca2+

to both β3 recombinant fragments and to the intact αIIbβ3 was

assessed by equilibrium dialysis (Fig. 3). Ca2+

binding to αIIbβ3 was measured in the

range of 0.1-750 µM added cation. The binding isotherm obtained was complex (Fig.

3A), indicative of multiple classes of binding sites. By curve fitting analyses, the best fit

of these experimental data points corresponded to two classes of sites with different

affinities for Ca2+

. There was a single high-affinity binding site (N1 = 1.0 ± 0.15

mole/mole) described by a Kd of 98 ± 19 nM, and there were 4-5 binding sites (N2 = 4.45

± 0.89 mole/mole) of intermediate affinity (Kd = 42.0 ± 8.4 µM). These results are

similar to those obtained by others using equilibrium dialysis (34), as well as by other

approaches (35,57), to measure cation binding to the receptor. Therefore, these same

conditions were used to measure the interaction of the two β3 recombinant fragments

with Ca2+

(Fig. 3B). When the longer recombinant fragment, β3(95-373), was tested, the

best fit to the experimental data (average of three experiments) suggested the presence of


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two binding sites (N = 2.01 ± 0.25 mole/mole). Their Kd of 78 ± 12 µM corresponded

well to that of the intermediate affinity binding sites found in the intact receptor. β3(95-

301) bound only one Ca2+

(N = 1.21 ± 0.15 mole/mole), also with an intermediate affinity

(Kd of 98 ± 25 µM). The Kd values of the two fragments for Ca2+

were not statistically

significant (p> 0.05). Thus, the shorter β3 fragment appeared to lack one of the two Ca2+

binding sites present in β3(95-373).

Expression of mutant forms of the ββββ3 mid-segment and their interaction with Ca2+

.

To probe the structural requirements of the two cation binding sites within β3(95−373),

two sets of mutant fragments were developed. The first set consisted of a series of single

point mutations in which the cation coordinating residues of the putative MIDAS motif

were changed to alanines. In the β3 subunit, these residues are: D119, S121, and S123,

which comprise the DxSxS component; either T182 or T183, which constitutes the fourth

coordination site; and D217, which is predicted to provide the fifth coordinating group

(29,58). In addition, E220, which resides in close proximity to last coordinating residue,

also was mutated to A. The second set of mutants consisted of β3(95-373) fragments in

which short segments were replaced with the homologous segments of the β2 subunit. As

shown in Table 1, these regions of the β3 and β2 subunits are highly homologous to one

another (43% identity and an additional 10% conservative substitutions). They are both

predicted to contain the putative MIDAS motif and to have similar secondary structures.

Therefore, segment swaps between the β3 and β2 segments should not cause gross

perturbations in conformation. Nevertheless, β3 integrins display a high affinity RGD

recognition specificity, whereas β2 integrins do not. Furthermore, in attempt to stress


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functional differences without perturbing structure, in introducing the swaps, we utilized

the mouse β2 sequence, which introduced still further changes in specific amino acids but

should not perturb conformation. All mutants that were developed are illustrated in Fig.

4A, and the amino acid sequences of peptide segments changed are summarized in the

lower portion of Table 1. All mutants of β3(95-373) were expressed in Escherichia coli

as His-tag fusion proteins; solubilized from inclusion bodies in 6M urea; purified by

chromatography on Chelating-Sepharose substituted with nickel; and refolded by dialysis

to remove urea. When analyzed by SDS-PAGE, each migrated as single major band (see

Fig. 4B), and their identity was confirmed by immunoblotting (Fig. 4C). The CD spectra

of six representative mutants, including several with altered Ca2+

binding properties (see

below), were performed. Their helical content estimated from their CD spectra were not

statistically different (range = 16.1 ± 2.5 % to 16.9 ± 1.9 %) from that of the wild-type

recombinant fragment (16.5 ± 3.1%), suggesting that the mutational strategy selected was

appropriate and did not significantly perturb conformation.

