The Crystal Structure of Placental Growth Factor in Complex with Domain 2 of Vascular Endothelial Growth Factor Receptor-1
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m313237200
ISSN1083-351X
AutoresHans W. Christinger, Germaine Fuh, Abraham M. de Vos, Christian Wiesmann,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoPlacental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and plays an important role in pathological angiogenic events. PlGF exerts its biological activities through binding to VEGFR1, a receptor tyrosine kinase that consists of seven immunoglobulin-like domains in its extracellular portion. Here we report the crystal structure of PlGF bound to the second immunoglobulin-like domain of VEGFR1 at 2.5 Å resolution and compare the complex to the closely related structure of VEGF bound to the same receptor domain. The two growth factors, PlGF and VEGF, share a sequence identity of ∼50%. Despite this moderate sequence conservation, they bind to the same binding interface of VEGFR1 in a very similar fashion, suggesting that both growth factors could induce very similar if not identical signaling events. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and plays an important role in pathological angiogenic events. PlGF exerts its biological activities through binding to VEGFR1, a receptor tyrosine kinase that consists of seven immunoglobulin-like domains in its extracellular portion. Here we report the crystal structure of PlGF bound to the second immunoglobulin-like domain of VEGFR1 at 2.5 Å resolution and compare the complex to the closely related structure of VEGF bound to the same receptor domain. The two growth factors, PlGF and VEGF, share a sequence identity of ∼50%. Despite this moderate sequence conservation, they bind to the same binding interface of VEGFR1 in a very similar fashion, suggesting that both growth factors could induce very similar if not identical signaling events. Angiogenesis, the process of new blood vessel formation, is a complex process that involves a number of different growth factors. It is essential for a variety of physiologically important events such as embryogenesis, wound healing, and tissue repair but is also critical in a number of diseases such as tumor progression, psoriasis, rheumatoid arthritis, and diabetic retinopathy (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7153) Google Scholar, 2Ferrara N. Curr. Opin. Biotechnol. 2000; 11: 617-624Crossref PubMed Scopus (351) Google Scholar). Consequently, the molecules that induce or mediate angiogenic events are potentially important targets for the treatment of these diseases. VEGF-A (or VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; PlGF, placental growth factor; BTP, bis-Tris propane (1,3-bis[tris(hydroxymethyl)methylamino]propane); r.m.s.d., root mean square deviation.1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; PlGF, placental growth factor; BTP, bis-Tris propane (1,3-bis[tris(hydroxymethyl)methylamino]propane); r.m.s.d., root mean square deviation. the most important inducer of angiogenesis, is also the founding member of the family of vascular endothelial growth factors (3Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K.S. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3003) Google Scholar). This family of structurally and functionally related growth factors includes VEGF-A (VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF). The members of the VEGF family are responsible for an array of angiogenic, vasculogenic, and lymphangiogenic processes and mediate their function mainly through differential binding to the three homologous receptor tyrosine kinases VEGFR1 (also called flt-1), VEGFR2 (or KDR), and VEGFR3. These receptors are a subclass of the platelet-derived growth factor (PDGF) receptor family, but whereas the extracellular domain of the PDGF receptors have five Ig-like domains, the ectodomains of the VEGFRs contain a total of seven Ig-like domains. The extracellular portion of all receptor tyrosine kinases is connected to the intracellular tyrosine kinase domain via a single transmembrane helix. VEGF, the most potent angiogenic factor of the VEGF family, signals through binding to VEGFR1 and VEGFR2. A number of studies have identified VEGFR2 as the key signaling receptor mediating the proliferative effects of VEGF (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7153) Google Scholar, 3Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K.S. