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• W1978532711 abstract "Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX3C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX3CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 Å resolution. The CDF monomers form a dimer through an intermolecular β-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX3C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivois not well understood. Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX3C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX3CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 Å resolution. The CDF monomers form a dimer through an intermolecular β-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX3C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivois not well understood. glycosaminoglycan chemokine domain of fractalkine macrophage inflammatory protein macrophage chemoattractant protein nuclear magnetic resonance Chemokines, or chemoattractant cytokines, are small (5–20 kDa), basic, heparin-binding proteins that show 20–70% identity in their amino acid sequences (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1985) Google Scholar). Chemokines provide a chemotactic gradient toward the site of inflammation as they direct the trafficking of a number of leukocytes, accomplishing this by binding to and activating 7-transmembrane-spanning G-protein-coupled receptors on the surface of the leukocytes. Activation of these receptors then leads to an increase in integrin adhesiveness and stable arrest (2Bargatze R.F. Butcher E.C. J. Exp. Med. 1993; 178: 367-372Crossref PubMed Scopus (252) Google Scholar, 3Honda S. Campbell J.J. Andrew D.P. Engelhardt B. Butcher B.A. Warnock R.A. Ye R.D. Butcher E.C. J. Immunol. 1994; 152: 4026-4035PubMed Google Scholar, 4Campbell J.J. Hedrick J. Zlotnik A. Siani M.A. Thompson D.A. Butcher E.C. Science. 1998; 279: 381-384Crossref PubMed Scopus (837) Google Scholar). Chemokines are secreted onto the cell surface and immobilized by binding to GAGs1 (5Witt D.P. Lander A.D. Curr. Biol. 1994; 4: 394-400Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). This immobilization is accompanied by oligomerization, which seems to enhance the affinity of the chemokines for their receptors (6Hoogewerf A.J. Kuschert G.S. Proudfoot A.E. Borlat F. Clark-Lewis I. Power C.A. Wells T.N. Biochemistry. 1997; 36: 13570-13578Crossref PubMed Scopus (437) Google Scholar, 7Appay V. Brown A. Cribbes S. Randle E. Czaplewski L.G. J. Biol. Chem. 1999; 274: 27505-27512Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To date, more than 40 different human chemokines have been identified (8Proudfoot A.E. Eur. J. Dermatol. 1998; 8: 147-157PubMed Google Scholar). They are mainly characterized by the presence and conserved relative position of the first two cysteine residues. The intramolecular disulfide bridges stabilize the fold of the peptide, ensuring its binding to specific receptors and its functional activity. Chemokines are classified into four categories: C, CC, CXC, and CX3C. CC chemokines contain two N-terminal adjacent cysteines, whereas the same cysteines in CXC chemokines are separated by one residue. C chemokines contain only one N-terminal cystine residue, and only one member, lymphotactin, has been identified so far (9Kelner G.S. Kennedy J. Bacon K.B. Kleyensteuber S. Largaespada D.A. Jenkins N.A. Copeland N.G. Bazan J.F. Moore K.W. Schall T.J. Science. 1994; 266: 1395-1399Crossref PubMed Scopus (627) Google Scholar). Recently, a new chemokine was discovered that contains the cysteine motif CX3C. This 372-residue protein, known as fractalkine (10Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1700) Google Scholar, 11Coulin F. Power C.A. Alouani S. Peitsch M.C. Schroeder J.M. Moshizuki M. Clark-Lewis I. Wells T.N. Eur. J. Biochem. 1997; 248: 507-515Crossref PubMed Scopus (52) Google Scholar) or neurotactin (12Pan Y. Lloyd C. Zhou H. Dolich S. Deeds J. Gonzalo J.A. Vath J. Gosselin M. Ma J. Dussault B. Woolf E. Alperin G. Culpepper J. Gutierrez-Ramos J.C. Gearing D. Nature. 