When tested by equilibrium dialysis, all mutants but β3(N279-S291) showed some

alteration in their Ca2+

binding properties relative to wild type β3(95-373). Based upon

their binding isotherms, the mutants could be classified into two groups (Table 2). The

first group retained the same number of Ca2+

binding sites as the parent β3 fragment but

exhibited a significantly reduced binding affinity for the cation. This group consisted of

β3(L128-Q141), β3(K159-P170), β3(C177-C184), and β3(L194-E206). The second group

expressed only one Ca2+

binding site, with or without a significant change in affinity

relative to the β3(95-373). This group was composed of the following homolog scanning


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and point mutants: β3(Q210-D217), β3(Q301-E312), β3(T311-S323), D119A, S121A,

S123A, D217A and E220A.

The following description of the behavior of the individual mutants is organized

to proceed from the N- to the C-terminus of the β3 fragment. A switch of the

β3(F100)SIQVRQVED(109) sequence in the region N-terminal to the conserved

D119xSxS motif to the corresponding sequence in the β2 subunit, FNVTFRRAKG,

resulted in a significant decrease in binding affinity for Ca2+

. A similar effect was

observed when the C-terminal flanking sequence, β3(L128)WSIQNLGTKLAT(140), was

switched to the β2 (LNNVKKLGGDLL) sequence. However, the decrease both in

affinity and in number of Ca2+

binding sites was observed with the D119A, S121A and

S123A point mutations. Thus, the oxygenated residues in the DxSxS motif contribute to

one of the Ca2+

binding sites of the β3(95-373) fragment. Replacement of the

β3(C177)YDMKTT(183) sequence with the CPNKEKA segment of β2, thus eliminating

the T residues (T182 and T183) predicted to serve as a fourth cation coordination site in a

β3 MIDAS motif (22), altered the affinity but not the number of Ca2+

binding sites in the

mutant β3(95-373) fragment. Similarly, the Ca2+

binding affinity but not the number of

sites was altered by replacement of the two flanking segments,

β3(K159)PVSPYMYIS(168) and β3(L194)TLTDQVTRFNE(205) with corresponding β2

sequences, KTVLPFVNTHPE and LKLTDNSNQFQTE, respectively. Thus, this mid-

region behaved as a functional unit with respect to its contribution to the cation binding

properties of β3(95-373). A switch of the β3(Q210)SVSRNR(216) sequence in β3 to the

QLISGNLD sequence of β2, directly preceding D217 which is predicted to be the fifth

cation coordinating residue in the MIDAS motif, destroyed one of Ca2+

binding sites


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without affecting the binding affinity of the remaining one. The same change in binding

characteristics was observed when point mutation of E220A was introduced into β3(95-

373). This single substitution caused a disappearance of one cation binding site. The

similar effect was observed for the mutant in which the β3(Q301)KNINLIFAVT(311)

sequence was switched to the β2 sequence, ESNIQPIFAVTK. This mutant bound only

one Ca2+

although the affinity of the remaining Ca2+

binding site was also significantly

perturbed. To summarize these data from the perspective of the number of Ca2+

binding

sites, mutation of β3 residues D119A, S121A, S123A, D217A, and E220A destroyed one

of the two cation binding sites in β3 (95-373). The same effect was also observed when

two β3 sequences located upstream to Q210 were switched with the corresponding

segments of β2 (see Table 2).

Specificity of the two cation binding sites in ββββ3(95-373).

The next series of equilibrium dialysis experiments was designed to examine the

specificity of the two cation binding sites in the recombinant β3 fragments. Aliquots of

β3(95-373) or β3(95-301) were equilibrated with 500 µM 45

Ca2+

in the presence of

increasing concentrations of Mg2+

, ranging from 0 to 100 mM. As shown in Fig. 5, only

one of two cation binding sites occupied by Ca2+

in the β3(95-373) could be displaced by

Mg2+

. The second site appears to be specific for Ca2+

even in the presence of 200-fold

excess of Mg2+

. Only a single cation binding site was present in the β3(95-301) fragment,

and this site could be filled with either Ca2+

or Mg2+

. Thus, deletion of the C-terminal

β3(301-373) segment from the β3(95-373) resulted in a loss/destruction of a cation

binding site with specificity for Ca2+

. In order to further locate these cation binding sites


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within the β3(95-373), several of the mutants described above were used in the

equilibrium dialysis experiments performed in the presence of a saturating concentration

of Mg2+

(Fig. 6). Under such conditions, one of two cation binding sites occupied by

Ca2+

in mutants β3(L128-Q141), β3(K159-P170), β3(C177-C184), and β3(L194-E206)

was displaced by Mg2+

. Thus, these fragments behaved similarly to the wild type β3(95-

373), containing both types of cation binding sites. There was no displacement of Ca2+

by Mg2+

in recombinant fragments bearing the D119A, S121A, S123A and D217A point

mutations. In contrast, Mg2+

almost completely inhibited the binding of Ca2+

to the

E220A, β3(Q210-D217) and β3(Q301-E312) fragments, indicating that these mutations

destroyed the cation binding site specific for Ca2+

.

