Identification and Characterization of Two Cation Binding Sites in the Integrin β3 Subunit
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m112388200
ISSN1083-351X
AutoresAleksandra Cierniewska‐Cieslak, Czesław S. Cierniewski, Kamila Blecka, Malgorzata Papierak, Lidia Michalec, Li Zhang, Thomas A. Haas, Edward F. Plow,
Tópico(s)Blood properties and coagulation
ResumoThe midsegment of the β3subunit 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. Whereas 0.1 mmCa2+ 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, whereas β3-(95–301) lacked the Ca2+-specific site. Mutant forms of β3-(95–373) suggested that the LC site is a MIDAS motif involving Asp119, Ser121, Ser123, Asp217, and/or Glu220 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). The midsegment of the β3subunit 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. Whereas 0.1 mmCa2+ 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, whereas β3-(95–301) lacked the Ca2+-specific site. Mutant forms of β3-(95–373) suggested that the LC site is a MIDAS motif involving Asp119, Ser121, Ser123, Asp217, and/or Glu220 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). αIIbβ3 is a typical member of the integrin family of cell adhesion receptors (1.Hynes R.O. Cell. 1987; 48: 549-550Abstract Full Text PDF PubMed Scopus (3104) Google Scholar), 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). 1The abbreviations used are: FgfibrinogenMIDASmetal ion-dependent adhesion siteHis tagthe MRGSHHHHHHGS sequence at the N terminus of expressed recombinant proteinsLC siteligand competent cation binding siteI siteinhibitory cation binding sitemAbmonoclonal antibody 1The abbreviations used are: FgfibrinogenMIDASmetal ion-dependent adhesion siteHis tagthe MRGSHHHHHHGS sequence at the N terminus of expressed recombinant proteinsLC siteligand competent cation binding siteI siteinhibitory cation binding sitemAbmonoclonal antibody Two sets of peptides (HHLGGAKQAGDV, corresponding to the sequence at the C terminus of the Fg γ-chain (2.Kloczewiak M. Timmons S. Hawiger J. Biochem. Biophys. Res. Commun. 1982; 107: 181-187Crossref PubMed Scopus (104) Google Scholar) 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 Ref. 3.Plow E.F. Marguerie G.A. Ginsberg M. Biochem. Pharmacol. 1987; 36: 4035-4040Crossref PubMed Scopus (68) Google Scholar). 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 Refs. 4.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (355) Google Scholar and 5.Byzova T.V. Rabbani R. D'Souza S. Plow E.F. Thromb. Haemostasis. 1998; 80: 726-734Crossref PubMed Scopus (162) Google Scholar). This integrin also exhibits an RGD recognition specificity but shows a much weaker recognition of Fg γ-chain peptides (6.Smith J.W. Ruggeri Z.M. Kunicki T.J. Cheresh D.A. J. Biol. Chem. 1990; 265: 12267-12271Abstract Full Text PDF PubMed Google Scholar). Numerous studies have suggested that binding of macromolecular ligands to αIIbβ3 as well as αVβ3 involves multiple contacts in each subunit (7.D'Souza S.E. Ginsberg M.H. Burke T.A. Plow E.F. J. Biol. Chem. 1990; 265: 3440-3446Abstract Full Text PDF PubMed Google Scholar, 8.D'Souza S.E. Ginsberg M.H. Lam S.C.T. Plow E.F. J. Biol. Chem. 1988; 263: 3943-3951Abstract Full Text PDF PubMed Google Scholar, 9.Smith J.W. Cheresh D.A. J. Biol. Chem. 1988; 263: 18726-18731Abstract Full Text PDF PubMed Google Scholar, 10.Loftus J.C. Smith J.W. Ginsberg M.H. J. Biol. Chem. 1994; 269: 25235-25238Abstract Full Text PDF PubMed Google Scholar, 11.Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem. 1996; 271: 2033-2039Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12.Cierniewski C.S. Byzova T. Papierak M. Haas T.A. Niewiarowska J. Zhang L. Cieslak M. Plow E.F. J. Biol. Chem. 1999; 274: 16923-16932Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 13.Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem. 1999; 274: 4633-4639Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Essential residues for ligand binding to αIIbβ3 reside in two major regions: a midsegment of β3, β3-(95–400), and the amino-terminal aspect of αIIb, αIIb-(1–334) (11.Loftus J.C. Halloran C.E. Ginsberg M.H. Feigen L.P. Zablocki J.A. Smith J.W. J. Biol. Chem. 1996; 271: 2033-2039Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), which contains seven structural repeats (14.Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (386) Google Scholar). The midsegment 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 nine integrin α subunits and play major roles in the ligand binding functions of their integrin heterodimers (15.Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 16.Diamond M.S. Garcia-Aguilar J. Bickford J.K. Corbı́ A.L. Springer T.A. J. Cell Biol. 1993; 120: 1031-1043Crossref PubMed Scopus (468) Google Scholar, 17.Randi A.M. Hogg N. J. Biol. Chem. 1994; 269: 12395-12398Abstract Full Text PDF PubMed Google Scholar, 18.Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 19.Leitinger B. Hogg N. Biochem. Soc. Trans. 1999; 27: 826-832Crossref PubMed Scopus (19) Google Scholar, 20.Oxvig C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2215-2220Crossref PubMed Scopus (122) Google Scholar). The relationship between the conserved β midsegments and α I domains was proposed based upon similarities in their hydropathy profiles, secondary structural predictions, and mutational analyses (18.Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 20.Oxvig C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2215-2220Crossref PubMed Scopus (122) Google Scholar, 21.Goodman T.G. Bajt M.L. J. Biol. Chem. 1996; 271: 23729-23736Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 22.Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A central feature of I domains is a metal ion-dependent adhesion site, a MIDAS motif (15.Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 18.Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 19.Leitinger B. Hogg N. Biochem. Soc. Trans. 1999; 27: 826-832Crossref PubMed Scopus (19) Google Scholar, 23.Qu A. Leahy D.J. Structure. 1996; 4: 931-942Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In MIDAS motifs, three of the five cation coordination sites are provided by a D XS XS sequence and two other coordination sites are provided by oxygenated residues distant in the 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.Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 24.Loftus J.C. O'Toole T.E. Plow E.F. Glass A. Frelinger A.L. Ginsberg M.H. Science. 1990; 249: 915-918Crossref PubMed Scopus (325) Google Scholar, 25.Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). The β3 midsegment does contain a119D XS XS sequence, and these residues also have been implicated in the ligand binding functions of αIIbβ3 (24.Loftus J.C. O'Toole T.E. Plow E.F. Glass A. Frelinger A.L. Ginsberg M.H. Science. 1990; 249: 915-918Crossref PubMed Scopus (325) Google Scholar, 26.Charo I.F. Nannizzi L. Phillips D.R. Hsu M.A. Scarborough R.M. J. Biol. Chem. 1991; 266: 1415-1421Abstract Full Text PDF PubMed Google Scholar, 27.Bajt M.L. Ginsberg M.H. Frelinger III, A.L. Berndt M.C. Loftus J.C. J. Biol. Chem. 1992; 267: 3789-3794Abstract Full Text PDF PubMed Google Scholar, 28.Lanza F. Stierlé A. Fournier D. Morales M. Andre G. Nurden A.T. Cazenave J.-P. J. Clin. Invest. 1992; 89: 1995-2004Crossref PubMed Google Scholar). While there is broad consensus that the midsegment 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.Lin C.K.E. Ratnikov B.I. Tsai P.M. Gonzalez E.R. McDonald S. Pelletier A.J. Smith J.W. J. Biol. Chem. 1997; 272: 14236-14243Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). fibrinogen metal ion-dependent adhesion site the MRGSHHHHHHGS sequence at the N terminus of expressed recombinant proteins ligand competent cation binding site inhibitory cation binding site monoclonal antibody fibrinogen metal ion-dependent adhesion site the MRGSHHHHHHGS sequence at the N terminus of expressed recombinant proteins ligand competent cation binding site inhibitory cation binding site monoclonal antibody The presence of divalent cations, such as Ca2+ and Mg2+, is essential to the integrity of the heterodimeric structure of αIIbβ3 (reviewed in Ref. 30.Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar), its conformational state (31.Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5851) Google Scholar, 32.Gailit J. Ruoslahti E. J. Biol. Chem. 1988; 263: 12927-12932Abstract Full Text PDF PubMed Google Scholar), and the ligand binding functions of this as well as all integrins (33.Kirchhofer D. Grzesiak J. Pierschbacher M.D. J. Biol. Chem. 1991; 266: 4471-4477Abstract Full Text PDF PubMed Google Scholar). αIIbβ3has approximately five Ca2+-binding sites, falling into at least two distinct affinity classes as estimated by equilibrium dialysis (34.Rivas G.A. Gonzalez-Rodriguez J. Biochem. J. 1991; 276: 35-40Crossref PubMed Scopus (51) Google Scholar) and Tb3+ luminescence spectroscopy (35.Cierniewski C.S. Haas T.A. Smith J.W. Plow E.F. Biochemistry. 1994; 33: 12238-12246Crossref PubMed Scopus (37) Google Scholar). 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.D'Souza S.E. Haas T.A. Piotrowicz R.S. Byers-Ward V. McGrath D.E. Soule H.R. Cierniewski C.S. Plow E.F. Smith J.W. Cell. 1994; 79: 659-667Abstract Full Text PDF PubMed Scopus (204) Google Scholar). Dissection of the ligand binding reaction into association and dissociation steps and subsequent (37.Smith J.W. Piotrowicz R.S. Mathis D. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar) surface plasmon resonance experiments (13.Hu D.D. White C.A. Panzer-Knodle S. Page J.D. Nicholson N. Smith J.W. J. Biol. Chem. 1999; 274: 4633-4639Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 38.Hu D.D. Barbas III, C.F. Smith J.W. J. Biol. Chem. 1996; 271: 21745-21751Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) 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). Taking into consideration the significant role of the midsegment 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. αIIbβ3was isolated from outdated platelets by RGD affinity chromatography (39.Pytela R. Pierschbacher M.D. Ginsberg M.H. Plow E.F. Ruoslahti E. Science. 1986; 231: 1559-1562Crossref PubMed Scopus (661) Google Scholar, 40.Lam S.C.T. Plow E.F. Smith M.A. Andrieux A. Ryckwaert J.-J. Marguerie G. Ginsberg M.H. J. Biol. Chem. 1987; 262: 947-950Abstract Full Text PDF PubMed Google Scholar). Briefly, platelets were lysed in buffer containing 10 mm HEPES, 150 mm NaCl, 1 mmCaCl2, 1 mm MgCl2, 0.1 mm leupeptin, 10 mm N-ethylmaleimide, 1 mm phenylmethanesulfonyl fluoride, and 50 mm octyl glucoside, pH 7.3, centrifuged for 1 h at 30,000 × g, and applied onto a GRGDSPK-Sepharose column (12 × 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 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.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207048) Google Scholar). Prior to ligand binding experiments, samples of αIIbβ3 were dialyzed against 0.02m HEPES buffer, pH 7.3, containing 0.15m NaCl, 25 mm octyl glucoside, and selected divalent cations. Two recombinant β3 fragments, β3-(95–301) and β3-(95–373), were produced. Each contains the Val107–Ser292 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 BamHI and XhoI restriction sites was generated using the following primers: 5′-CTCCGCCTGggatccGATGATTCGAAG-3′ (upper primer) and 5′-CTCATTctcgagTCAGGTGGCATTGAAGGA-3′ (lower primer). To express β3-(95–301), 5′-CTCAATctcgagTCATTTCTGGGATAGCTTCTCAG-3′ replaced the lower primer. The PCR products were digested with BamHI and XhoI restriction enzymes and inserted into pRSETa (Invitrogen) for expression in Escherichia coli (DH5α; Invitrogen) as His tag fusion proteins 