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3003) Google Scholar). The role of VEGFR1, the only tyrosine kinase receptor for PlGF, is still less well understood. Mice devoid of VEGFR1 develop endothelial cells but die because of severe disorganization of the vascular system (4Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2184) Google Scholar). In contrast, mice that express only a tyrosine kinase-deficient variant of the receptor are viable and do not display impaired embryogenesis (5Hiratsuka S. Minowa O. Kuno J. Noda T. Shibuya M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9349-9354Crossref PubMed Scopus (882) Google Scholar). PlGF is specific for VEGFR1, and studies of PlGF isoforms showed that it has only limited mitogenic activity but that it augments and potentiates the activity of low concentrations of VEGF in vitro and in vivo (6Park J.E. Chen H.H. Winer J. Houck K.A. Ferrara N. J. Biol. Chem. 1994; 269: 25646-25654Abstract Full Text PDF PubMed Google Scholar). Therefore it has been suggested that VEGFR1 act as a nonsignaling reservoir for VEGF during development or at least, under certain conditions, as a decoy receptor (7Ferrara N. Am. J. Physiol. 2001; 280: C1358-C1366Crossref PubMed Google Scholar). Recent studies showed that VEGFR1 is expressed not only on vascular endothelial cells but is also present on the surface of pluripotent stem cells. PlGF is specific for VEGFR1 and can reconstitute hematopoiesis by recruiting these stem cells from the bone marrow (8Hattori K. Heissig B. Wu Y. Dias S. Tejada R. Ferris B. Hicklin D.J. Zhu Z. Bohlen P. Witte L. Hendrikx J. Hackett N.R. Crystal R.G. Moore M.A. Werb Z. Lyden D. Rafii S. Nat. Med. 2002; 8: 841-849Crossref PubMed Scopus (554) Google Scholar). It was further shown that selective activation of VEGFR1 induces proliferation of hepatocytes rather than endothelial cells (9LeCouter J. Moritz D.R. Li B. Phillips G.L. Liang X.H. Gerber H.P Hillan K.J. Ferrara N. Nature. 2003; 299: 890-893Google Scholar). Interestingly, PlGF is not required for embryogenic angiogenesis, as mice that lack PlGF are healthy and unaffected by the mutation (10Carmeliet P. Moons L. Luttun A. Vincenti V. Compernolle V. De Mol M. Wu Y. Bono F. Devy L. Beck H. Scholz D. Acker T. DiPalma T. Dewerchin M. Noel A. Stalmans I. Barra A. Blacher S. Vandendriessche T. Ponten A. Eriksson U. Plate K.H. Foidart J.M. Schaper W. Charnock-Jones D.S. Hicklin D.J. Herbert J.M. Collen D. Persico M.G. Nat. Med. 2001; 7: 575-583Crossref PubMed Scopus (1364) Google Scholar). However, PlGF-deficient mice display attenuated responses to VEGF in pathological angiogenesis (11Luttun A. Brusselmans K. Fukao H. Tjwa M. Ueshima S. Herbert J.M. Matsuo O. Collen D. Carmeliet P. Moons L. Biochem. Biophys. Res. Commun. 2002; 295: 428-434Crossref PubMed Scopus (77) Google Scholar), and PlGF is necessary for angiogenic events in adult tissues during ischemia, inflammation, wound healing, and cancer (10Carmeliet P. Moons L. Luttun A. Vincenti V. Compernolle V. De Mol M. Wu Y. Bono F. Devy L. Beck H. Scholz D. Acker T. DiPalma T. Dewerchin M. Noel A. Stalmans I. Barra A. Blacher S. Vandendriessche T. Ponten A. Eriksson U. Plate K.H. Foidart J.M. Schaper W. Charnock-Jones D.S. Hicklin D.J. Herbert J.M. Collen D. Persico M.G. Nat. Med. 2001; 7: 575-583Crossref PubMed Scopus (1364) Google Scholar). Antibodies against VEGFR1 suppress neovascularization in tumors and ischemic retina, angiogenesis, and inflammatory joint destruction in autoimmune arthritis (12Luttun A. Tjwa M. Moons L. Wu Y. Angelillo-Scherrer A. Liao F. Nagy