1997; 387: 611-617Crossref PubMed Scopus (574) Google Scholar), contains an extracellular 76-residue chemokine domain (CDF or chemokine domain offractalkine) tethered to a membrane-spanning domain via a mucin-like stalk. Because of this natural immobilization, fractalkine can withstand vascular flow to capture and activate leukocytes (13Fong A.M. Robinson L.A. Steeber D.A. Tedder T.F. Yoshie O. Imai T. Patel D.D. J. Exp. Med. 1998; 188: 1413-1419Crossref PubMed Scopus (592) Google Scholar,14Haskell C.A. Cleary M.D. Charo I.F. J. Biol. Chem. 1999; 274: 10053-10058Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). An extracellular fragment of fractalkine (containing both the chemokine domain and the mucin-like stalk) can be released by proteolysis, and this form of the protein has activities that are different from those of the membrane-bound form (10Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1700) Google Scholar, 12Pan Y. Lloyd C. Zhou H. Dolich S. Deeds J. Gonzalo J.A. Vath J. Gosselin M. Ma J. Dussault B. Woolf E. Alperin G. Culpepper J. Gutierrez-Ramos J.C. Gearing D. Nature. 1997; 387: 611-617Crossref PubMed Scopus (574) Google Scholar, 15Imai T. Hieshima K. Haskell C. Baba M. Nagira M. Nishimura M. Kakizaki M. Takagi S. Nomiyama H. Schall T.J. Yoshie O. Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). The fractalkine receptor, CX3CR1, is expressed in leukocytes and binds tightly (Kd ∼1–4 nm) to CDF (15Imai T. Hieshima K. Haskell C. Baba M. Nagira M. Nishimura M. Kakizaki M. Takagi S. Nomiyama H. Schall T.J. Yoshie O. Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar, 16Harrison J.K. Jiang Y. Chen S. Xia Y. Maciejewski D. McNamara R.K. Streit W.J. Salafranca M.N. Adhikari S. Thompson D.A. Botti P. Bacon K.B. Feng L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10896-10901Crossref PubMed Scopus (911) Google Scholar, 17Combadiere C. Salzwedel K. Smith E.D. Tiffany H.L. Berger E.A. Murphy P.M. J. Biol. Chem. 1998; 273: 23799-23804Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 18Combadiere C. Gao J. Tiffany H.L. Murphy P.M. Biochem. Biophys. Res. Commun. 1998; 253: 728-732Crossref PubMed Scopus (58) Google Scholar); the complex seems to play an anti-inflammatory role (19Boddeke E.W. Meigel I. Frentzel S. Biber K. Renn L.Q. Gebicke-Harter P. Eur. J. Pharmacol. 1999; 374: 309-313Crossref PubMed Scopus (67) Google Scholar). A detailed analysis of the structure of CDF would elucidate the differences between CX3C and other chemokines. Here, we describe the x-ray crystal structure of CDF, solved at 2.0 Å resolution. The crystal structure shows a novel quaternary structure for chemokines, which is directly dependent on the bulge produced by the introduction of three residues between the N-terminal cysteines. CDF can bind to heparin, as shown by heparin-agarose chromatography, with an affinity similar to MIP-1α. A comparison of the calculated electrostatic surface maps for CDF with those for other chemokines shows similarities of charge distribution, which is indicative of heparin-binding capability. CDF (residues 1–76 of fractalkine, with the N-terminal methionine numbered 0) and its selenomethionine form were overexpressed using the plasmid pLSM103 in Escherichia coli strain BL21/pLysS (20Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (126) Google Scholar). Both proteins were lyophilized after reverse phase-high pressure liquid chromatography and were dissolved in water just before crystallization. Crystals were grown by vapor diffusion, mixing 4 μl of protein (17 mg/ml) with 2 μl of well solution (4 m sodium formate, 100 mm disodium citrate, pH 3.0) and 10 μl of water. Hexagonal prisms formed quickly (within 1–2 days) and continued to grow for about 1 week. Although very large crystals could be grown (1–2-mm diameter), they diffracted poorly when exposed to a conventional x-ray source (a rotating anode powered by a Rigaku RU200 generator), on average showing a maximum resolution of 2.8–3.0 Å for a 1° oscillation of 10-min exposures. The crystals also deteriorated quickly because of radiation damage, as even the largest (2 mm) crystals mounted in capillaries stopped diffracting after several minutes of exposure. Rapid cooling of the crystals in a liquid nitrogen stream (100 K), after a brief (30 s) soaking in cryoprotectant (6m sodium formate, 100 mm disodium citrate, 10% glycerol, pH 3.0), allowed complete data sets to be collected. In addition, synchrotron radiation allowed for higher resolution data collection (2.0 Å). The space group was determined to be either P6122 or P6522, with unit cell dimensionsa = 110.47 Å, c = 123.99 Å. A data set for the native protein and data sets for the selenomethionine derivative at three wavelengths in the multiple anomalous diffraction experiment were collected (beamline X9B, NSLS) (Table I). The positions of the selenium atoms were determined using SHELXS (21Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-344Crossref PubMed Scopus (1884) Google Scholar) and were refined using SHARP (22de La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The phases were further improved by electron density modification and solvent flattening using DM (23Cowtan K. Joint CCP4 ESF-EACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar) and PHASES (24Furey W. Swaminathan S. Acta Crystallogr. 