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 (Amersham Biosciences, Inc.), loaded with nickel ions according to the manufacturer's instructions. Recombinant β3-(95–301) or β3-(95–373) fragments were eluted with 0.02 m Tris-HCl, pH 7.9, containing 1.0m imidazole, 0.5 m NaCl, and 6 murea. Refolding of β3-(95–301) and β3-(95–373) was performed by dialysis of dilute protein solutions (100–200 μg/ml) against 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. Afterward, 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 0.14m 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). 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 β3 fragments were produced. In the first set, individual segments from β3-(95–373) were replaced with the homologous segments from the integrin β2subunit. Nine mutants designated β3(F100-Y110), β3(L128-Q141), β3(K159-P170), β3(C177-C184), β3(L194-G206), 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 as follows: D119A (5′-ggacatctactacttgatggcactgtcttactccatg-3′ and 5′-catggagtaagacagtgccatcaagtagtagatgtcc-3′); S121A (5′-cttgatggacctggcatactccatgaaggatgatctg-3′ and 5′-cagatcatccttcatggagtatgccaggtccatcaag-3′), S123A (5′-ggacctgtcttacgcaatgaaggatgatctgtgg-3′ and 5′-ccacagatcatccttcattgcgtaagacagctac-3′), D217A (5′-gtcacggaaccgagcagccccagagggtggc-3′ and 5′-gccaccctctggggctgctcggttccgtgac-3′), and E220A (5′-ccgagatgccccagcaggtggctttgatgccatc-3′ and 5′-gatggcatcaaagccacctgctggggcatctcgg-3′). All mutations were confirmed by dideoxy sequencing and were subsequently subcloned into the pRSETa vector for expression. Human Fg was purified and characterized as described previously (42.Cierniewski C.S. Budzynski A.Z. J. Biol. Chem. 1987; 262: 13896-13901Abstract Full Text PDF PubMed Google Scholar). The binding of Fg to immobilized αIIbβ3, β3-(95–301), β3-(95–373), or its mutants was performed as described for intact receptor (35.Cierniewski C.S. Haas T.A. Smith J.W. Plow E.F. Biochemistry. 1994; 33: 12238-12246Crossref PubMed Scopus (37) Google Scholar). 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 postcoated with 4% bovine serum albumin overnight at 4 °C. In binding experiments, 20 μl of 125I-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 h 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 enzyme-linked immunosorbent assay 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 β3subunit (0.2 mg/ml) were mixed in a total volume of 4 ml of 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 h at room temperature, the mixtures were concentrated by centrifugation in 100 K Centricon tubes (Millipore Corp., Bedford, MA) and washed three times, each time followed by a concentration step in the Centricon tubes. The retentates were analyzed by electrophoresis on either nondenaturing 7.5% acrylamide gels (43.Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10471) Google Scholar) or 12% acrylamide gels containing SDS and 2-mercaptoethanol (41.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207048) Google Scholar). 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 mmTris, 0.14 m NaCl, pH 7.3, containing 50 mmoctyl glucoside (the equilibrium buffer). Before use, glassware, plasticware, and dialysis tubing were treated as described by Gulino et al. (44.Gulino D. Boudignon C. Zhang L.Y. Concord E. Rabiet M.J. Marguerie G. J. Biol. Chem. 1992; 267: 1001-1007Abstract Full Text PDF PubMed Google Scholar). Total Ca2+ concentrations were determined by spectrofluorimetry, using Fura-2 and Ca2+standard solutions from Molecular Probes, Inc. (Eugene, OR).45Ca2+ was measured in a scintillation counter (Beckman LS6000LL). Below 10 μm, the required free Ca2+ concentration was adjusted by the addition of 7 mm EGTA, as calculated according to Fabiato (45.Fabiato A. Methods Enzymol. 