J.A. Hooper A. Priller J. De Klerck B. Compernolle V. Daci E. Bohlen P. Dewerchin M. Herber J.M. Fava R. Matthys P. Carmeliet G. Collen D. Dvorak H.F. Hicklin D.J. Carmeliet P. Nat. Med. 2002; 8: 831-840Crossref PubMed Scopus (931) Google Scholar). These findings make PlGF and its receptor, VEGFR1, potentially attractive targets for modulation of inflammation and angiogenesis. All members of the VEGF family are secreted dimeric glycoproteins. As a result of alternative splicing events, PlGF and VEGF appear in a number of isoforms. So far three isoforms of mature human PlGF have been reported (13Maglione D. Guerriero V. Viglietto G. Ferraro M.G. Aprelikova O. Alitalo K. Del Vecchio S. Lei K.J. Chou J.Y. Persico M.G. Oncogene. 1993; 8: 925-931PubMed Google Scholar, 14Cao Y. Ji W.-R. Qi P. Rosin A. Cao Y. Biochem. Biophys. Res. Commun. 1997; 235: 493-498Crossref PubMed Scopus (153) Google Scholar). The shortest isoform of PlGF, PlGF-1, comprises 131 residues in its mature form and, like the corresponding isoform VEGF121 of VEGF, entails the receptor-binding domain. PlGF-2 has an insertion of 21 residues near its C terminus and like the longer VEGF-isoforms is able to bind heparin. PlGF-3 has an insertion of 72 amino acids near the C terminus of PlGF-1 but, unlike PlGF-2, does not bind to heparin (14Cao Y. Ji W.-R. Qi P. Rosin A. Cao Y. Biochem. Biophys. Res. Commun. 1997; 235: 493-498Crossref PubMed Scopus (153) Google Scholar). Although the structures of the VEGF (15Muller Y.A. Christinger H.W. Keyt B.A. de Vos A.M. Structure. 1997; 5: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and PlGF (16Iyer S. Demetres D. Leonidas G. Swaminathan J. Maglione D. Battisti M. Tucci M Persico G. Acharya K.R. J. Biol. Chem. 2001; 276: 12153-12161Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) receptor-binding domains in their free forms have been reported previously, only the complex between VEGF and VEGFR1 has been available until now (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Here we describe the crystal structure of the minimal ligand binding fragment of VEGFR1 in complex with the receptor-binding domain of PlGF and compare it with the complex between VEGFR1-d2 and VEGF. The structure and comparison of both complexes should shed light on the differences in the downstream effects of VEGFR1 activation via PlGF and VEGF (18Autiero M. Waltenberger J. Communi D. Kranz A. Moons L. Lambrechts D. Kroll J. Plaisance S. De Mol M. Bono F. Kliche S. Fellbrich G. Ballmer-Hofer K. Maglione D. Mayr-Beyrle U. Dewerchin M. Dombrowski S. Stanimirovic D. Van Hummelen P. Dehio C. Hicklin D.J. Persico G. Herbert J.M. Communi D. Shibuya M. Collen D. Conway E.M. Carmeliet P. Nat. Med. 2003; 9: 936-943Crossref PubMed Scopus (640) Google Scholar). Expression, Refolding, and Purification—VEGFR constructs were expressed, purified, and tested for functional integrity as described (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 19Fuh G. Li B. Crowley C. Cunningham B. Wells J.A. J. Biol. Chem. 1998; 273: 11197-11204Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). VEGFR-d1–7 and -d23 were expressed in mammalian cell by transient transfection (19Fuh G. Li B. Crowley C. Cunningham B. Wells J.A. J. Biol. Chem. 1998; 273: 11197-11204Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), and VEGFR-d2 was expressed in Escherichia coli and refolded as reported (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). A construct comprising residues 19–116 of PlGF-2, corresponding to the receptor-binding domain, was expressed as insoluble protein in E. coli. Inclusion bodies were isolated by passing homogenized cells in 20 mm Tris-HCl (pH 7.5) through a French pressure cell and centrifuging the homogenate for 15 min at 4000 × g. The inclusion bodies were resuspended and the centrifugation repeated. The pellet, consisting primarily of PlGF, was dissolved in 6 m urea, 20 mm Tris-HCl, pH 7.5, and stirred for 1 h in 20 mm dithiothreitol. The protein solution was diluted to a concentration of 0.75 mg/ml