Effect of mutations on fibrinogen binding of ββββ3(95-373).

The Fg binding activity of the various β3(93-373) mutants was examined in the presence

of Ca2+

ions (0.1 mM and 1.0 mM), utilizing 1 mM EDTA to define the non-specific

binding. In these analyses, 50 nM 125

I-Fg was added to the immobilized recombinant

fragments. Introduction of mutations D119A, S121A, S123A, D217A and E220A

reduced Fg binding to background levels. Based on the binding of Fg, all of the mutants

could be divided into three groups. Group I is represented by L128-Q141 and shows

changes in binding properties similar to those observed with the mutants with single

amino acid substitutions; i.e., nearly complete loss of binding activity. Mutants K159-

P170, C177-C184, and L193-E206 form group II, which showed only slight differences

in their Fg binding characteristics compared to the wild type β3 fragment. Group III

consists of Q210-D217 and Q301-E312; these mutants bound fibrinogen to a similar


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extent at both Ca2+

concentrations, thereby behaving like the shorter β3 (95-301) fragment

(see Fig. 2).


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DISCUSSION

In this study, we have examined the ligand and divalent cation binding properties

of the mid-segment of the β3 subunit, β3(95-373). This segment is shown to bind a major

protein ligand, Fg, of the two β3 integrins, αIIbβ3 and αVβ3, in a divalent cation dependent

manner. β3(95-373) also is shown to harbor not only the one cation binding site predicted

in numerous previous analyses (24,29,59) but also a second cation binding site. These

two cation binding sites display distinct specificities: one site is capable of binding Ca2+

,

Mg2+

and Mn2+

, whereas the second site is specific for Ca2+

. Furthermore, these two

cation binding sites differentially influence ligand binding to the β3 mid-segment: one site

supports ligand binding and the second is suppressive. Thus, these sites fulfill the

characteristics of the ligand competent (LC) and the inhibitory (I) sites, which had been

previously defined on a functional basis (38) but not located within the structure of the

receptor. The existence of two functionally distinct divalent cation binding sites in this

mid-segment necessitates a fundamental rethinking about the relationship between cation

and ligand contact sites in the β3 integrins. Furthermore, since this mid-segment is highly

conserved among integrin β subunits, the conclusions, particularly with regard to the

divalent cation binding sites in this segment, may be applicable to the entire family.

Like all integrins, the two β3 integrins contain several cation binding sites, and

bound divalent ions can act as either effectors or antagonists of ligand binding, [reviewed

in (30)]. There has been considerable discussion in the literature as to the precise

number and location of metal-binding sites in αIIbβ3. In 1984, Brass and Shattil (57) used

the difference in the Ca2+

-binding to the surface of normal and thrombasthenic platelets


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(lack αIIbβ3) to estimate that each αIIbβ3 has two high-affinity (Kd = 9 ± 2 nM) and six

intermediate-affinity (Kd = 0.4 ± 0.1 µM) Ca2+

binding sites. Rivas and Gonzalez-

Rodriguez (34) performed direct equilibrium binding measurements using purified αIIbβ3

and its αIIb and β3 subunits in Triton X-100 and found that isolated αIIbβ3 contained five