1990; 18: 73-83Google Scholar). Interestingly, interpretable maps could also be generated using phases derived from data belonging to the pseudocell of length c/2 (l = 2n+1 removed). In both cases, the density for the C-terminal helices and internal β-strands could be identified only when the symmetry for space group P6122 or P6222 was applied.Table ICrystal data and refinement statisticsNativeSeMet λ1SeMet λ2SeMet λ3Wavelength (Å)0.980.979150.979460.96483Resolution range (Å)20–2.020–3.020–3.020–3.0Number of reflections (total/unique)345,321/30,36653,284/9,24842,954/9,22843,047/9,245Unit cell (Å)a = 110.47a = 110.27c = 123.99c = 121.86Space groupP6122P6122Completeness (overall/last shell1-aUsing only those reflections within the shell of 2.07–2.0 Å (native) or 3.11–3.0 Å resolution (SeMet).)98.8%/98.4%100%/100%99.9%/99.9%99.9%/99.9%R merge1-bR merge = Σ‖I n − 〈I〉‖/Σ〈I〉.(overall/last shell1-aUsing only those reflections within the shell of 2.07–2.0 Å (native) or 3.11–3.0 Å resolution (SeMet).)3.9%/49.1%9.8%/50.0%8.7%/40.1%8.9%/50.3%I/ςI (overall/last shell1-aUsing only those reflections within the shell of 2.07–2.0 Å (native) or 3.11–3.0 Å resolution (SeMet).)61.5/3.914.1/3.313.8/3.714.9/3.0R free/R work1-cR work = Σ‖{‖Fo(h)‖−k‖Fo(h)‖}‖/Σ‖Fo(h)‖;R free = Σ(h) ·T‖{‖Fo(h)‖−k‖Fc(h)‖}‖/Σ(h) · T‖Fo(h)‖, whereT represents a test set of reflections (10% of total, chosen at random) not used in the refinement.32.1%/23.8%RMSD: bonds1-dRMSD, root mean square deviation.0.012 ÅRMSD: angles1-dRMSD, root mean square deviation.1.927°B-factor (Å2; protein atoms/all atoms)46.4/49.8Number of residues262Number of waters2641-a Using only those reflections within the shell of 2.07–2.0 Å (native) or 3.11–3.0 Å resolution (SeMet).1-b R merge = Σ‖I n − 〈I〉‖/Σ〈I〉.1-c R work = Σ‖{‖Fo(h)‖−k‖Fo(h)‖}‖/Σ‖Fo(h)‖;R free = Σ(h) ·T‖{‖Fo(h)‖−k‖Fc(h)‖}‖/Σ(h) · T‖Fo(h)‖, whereT represents a test set of reflections (10% of total, chosen at random) not used in the refinement.1-d RMSD, root mean square deviation. Open table in a new tab Because only four sites were located clearly, only one of the three selenomethionines present in the monomer was ordered. From its location in the electron density as well as by comparison to the NMR model of CDF (20Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (126) Google Scholar), Met62 was identified as the ordered selenium site. A relatively good fit to the electron density was achieved for the regions corresponding to the β-strands and C-terminal α-helix with the sulfur of Met62 placed at the selenium sites. Because of the strong noncrystallographic symmetry, two monomers could be placed by fitting the NMR model to the density, whereas the remaining two were positioned by translating the first two monomers along the c axis by c/2. Although significant fragments of the main chain of all four monomers were located in the electron density, an extensive remodeling followed by refinement was necessary for most of the model. The model was truncated to residues 7–68, and regions 7–19 and 28–36 were rebuilt based on initial maps using the program O (25Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar). The model was refined using X-PLOR (26Brünger A.T. X-PLOR: A System for X-Ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar) and SHELXL (21Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-344Crossref PubMed Scopus (1884) Google Scholar), giving a final R-factor of 0.238 and a freeR-factor of 0.321. See Table Ifor further details. The chemokines RANTES (regulated on activation normal T cell expressed), MCP-1, MCP-3, MCP-4, MIP-1α, and MIP-1β were loaded onto a 1-ml HiTrap heparin-Sepharose column (Amersham Pharmacia Biotech) in 20 mm HEPES buffer (pH 7.5) and eluted with a 15-ml linear gradient (0–2 mNaCl in 20 mm HEPES buffer, pH 7.5) at a flow rate of 1.0 ml/min. The presence of