and then dialyzed against 20 mm Tris-HCl, pH 8.0, 1 mm cysteine at 4 °C for 24 h. Refolded PlGF was purified in consecutive chromatography steps using ion exchange (Q-Sepharose, Amersham Biosciences), hydrophobic interaction (Phenyl Toyopearl, Tosohaas), and size exclusion (S-200, Amersham Biosciences). The purity of the protein was assessed by SDS-PAGE and mass spectrometry. Purified PlGF was mixed in a 1:2 molar ratio with VEGFR1-d2. The resulting complex was purified using size exclusion chromatography (S-200, Amersham Biosciences) and concentrated to 6.5 mg/ml. The composition of the complex was confirmed by size exclusion chromatography and reverse phase HPLC (Vydac, 214 nm). Binding Assays—For BIAcore competition assays, VEGF8–109 was conjugated at a high density of 800–1000 response units (RU) to a B1 BIAcore chip using an amine coupling kit (BIAcore, Piscataway, NJ). First, the concentrations of a VEGFR1 fragment that produced a signal on a VEGF-conjugated chip over a blank chip in the range of 30 to 70 response units in a 2-min injection at 30 μl/min were determined. Approximately 0.1–0.15 nm VEGFR1-d1–7 or -d23 and 2 nm VEGFR1-d2 were chosen for the binding experiments and incubated with increasing concentrations of PlGF and VEGF at 4 °C overnight. The mixtures were then injected onto the chip where unbound forms of the VEGFR1 fragment were captured on the VEGF-coated chip. The amount of captured RU was plotted against the concentrations of VEGF or PlGF in the initial incubation stage, and IC50 levels were determined. This assay was restricted by the lowest concentrations of VEGFR1 fragments sufficient to produce a reasonable signal. In these instances we report the affinity derived from other assays such as radioimmunoreceptor competition binding assays or biotinylated protein enzyme-linked immunosorbent assays with a more sensitive readout as described previously (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Crystallization—Crystals were grown by vapor diffusion at room temperature using the hanging drop method. Crystallization buffer containing 25% polyethylene glycol 4000, 0.1 m ammonium sulfate, 0.02% NaN3 and 0.1 m bis-Tris propane (BTP), pH 6.5, was mixed with an equal volume of protein solution. The resulting crystals belonged to space group P212121 with cell dimensions of a = 54.25 Å, b = 71.97 Å, c = 115.34 Å and with 1 complex/asymmetric unit. Crystals were dipped into a drop containing reservoir plus 10% glycerol and then flash-frozen in liquid nitrogen. Data Collection, Processing, Phasing, and Refinement—Data were collected at Stanford Synchrotron Radiation Laboratory to 2.45 Å resolution and processed using the HKL package (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38246) Google Scholar). The structure was solved by molecular replacement using the program AMoRe (21Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar) and the crystal structure of VEGF in complex with VEGFR1-d2 (Protein Data Bank code 1FLT (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar)). Using data from 12 to 6 Å yielded a clear solution in the rotation and translation functions, and the best solution was subjected to rigid body refinement using the program X-PLOR (22Brünger A.T. X-PLOR Manual, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar). Alternate rounds of model building with the program O (23Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar) and further refinement with the program Refmac (24Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19667) Google Scholar) resulted in the final model with an Rfree and Rcryst of 26.0 and 19.8% for all data between 25 and 2.45 Å resolution, respectively. Affinity of the PlGF Binding Domain to VEGFR1—Based on sequence alignments and the crystal structure of VEGF in complex with VEGFR1-d2 (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar) a construct that included only the minimal receptor-binding domain of PlGF was designed. The resulting protein, PlGF19–116, consists of residues 19–116. VEGFR1 