Ca2+

binding sites at room temperature, one of high-affinity (Kd of 80 ± 30 nM) and 4

sites of intermediate affinity (Kd of 40 ± 15 µM). After incorporation of αIIbβ3 into

liposomes, the total number of Ca2+

-binding sites did not change, but the Kd of the high

affinity site decreased by an order of magnitude. The isolated αIIb and β3 subunits

contained five intermediate- and two low-affinity Ca2+

binding sites (Kd = 0.2 ± 0.3 mM),

respectively. Binding of 45

Ca2+

to both subunits also was demonstrated in blotting

experiments (34). Equilibrium dialysis experiments were used by Gulino et al (44) to

show that the 4 EF-hand-like structures in recombinant αIIb (171-464) were functional

cation binding sites. Formation of an exchange-inert cation:receptor complex using the in

situ oxidation of cobalt identified five Co3+

binding sites in αVβ3. Thus, overall, our

equilibrium dialysis experiments indicating the presence of one high- and 4-5

intermediate-affinity binding sites for Ca2+

in intact αIIbβ3 are consistent with published

results. Furthermore, our data suggest that two of these sites are located within the mid-

segment of the β3 subunit. The high-affinity cation binding site in αIIbβ3 may correspond

to the one residing in the cytoplasmic tail of αIIb (48) although caution must be exercised

in extrapolating the results obtained with peptides and recombinant fragments to the

intact receptor.

Multiple lines of evidence have implicated the mid-segment of the β3 subunit in

ligand binding to the β3 integrins. Both naturally-occurring and induced mutations


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(24,27,28,60,61) in this segment can abolish Fg binding to αIIbβ3; RGD ligand peptides

are cross-linked to this region of the receptor (9,62), and peptides from this segment can

bind ligands (26,36). Alemany et al. (56) have previously expressed β3(274-368) and

reported that it bound Fg but in a divalent ion independent manner. We also observed

divalent cation independent binding of Fg to β3(95-373) but found that the majority of the

binding to this recombinant fragment was divalent cation dependent. This difference in

results may reflect the particular recombinant fragments used or their folding or could be

a consequence of the high Ca2+

concentration used by Alemany et al. (56), which might

have suppressed the divalent ion dependent component of binding. We previously

reported that a cyclic RGD peptide bound to β3(95-373) in the divalent cation-dependent

manner but a cyclic peptide mimetic of the γ-chain of Fg did not (12). Of note, higher

concentrations of Ca2+

do not inhibit Fg binding to αIIbβ3 (63,64) but do inhibit its

binding to αVβ3 (6); and the binding of Fg to αIIbβ3 requires the γ-chain sequence (65),

whereas its binding to αVβ3 is dependent upon its RGD sequences (66). Thus, the

suppressive effect of Ca2+

on Fg binding to β3(95-373) may be a reflection of the more

prominent role of this mid-segment in ligand binding to αVβ3.

To probe the structural requirements of these two cation binding sites, two series

of mutants were produced and utilized. Substitution of single amino acid in β3(95-373)

which correspond to residues known to coordinate cations in I domains, namely D119,

S121, S123, D217 to A, destroyed the LC cation binding site and reduced Fg binding.

These observations are consistent with the proposal that that the structure of the LC site

in β3(95-373) involves D119xSxS and D217 and is similar to the MIDAS motifs in the

authentic I domain (15,18). The fourth coordination residue is probable neither T182 nor


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T183 residues as mutant β3(C177-C184) retained cation (albeit at reduced affinity) and

Fg binding properties. The lack of essential involvement of these T are consistent with

the data of Lin et al. (29), who analyzed their involvement in the function of β3 and β5

integrins. However, as discussed below, our data are consistent with D251 providing a

fifth coordination site for a cation bound in the β3 MIDAS motif. The second cation

binding site, the I site, requires the presence of 3(301-373) in β3(95-373) and amino acid

residues β3(301-323) are critical to its function. This conclusion is based on the

following observations: (a) deletion of the C-terminal β3(301-373) segment from β3(95-

373) resulted in a loss of the cation binding site specific for Ca2+

; and (b) mutants Q301-

E312 and T311-S323 contain an only single cation binding site that was no longer Ca2+

-

specific; i.e., could be almost completely inhibited by Mg2+

. These data indicate that

β3(301-373) is necessary for the function of the I site although we recognize that the data

do not necessarily confine this site in this specific segment.

In attempt to begin to place these data in a structural framework, we have

developed a molecular model of the mid-segment of β3. Several models for this region of

integrin β3 subunits have been proposed recently (22,67), but we sought to construct one

independent of the previous proposals. Thus, a model of the β3(81-373) was built based

on its homology to known crystal structure of the I domains of α2, αM and αL subunits

(50-52); secondary structural predictions for β3(1-405) using Chou-Fasman, jpeg and

Gor II predictive algorithms (Table 4); and recent epitope mapping of several mAb to the

mid-segment of the β2 subunit (67). The model in Fig. 8 shows the prediction for β3(110-

353) and fits well to the structure of the I-domain present in αM and αL with two

insertions, one between β-strand 2 and 3 and the other between β-strand 4 and α-helix 4.