protein was monitored by absorbance at 280 nm, and the concentration of NaCl was determined by using an in-line conductivity meter calibrated using the same gradient without protein. RANTES was a gift from Dr. Amanda Proudfoot (27Proudfoot A.E. Power C.A. Hoogewerf A.J. Montjovent M.O. Borlat F. Offord R.E. Wells T.N. J. Biol. Chem. 1996; 271: 2599-2603Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). MCP-1, MCP-3, and MCP-4 were overexpressed in E. coli and purified using standard protocols. MIP-1α and MIP-1β were purchased from PeproTech (Princeton, NJ). The interactions between monomers of CDF in the crystal are limited, and as a result the average B-factor of the molecule is relatively high (46 Å2) for a frozen crystal. This is not the first such example for chemokines; however, for instance, the crystal structure of viral MIP-2 solved using multiple anomalous diffraction also shows a high average B-factor (49 Å2) (PDB accession code 1cm9). The loose packing of the molecules reflects the high solvent content of 65–70%. Four monomers of CDF are found within the asymmetric unit arranged as layers along the c-parameter (Fig. 1). This arrangement gives rise to large (70 Å) channels along this direction. The four monomers in the asymmetric unit are arranged as two asymmetric dimers related by a noncrystallographic 2-fold rotation. This 2-fold axis lies along the (1 -1 0) vector, translated to approximatelyc = 1/6. Because of slight translational misalignments and packing anomalies between the two dimers (see below), the exact symmetry for P6222, with c = 62 Å (the pseudo half-cell), is broken, forcing four monomers rather than two into the asymmetric unit of P6122, with c = 124 Å. When the data were scaled as the pseudo half-cell (l = 2n+1 removed) and the model was refined against these data, the free R-factor did not drop below 0.40, confirming the correct space group as P6122. There is no direct contact seen between these two created dimers, but rather through bound solvent molecules. The closest contact (∼4 Å) between the dimers is at the C terminus of monomer D, the α-helix of which is ordered for an extra 1½ turns (residues 68–74), and of the symmetry-related monomer B. There is a presumed contact between the disordered C termini of monomers A and C, whose helices, if ordered past residue 68, would overlap at the site of the noncrystallographic 2-fold axis in the crystal. The two monomers that create the dimers (monomers A and D, and B and C) make close contact with each other through an asymmetric interaction of residues 8–14. The dimer is stabilized by a β-sheet formed by residues Cys8 to Thr11 (monomers A and C) and residues Thr11 to Lys14 (monomers B and D) and additional hydrogen bonds between loops 26–28 and 46–48. Note that CDF has not been found to dimerize in solution at any concentration (20Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (126) Google Scholar, 28Hoover, D. M., Shaw, J. P., Gryczynski, I., Proudfoot, A. E. I., Wells, T. N., and Lubkowski, J. (2000)Prot. Pept. Lett., in pressGoogle Scholar). However, this does not rule out dimer formation in vivo, in the presence of the receptor and other participating molecules. It has been observed that at high concentrations, as well as in crystalline environments, chemokines frequently form well defined dimers (or tetramers (29Czaplewski L.G. McKeating J. Craven C.J. Higgins L.D. Appay V. Brown A. Dudgeon T. Howard L.A. Meyers T. Owen J. Palan S.R. Tan P. Wilson G. Woods N.R. Heyworth C.M. Lord B.I. Brotherton D. Christison R. Craig S. Cribbes S. Edwards R.M. Evens S.J. Gilbert R. Morgan P. Randle E. Schofield N. Varley P.G. Fisher J. Waltho J.P. Hunter M.G. J. Biol. Chem. 1999; 274: 16077-16084Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 30Lubkowski J. Bujacz G. Boqué L. Domaille P.J. Handel T.M. Wlodawer A. Nat. Struct. Biol. 1997; 4: 64-69Crossref PubMed Scopus (140) Google Scholar)) but remain monomeric at lower concentrations in solution (31St. Charles R. Walz D.A. Edwards B.F. J. Biol. Chem. 1989; 264: 2092-2099Abstract Full Text PDF PubMed Google Scholar). The modes of dimerization of chemokines can be classified into two general categories, one observed for most of the CXC chemokines and the other found for CC chemokines. The dimer formed by CDF does not resemble either of these modes, except that the dimeric interaction is formed via an intermolecular β-sheet, similar to