1988; 157: 378-417Crossref PubMed Scopus (974) Google Scholar) 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 h at 22 °C against the equilibrium buffer containing enough EGTA to reduce the free Ca2+ to below 1 nm as described (34.Rivas G.A. Gonzalez-Rodriguez J. Biochem. J. 1991; 276: 35-40Crossref PubMed Scopus (51) Google Scholar). Equilibrium dialysis experiments were then performed as follows. Various amounts of 10 mm CaCl2 were added in the equilibrium buffer containing 45Ca2+ (3 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+.45CaCl2 (0.48 μCi) was injected into one half-cell, and the proteins were injected into the other one. After dialysis for 24 h at 21 °C, aliquots were removed for45Ca 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 and 30–40 μm, respectively. To test the effects of Mg2+ on Ca2+ binding, the equilibrium dialysis was performed in the presence of 45CaCl2 (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. The CD spectra of β3-(95–301), β3-(95–373), and various mutants were measured in the 200–260-nm range using a CD6 JOVIN YVON spectropolarimeter at protein concentrations of 80 μmusing 0.1-mm path length cells. The secondary structural content of the fragments was estimated by published protocols (46.Chen Y.H. Yang J.T. Martinez H.M. Biochemistry. 1972; 11: 4120-4131Crossref PubMed Scopus (1904) Google Scholar, 47.Sreerama N. Woody R.W. J. Mol. Biol. 1994; 242: 497-507PubMed Google Scholar, 48.Haas T.A. Plow E.F. J. Biol. Chem. 1996; 271: 6017-6026Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Na125I (specific activity 15–17 μCi of 125I/mg of iodine) from Amersham Biosciences was used for radioiodination. Fg was labeled using IODO-GEN (Pierce). The iodinated protein was separated from free Na125I by gel filtration on a Bio-Gel P2 column (Bio-Rad). The specific radioactivity of the 125I-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 2 weeks before use. 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 work station as previously described (49.Haas T.A. Plow E.F. Protein Eng. 1997; 10: 1395-1405Crossref PubMed Scopus (42) Google Scholar). The I domain structures of αL (CD11a) 1zon (50.Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar); αM (CD11b), 1jlm (51.Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar); and α2, 1aox (52.Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) were structurally aligned based on homology, and the sequence of β2-(75–397) was then aligned to these known structures as previously described (49.Haas T.A. Plow E.F. Protein Eng. 1997; 10: 1395-1405Crossref PubMed Scopus (42) Google Scholar). 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. Monoclonal antibody (mAb) P37 was obtained from Dr. Rodriguez-Gonzalez and reacts with β3-(101–109) (53.Calvete J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar), and mAb 25E11 was originally described by Burns et al.(54.Burns G.F. Cosgrove L. Triglia T. Beal J.A. Lopez A.F. Nerkmeisler J.A. Begley C.G. Haddad A.P. d'Apice A.J.F. Vadas M.A. Cawley J.C. Cell. 1986; 45: 269-280Abstract Full Text PDF PubMed Scopus (42) Google Scholar) and was purchased from Chemicon (Temecula, CA) as mAb 1957. A polyclonal antibody was raised to β3-(95–373) by immunizing rabbits with the purified recombinant fragment. All of 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.Zar J.H. Biostatistical Analysis. 2nd Ed. Prentice-Hall, Englewood Cliffs, NJ1984Google Scholar) were employed to assess the significance of differences (pvalues) among groups. 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 E. coli as His tag fusion proteins, purified on nickel chelating columns in 6 m 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 β3fragments reacted with mAb 937 to β3-(101–109) (53.Calvete J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar) and mAb 25E11 to β3-(237–248) (54.Burns G.F. Cosgrove L. Triglia T. Beal J.A. Lopez A.F. Nerkmeisler J.A. Begley C.G. Haddad A.P. d
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