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The docking of Ca2+

cations within the model for β3(110-353) in Fig. 8 was performed

using an electrostatic surface grid and imposing the mutational data developed in our

study. As expected, the D119xSxS component, together with E220 and D251, was

predicted to be a cation binding site. The predicted distances of the coordination sites in

these residues are indicated in Table 3. All of these coordination sites are at the same

locations as in the MIDAS of the αM I-domain and would correspond to the LC site.

Mutation of D217 also caused a loss in the function of this motif. The basis for this

change can be explained by changes in the salt-bridge coordination between D217 and

R216. Mutation D217A would break this salt-bridge and R216 may then form a salt-

bridge with E220 or hydrogen bond with S121/S123, which would perturb one or more

MIDAS coordinating atoms. A second cation binding site can be predicted to reside in a

distance of 1.27 nm from the MIDAS cation. The coordinating residues comprising this

site, predicted to be the I site, are V310, T311, E312, Q319, G331, V332, L333 and S334

(see Table 3), which are included in the loops and sheets formed by β3(311-315), β3(329-

333) and β3(334-339). These coordinating residues are consistent with the behavior of

mutants β3(Q301-E312) and β3(T311-S323). It should be noted that these two mutants

were among those evaluated by CD spectroscopy and their helical content was the same

as that of the wild-type fragment, suggesting that overall conformation had been

preserved. Based on energies calculated for the two cation sites, the binding of Ca2+

to

the MIDAS is predicted to be stronger than to the I site. These results are also consistent

with location and structure of the two cation sites, with the LC site located in a MIDAS

formed by the multiple helices and strands, while the I site is located within flexible loop

regions, also located on the surface of β3(95-373) (Fig. 8). The flexibility of this loop


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might allow for allosteric effects to be transmitted across the β3 mid-segment. The

docking program used does not allow comparison of Ca2+

to other cations and so the

basis for the different specificities for the two divalent cation binding sites cannot not be

predicted.

A primary strategy used in our mutational analyses was to replace a series of

small segments within β3(95-373) with the corresponding segments from the mouse β2

subunit. Since the β2 integrins do not bind RGD ligands, this approach should perturb the

ligand binding function of the β3 fragment but it also led to the loss of one of the two

divalent cation binding sites. This fortutious outcome implies that the β2 subunit does not

possess the same two cation binding sites within the region corresponding to β3(95-373).

It is likely that the MIDAS site is functional within all the integrin β subunits since the

residues implicated by the single point mutations, DxSxS and the downstream E and D,

are conserved. Thus, it would be the I site that is not functional in the β2 subunit. The

molecular model for the β3 mid-segment that is displayed in Fig. 8 is similar to the one

recently published for β2(75-397) (68) but there are some notable exceptions: 1) the

position of loop 3 between β-strand 2 and 3 is positioned so that the N-terminus of β-

strand 3 and the C-terminus of α-helix-2 are surface accessible; 2) a disulfide bond is

formed between C177 and C184 in loop 3; 3) α-helix-4 resides closer to MIDAS motif;

and 4) loop 7, located between β-strand 4 and α-helix 4 is positioned so that the N-

terminus of helix 4 can be involved in ligand binding consistent with our Fg binding data.

These differences arise in part from the differences in the primary structures of the two β

subunits and in part for the differences in the modeling programs utilized. In the β2


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subunit, the flexible loop that is predicted to form the I site in β3(95-373) is still present

but may not be positioned appropriately to form the I site and also lacks some of the

residues that are predicted to coordinate the Ca2+

in the I site (e.g. E312 is replaced with a

S). Nevertheless, high Ca2+

does suppress ligand binding to the β2 integrins, suggesting

the existence of a functional I site. One possibility is that the I site in β2 integrins may be

associated with the α, more specifically the MIDAS motif within the I-domain, rather

than with the β subunit. Ca2+

and Mn2+

have differential effects on ligand binding to

αMβ2, and the crystal structure of the αMI-domain with Ca2+

or Mn2+

bound in the

MIDAS motif is strikingly different. With regard to the flexible loop region, it would

appear that the coordinating residues for the I site are probably conserved within the β1

subunit. However, such speculations are best left to direct experimentation. The

approaches and the model shown in Fig. 8 provide guide for future mutational and

functional analyses of the mid-segment of the integrin β subunits. 2


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Acknowledgments

This work was supported in part by NIH Grant HL54924 and Grant PAN/NIH - 98 - 326

from the U.S.-Polish Maria Sklodowska-Curie Joint Fund II and by Grant 004/PO4/98

from Polish Committee for Scientific Research.