consists of seven immunoglobulin-like domains in its extracellular portion and binds to PlGF with sub-nanomolar affinity (6Park J.E. Chen H.H. Winer J. Houck K.A. Ferrara N. J. Biol. Chem. 1994; 269: 25646-25654Abstract Full Text PDF PubMed Google Scholar). To establish the minimal ligand binding domain toward PlGF, we tested three constructs of VEGFR1 entailing the entire ectodomain (VEGFR1-d1–7), domains 2 and 3 (VEGFR1-d2–3), and domain 2 only (VEGFR1-d2) in Biacore competition assays. In these assays, the longest construct, entailing the entire ectodomain of the receptor, binds PlGF19–116 with an IC50 of 0.5 nm. The affinity of PlGF19–116 toward the VEGFR1-d2-3 construct is only about 2-fold weaker, whereas VEGFR1-d2, the shortest construct tested, bound to the ligand with an IC50 of about 270 nm and therefore binds PlGF19–116 more than 500-fold weaker than the full-length receptor (Table I).Table IBinding affinitiesIC50aBinding affinities were measured as IC50 using a solution binding assay coupled with BIAcore competition capturing of unbound VEGFR1 as described under "Experimental Procedures." Binding affinities of VEGFR1 fragments for VEGF-(8-109) were higher than PlGF and were measured with other assay formats because BIAcore assays were restricted by limited accuracy at that rangeVEGFR1-d2VEGFR1-d2-3VEGFR1-d1-7nMVEGF-(8-109)3.00.141.3bEnzyme-linked immunosorbent assay competition assay using biotinylated VEGF (17)0.054cData were derived from a radioimmunoreceptor competition binding assay as described in Ref. 17, using iodinated VEGF as the tracer0.024cData were derived from a radioimmunoreceptor competition binding assay as described in Ref. 17, using iodinated VEGF as the tracerPlGF-(19-116)2751.10.435a Binding affinities were measured as IC50 using a solution binding assay coupled with BIAcore competition capturing of unbound VEGFR1 as described under "Experimental Procedures." Binding affinities of VEGFR1 fragments for VEGF-(8-109) were higher than PlGF and were measured with other assay formats because BIAcore assays were restricted by limited accuracy at that rangeb Enzyme-linked immunosorbent assay competition assay using biotinylated VEGF (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar)c Data were derived from a radioimmunoreceptor competition binding assay as described in Ref. 17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, using iodinated VEGF as the tracer Open table in a new tab Quality of the Model—The crystal structure of the PlGF·VEGFR1-d2 complex was determined at 2.5 Å resolution. The asymmetric unit contains one full complex comprising two molecules of VEGFR1-d2 bound to a PlGF homodimer, one molecule of BTP, and 161 solvent molecules. The model of PlGF comprises residues 22–115 for one molecule and residues 21–115 for the second (residue numbers refer to the sequence of the mature protein and differ by 1 compared with the numbering used by Iyer et al. (16Iyer S. Demetres D. Leonidas G. Swaminathan J. Maglione D. Battisti M. Tucci M Persico G. Acharya K.R. J. Biol. Chem. 2001; 276: 12153-12161Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar)). Both copies of VEGFR1-d2 contain residues 133–224. The structure was refined to an R-value of 19.4% (Rfree 26.0%) using all reflections between 20 and 2.45 Å resolution (Table II). Of the 323 non-glycine and non-proline residues, 89.2% have their main chain torsion angles in the "most-favorable" and 10.5% in the "additionally allowed" regions of the Ramachandran plot (25Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). A single residue lies in the "generously allowed" region.Table IIData collection and refinement statisticsData collection Resolution (Å)25-2.45 (2.54-2.45)aNumbers in parentheses refer to the highest resolution shell RsymbRsym = Σ | I - 〈I〉 | /ΣI. 