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Footnotes:

1 Abbreviations used: Fg, fibrinogen; MIDAS, metal ion dependent adhesion site; His-

tag, the MRGSHHHHHHGS sequence at the N-terminus of expressed recombinant

proteins; LC, ligand competent cation binding site; I-, inhibitory cation binding site;

mAb, monoclonal antibody.

2 While this work was under revision, the crystal structure of αVβ3 was published (69).

Two cations may have been present, consistent with our data, but the cation coordination

sites were not entirely consistent with our mutational data. As we know from previous

crystal structures of I(A) domains (70), cations can be coordinated in different

conformers leading to different activation states, and this is one potential explanation for

the apparent differences in results.


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Figure Legends

Fig. 1. Formation of soluble complexes between recombinant β3 fragments and

fibrinogen. Fg (1 mg/ml) was incubated with β3 (93-301) or β3 (95-373) for 4 hr at room

temperature and then the unbound recombinant peptides were removed by centrifugation

in Centricon filters with a 100,000 mol. wt. cut-off. The complexes were washed three

times with 50 mM Tris buffer, pH 7.5, containing 100 mM NaCl, 5 mM CaCl2 and 0.5 M

glucose, followed each time by ultrafiltration and separated by electrophoresis in native

7.5% polyacrylamide gel (panel A) or in 12 % polyacrylamide gels containing SDS under

reducing conditions (panels B). Panel A shows the complexes formed between Fg and

β3(93-301) (lane 1) or β3(95-373) (lane 2). After reduction (Panel B), the three

polypeptide chains, Aα, Bβ, and γ, of Fg and the β3(93-301) (Panel B, lane 4) or β3(95-

373) (panel B, lane 5) are seen. Molecular weight markers are shown in Panel B, lane 3.

Fig. 2. Binding of fibrinogen to β3(93-301) and β3(95-373). 125

I-Fg (50 nM) was added

to microtiter wells coated with β3(93-301) or β3(95-373). The binding was performed in

the presence of CaCl2 (0.1 or 1.0 mM) and EDTA (1 mM). Binding observed in the

presence of EDTA was subtracted and data represent the means ± S.D. of three

determinations expressed in cpm.

Fig. 3. Ca2+

binding to the IIbβ3 and the recombinant fragments of β3 integrin subunit as

a function of the added Ca2+

concentration. Binding of Ca2+

to the purified αIIbβ3 (panel

A) and β3(95-301) or β3(95-373) (panel B) was determined by equilibrium dialysis.


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EGTA was used to regulate the free Ca2+

concentration of the proteins analyzed in 50

mM Tris, 0.14 M NaCl, pH 7.3, containing 50 mM octyl-glucoside. αIIbβ3 and

recombinant β3(95-373) mutants were used at the concentration of 5-10 µM and 30-40

µM. Each point represents the means ± S.D. obtained in three separate experiments

performed in duplicate.

Fig. 4. Mutants of β3(95-373) constructed based upon homology scanning mutagenesis.

The individual homologous segments from the β2 subunit, which does not form RGD-

recognizing integrins, were swapped for the corresponding segment in β3(95-373) (Panel

A). The mutants were separated by chromatography on the chelating Sepharose column,

and their purity assessed by SDS-PAGE followed either by staining with Coomassie blue

(Panel B) or western immunoblotting a rabbit antiserum raised to β3(95-373) (Panel C).

Fig. 5. Inhibition of Ca2+

binding to the recombinant β3(95-301) and β3(95-373)

fragments by Mg2+

. Equilibrium dialysis was performed using 45

Ca2+

at the concentration

(500 µM) sufficient to saturate its binding sites in β3(95-301) and β3(95-373) in the

presence of Mg2+

at concentrations ranging from 0 to 100 mM. Data are expressed in

number of Ca2+

bound to the recombinant fragment and represent the means ± S.D. of

three separate experiments, each performed with duplicates.