〈I〉 is the average intensity of symmetry-related observations of a unique reflection0.046 (0.161)aNumbers in parentheses refer to the highest resolution shell No. of observations72,785 Unique reflections17,048 Completeness (%)99.0 (99.2)aNumbers in parentheses refer to the highest resolution shellRefinement Resolution (Å)20-2.45 No. of reflections15482 Final RcR = Σ | Fo - Fc | /ΣFo. Rfree is calculated as R but for 10% of the reflections excluded from all refinement, Rfree (F > 0)0.198, 0.260 No. of residues373 No. of solvent molecules161 No. of non-H atoms3164 r.m.s.d. bonds (Å)0.013 r.m.s.d. angles (°)1.6a Numbers in parentheses refer to the highest resolution shellb Rsym = Σ | I - 〈I〉 | /ΣI. 〈I〉 is the average intensity of symmetry-related observations of a unique reflectionc R = Σ | Fo - Fc | /ΣFo. Rfree is calculated as R but for 10% of the reflections excluded from all refinement Open table in a new tab Overall Structure of PlGF and VEGFR1-d2—The overall architecture of PlGF in complex with VEGFR1-d2 strongly resembles the complex between VEGF and same receptor fragment. The center of the complex is formed by the PlGF homodimer. Both receptor fragments are bound to the distant poles of this dimer giving the entire complex the dimensions 85 × 40 × 35 Å (Fig. 1). The overall structure of the receptor-binding fragment of PlGF in complex with VEGFR1-d2 is very similar to previously reported structures of PlGF or VEGF (15Muller Y.A. Christinger H.W. Keyt B.A. de Vos A.M. Structure. 1997; 5: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 16Iyer S. Demetres D. Leonidas G. Swaminathan J. Maglione D. Battisti M. Tucci M Persico G. Acharya K.R. J. Biol. Chem. 2001; 276: 12153-12161Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Like all members of the cystine knot family of growth factors, the PlGF monomer has an elongated shape with two pairs of twisted, antiparallel, two-stranded β-sheets in its central portion (strands A, B, C, and D) and the characteristic cystine knot motif, as well as the N and C termini on one end of the molecule (26Sun P.D. Davies D.R. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 269-291Crossref PubMed Google Scholar). The cystine knot is formed by three disulfide bridges. In this motif, two disulfide bridges together with the protein backbone form a closed ring that is penetrated by the third disulfide bridge. Like the related VEGF, PlGF has an N-terminal helix (α1). The protein segment connecting strands A and B includes residues 43–58 and contains a single turn of α-helix (α2) as well as a short additional β-strand (A′) that forms hydrogen bonds with strand C. The hydrogen bonding pattern between strand C and D is interrupted so that strand D is broken into two shorter β-strands, named strands D and D′. The BC loop and the CD loops are both relatively short and span residues 65–74 and 91–97, respectively (Fig. 1). In the biologically active dimer, two PlGF monomers are assembled in an antiparallel manner and are covalently connected through the formation of two disulfide bonds between residues Cys59 and Cys68. This homodimer, which has a rather unusual shape, can be described as a curved sheet that is about 70 Å long and 35 Å wide but less than 15 Å thick in its central portion (Fig. 1). A total of 2700 Å2 is buried in the interface between the two PlGF monomers. A large portion of this interface is formed by the N-terminal helices of PlGF, which pack on top of the respective other dimer within the complex and thus stabilize the dimeric assembly. The overall architecture of the complex between ligand and receptor is dictated by the internal 2-fold symmetry of the PlGF dimer. The two receptor fragments of VEGFR1 bind on the opposing edges of the PlGF dimer with the closest distance between any two atoms between both receptor fragments of about 30 Å. VEGFR1-d2 is a member of the I-set of Ig-like domains (17Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Generally, Ig-like domains consist of about 100 residues, which form two β-sheets that fold against each other to form a β-sandwich. In VEGFR1-d2 one β-sheet is formed by strands A′,G,F,C,andC′ and the other contains strands B, E, and D. One disulfide bond forms part of the hydrophobic core and connects strands B and F, thus stabilizing the fold of the domain. The two halves of the complex are almost identical and superimposing the entire complex onto itself according to the internal 2-fold symmetry results in an r.m.s.d. of 0.75 Å for 372 Cα positions with the largest deviations occurring in regions that are also flexible in the unbound VEGFR1-d2 (27Starovasnik M.A. Christinger H.W. Wiesmann C. Champe M.A. de Vos A.M. Skelton N.J. J. Mol. Biol. 2000; 293: 531-544Crossref Scopus (54) Google Scholar). Interface—The total surface area buried in each of the interfaces between PlGF and VEGFR1-d2 amounts to ∼1650 Å2. Both PlGF monomers participate in the binding of each receptor with one monomer contributing about 70% and the other 30% of the buried surfaces. Each receptor molecule is in contact with five segments of the PlGF dimer. Three of these, including the N-terminal helix of PlGF (residues 24–33), the loop connecting strands B and C, and the C-terminal residues 110–114, stem from one PlGF monomer; the other two segments, including residues of the AB loop (residues 54–56) and the CD loop (residues 87–99), stem from the other PlGF monomer. On the VEGFR1-d2 side, residues from four distinct segments of amino acids form the interface. They include residues 140–147 from the N-terminal strand A′, residues 171–175 from strand C and the loop connecting to strand C′, residues 199–204 from strand F and the short helix following it, as well as residues 217–224 from strand G (Fig. 1). The C terminus of the last VEGFR1-d2 residue with defined electron density, Arg224, projects toward a groove formed by the AB loop of one PlGF monomer and the CD loop of the second monomer suggesting that the linker connecting VEGFR1-d2 to the third domain of the intact ectodomain might also contribute to the formation of the complex. The interface is largely of hydrophobic nature with no apparent "knob-into-hole" interactions. Hydrophobic residues constitute ∼50% of the interface, with nine leucine and isoleucine residues being responsible for almost one-third of the buried surface. There are only three direct polar interactions; one hydrogen bond is formed between the side chain of PlGF residue Gln26 and the main chain carbonyl of Glu141 of VEGFR1-d2, and two charged interactions occur between Asp71 of PlGF and Arg224 of VEGFR1-d2. In addition several hydrogen bonds are mediated by a bound BTP molecule and a number of water molecules. bis-Tris Propane Bound in the Interface—In addition to one full complex between PlGF and VEGFR1-d2, the asymmetric unit contains a single molecule of BTP. The buffer molecule is near the interface between PlGF and VEGFR1-d2 and located on a 2-fold noncrystallographic symmetry (NCS) axis, with the NCS axis going through the central atom of the propane unit. Thus both of the tris(hydroxymethyl)methylamino head groups form nearly identical interactions with the proteins. Each head group is involved in hydrogen bond formation to Glu141 of the receptor domain and to Gln87 and Glu101 of VEGF (Fig. 2). Interestingly, the BTP molecule is therefore in contact with both PlGF and both VEGFR1-d2 molecules of the asymmetric unit, thus presumably greatly stabilizing the packing arrangement in these crystals (Fig. 2). Structure of Bound Versus Unbound PlGF—Because of the dimeric nature of the PlGF complex, there are two ways to superimpose the PlGF dimer in its free form (Protein Data Bank code 1FZV) and the PlGF in complex with VEGFR1-d2 on top of each other. In either case, the molecules have an r.m.s.d. of ∼2.1 Å for a total of 188 Cα atoms (residues 22–115). Even the superposition of a single PlGF monomer in its unbound form onto a PlGF monomer in complex with VEGFR1-d2 results into a rather large r.m.s.d. of 1.6 Å for 94 atoms. This deviation is the result of a number of changes. First, the overall shape of the PlGF dimer is slightly altered when bound to the receptor (Fig. 3A). The CD loops form the far end of the sheet-like PlGF dime
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