Fig. 6. Sensitivity of the Ca2+

binding sites in the β3(95-373) mutants to Mg2+

. The

β3(95-373) mutants were used in equilibrium dialysis at a concentration of 30 - 40 µM.

Binding of 45

CaCl2 (500 µM) was tested in the presence of 100-fold molar excess of


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Mg2+

. Data represents the means ± S.D. of three separate experiments. The closed bars

are the binding of Ca2+

to the mutants, and the open bars are binding in the presence of

Mg2+

.

Fig. 7. Cation-dependent binding of fibrinogen to the β3(95-373) mutants. 125

I-Fg (50

nM) was added to microtiter wells coated with the mutants in the presence of CaCl2 (0.1

mM and 1 mM). Binding observed in the presence of 1 mM EDTA was subtracted, and

values displayed are the means ± S.D. of three determinations.

Fig. 8. Molecular model of the β3(95-380). Molecular modeling was done using

InsightII and Homology Programs on a Silicon Graphics Indigo workstation as

previously described (49). An initial model was constructed by sequence alignment

followed by homology modeling using the crystal structure of the I-domain of αM as a

starting template. Loops were generated to fill gaps in structure. A minimized-energy

conformation was obtained by gradually minimizing the protein structure. Calcium ions

were docked and then the structure was subjected to a series of molecular dynamic

simulations and energy minimizations. The designations of the α-helices and β-strands

are based upon the model developed by Huang et al. (68).


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Table 1

Mutants of β3(95-373) produced by swapping of β2 segments in the corresponding

segments of β3 by homologous scanning mutagenesis. Upper table shows position of

swaps between aligned β3 and β2 sequences. Lower table identifies sequence and

numbering nomenclature of each swap.

β3 95 DDSKNFSIQVRQVEDYPVDIYYLMDLSYSMKDDLWSIQNLGTKLATQMRK 144β2 88 GQAAAFNVTFRRAKGYPIDLYYLMDLSYSMLDDLRNVKKLGGDLLRALNE 137

β3 145 LTSNLRIGFGAFVDKPVSPYMYISPPEALENPCYDMKTTCLPMFGYKHVL 194β2 138 ITESGRIGFGSFVDKTVLPFVNTHP-EKLRNPCPNKEKACQPPFAFRHVL 186

β3 195 TLTDQVTRFNEEVKKQSVSRNRDAPEGGFDAIMQATVCDEKIGWRNDASH 244β2 187 KLTNNSNQFQTEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRN-VTR 235

β3 245 LLVFTTDAKTHIALDGRLAGIVQPNDGQCHVGSDNHYSASTTMDYPSLGL 294β2 236 LLVFATDDGFHFAGDGKLGAILTPNDGRCHL-EDNMYKRSNEFDYPSVGQ 284

β3 295 MTEKLSQK-NINLIFAVTENVVNLYQNYSELIPGTTVGVLSMDSSNVLQL 343β2 285 LAHKLAE-NNIQPIFAVTKRMVKTYEKLTEIIPKSAVGELSEDSSNVVHL 333

β3 344 IVDAYGKIRSKVELEVRDLPEELSLSFNAT 373β2 334 IKNAYNKLSSRVFLDHNALPDTLKVTYDSF 363

Swap Nomenclature

Mutant β3 sequence β2 sequence

β3(F100-Y110) FSIQVRQVEDY FNVTFRRAKGY

β3(L128-Q141) LWSIQNLGTKLATQ LNNVKKLGGDLLQ

β3(K159-P170) KPVSPYMYISPP KTVLPFVNTHPE

β3(C177-C184) CYDMKTTC CPNKEKAC

β3(L194-E206) LTLTDQVTRFNEE LKLTDNSNQFQTE

β3(Q210-D217) QSVSRNRD QLISGNLD

β3(N279-S291) NHYSASTTMDYPS NMYKRSNEFDYPS

β3(Q301-E312) QKNINLIFAVTE ESNIQPIFAVTK

β3(T311-S322) TENVVNLYQNY TKKMVKTYEKLT


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Table 2

Apparent dissociation constants of β3 mutants for Ca2+

ions. The purified mutants of

β3(95-373) were allowed to bind Ca2+

ions in equilibrium dialysis experiments. The

apparent dissociation constants (Kd) and the number of Ca2+

binding sites were estimated

by Scatchard analysis.

No Mutant

Dissociation constant

(Kd) [µM]

Number of sites

(N)

Significance

(p values)

1 β3(95-373) 78 + 12 2.01 + 0.25 -

2 F100-Y110 100 + 25 1.95 + 0.21 >0.02

L128-Q141 300 + 45 1.85 + 0.30 >0.02

K159-P170 328 + 39 1.81 + 0.26 >0.02

C177-C184 320 + 61 1.75 + 0.35 >0.02

L194-E206 450 + 55 2.00 + 0.15 >0.02

Q210- D217 85 + 41 1.15 + 0.21 <0.0001

N279-S291 91 + 19 1.99 + 0.31 >0.02

Q301-E312 200 + 33 0.95 + 0.14 <0.0001

T311-S322 185 + 38 0.89 + 0.16 <0.0001

3 D119A 250 + 60 1.35 + 0.22 <0.0001

S121A 260 + 65 1.25 + 0.17 <0.0001

S123A 200 + 40 0.89 + 0.15 <0.0001

D217A 190 + 25 1.11 + 0.21 <0.0001

E220A 280 + 53 0.75 + 0.18 <0.0001


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Table 3

Distances between the cations and their coordinating β3 atoms as calculated based on the

molecular model presented in Figure 8.

Cation β3 Atom Distance (nm)

MIDAS Cation

Inhibitory Cation

D119:OD1D119:OD2S121:OGS123:OGE220:OE1E220:OE2D251:OD1D251:OD2 V310:OT311:OE312:OE1E312:OE2Q319:OE1G331:OV332:OL333:OS334:OG

2.502.622.644.352.582.712.632.68

2.532.622.602.633.984.312.842.782.62


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Table 4

Alignment and secondary structural prediction for β3 I-domain. The secondary structural

predictions for β3 are displayed above the β3 sequence (S: β-sheet; H: α-helix), and the β-

sheet and α-helical regions of β3 that are homologous to I-domain structures of MAC-1

(1-jlm), are underlined and numbered according to Huang et al (64). Below the β3

sequence, the β-sheet and α-helical regions identified in the crystal structure of αVβ3 are

underlined and number accordingly (65). Residues forming the MIDAS site and the

inhibitory site are highlighted with a dark grey box and a light grey box, respectively.

β1 α1SSS SSSSSS SSSSSSS SSSSSS HHH

B3 81 TQVSPQRIALRLRPDDSKNFSIQVRQVEDYPVDIYYLMDLSYSMKDDLWS 130A A’ B βA bβA’ α1

α1 β2HHHHHHHHHHHHHHH SS SSS

B3 131 IQNLGTKLATQMRKLTSNLRIGFGAFVDKPVSPYMYISPPEALENPCYDM 180α1’ βB

β3 α2 α3B3 181 KTTCLPMFGYKHVLTLTDQVTRFNEEVKKQSVSRNRDAPEGGFDAIMQAT 230

SSSSS HHHHHHHHHH HHHHHHHHβC α2 βC’ α3

β4HHH SSSSSSS SSSS

B3 231 VCDEKIGWRNDASHLLVFTTDAKTHIALDGRLAGIVQPNDGQCHVGSDNH 280α4 βD α310 βD’

α4 β5 α5HHHHHHHHHHH SSSSSS HHHHHHHHH S

B3 281 YSASTTMDYPSLGLMTEKLSQKNINLIFAVTENVVNLYQNYSELIPGTTV 330α5 βE α6 βF

β6 α6SSS HHHHHHHHH SSSSSSS SSSSSSSS

B3 331 GVLSMDSSNVLQLIVDAYGKIRSKVELEVRDLPEELSLSFNATCLNNEVI 380βF α7 C D D’


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Figure 1


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Figure 8


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Papierak, Lidia Michalec, Li Zhang, Thomas A. Haas and Edward F. PlowAleksandra Cierniewska-Cieslak, Czeslaw S. Cierniewski, Kamila Blecka, Malgorzata

subunit{sub}3βIdentification and characterization of two cation binding sites in the integrin

published online January 16, 2002J. Biol. Chem.

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