CC chemokines. However, the monomers in CDF form a more compact dimer than that of CC chemokine dimers. This motion is facilitated by the 3-residue insertion between the disulfides. Although the second disulfide of CDF (Cys12-Cys50) can be superimposed onto the second disulfide of two CC chemokines (MCP-1 (30Lubkowski J. Bujacz G. Boqué L. Domaille P.J. Handel T.M. Wlodawer A. Nat. Struct. Biol. 1997; 4: 64-69Crossref PubMed Scopus (140) Google Scholar) and RANTES (PDB accession code 1b3a)), the first disulfide of CDF (Cys8-Cys34) forces the N terminus to remain close to the core of the monomer. This arrangement causes the second monomer to twist around the peptide Ser13-Lys14to form the intramolecular β-sheet. The first disulfide also causes a bulge in the β-strands formed between the two CDF monomers, further distorting the dimeric interface and possibly weakening the dimer formation. The asymmetry of the dimerization is evident when the monomers are superimposed onto each other (Fig.2). Overall, the structure of the CDF monomer (Fig. 3) is similar to that of other chemokine monomers, particularly MCP-1 and RANTES. This similarity is not surprising, because CDF has the highest identities with these two chemokines (35% and 21%, respectively). The C-terminal α-helix on monomer D, however, extends out to Ala71, with the remaining residues in an extended conformation (Leu72-Arg74). The extension of the helical conformation is probably weakly stable, because there are no direct contacts between this extension and other protein atoms. Also, the extension of C termini for the other three monomers is blocked by the presence of protein crystal contacts. Almost all residues are clearly defined by electron density. Residues 42–47, which form a loop and are completely solvent accessible, are the most disordered within the chain, except for the extreme N and C termini. Several solvent-accessible side chains also appear to be disordered, such as Lys14 and Arg44. Met15, although clearly visible in the electron density, has a higherB-factor than Met62, which might be the reason why only one selenium site was determined by the multiple anomalous diffraction experiment. Superpositions of CC chemokine monomers MCP-1 and RANTES onto the CDF monomer show significant differences where the sequence alignment deviates (Fig. 4). The crystal structures of MCP-1 and RANTES superimpose on the crystal structure of CDF with fewer deviations than the superposition of the CDF NMR model (20Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (126) Google Scholar) on the crystal structure of CDF. This is seen clearly within the 40s loop (Thr43-Arg47) and in the bulge between Cys8 and Cys12. The N terminus, from Val5-Cys12, and the loop encompassing residues 28–38 are shifted unidirectionally relative to the crystal structure of CDF. This shift is considerable, because the superpositions show a maximal difference of 7 Å between the NMR model and crystal structure of CDF. The N terminus, which is attached via the disulfide between Cys8 and Cys34, is pulled along with this loop into its new position. This unidirectional shift between the NMR model and the crystal structure is partly because of crystal contacts at the edge of the 30s loop (Ser33), stabilizing this region of the molecule and perhaps twisting the chain fragments away. However, the crystal structures of MCP-1 and RANTES, although determined in completely different crystallographic conditions, are closer in structural alignment to CDF. Most chemokines are highly positively charged. This property facilitates the binding to GAGs on the surface of cells, which immobilizes and presents the chemokines for interaction with their receptors on mobile lymphocytes (5Witt D.P. Lander A.D. Curr. Biol. 1994; 4: 394-400Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). The free, proteolytically cleaved fractalkine form can chemoattract neutrophils and T lymphocytes, whereas the tethered fractalkine form can only chemoattract neutrophils after posttranslational processing in vivo (12Pan Y. Lloyd C. Zhou H. Dolich S. Deeds J. Gonzalo J.A. Vath J. Gosselin M. Ma J. Dussault B. Woolf E. Alperin G. Culpepper J. Gutierrez-Ramos J.C. Gearing D. Nature. 1997; 387: 611-617Crossref PubMed Scopus (574) Google Scholar). This finding raises the question of whether free or tethered CDF can interact and bind to GAGs similar to other freely diffusible chemokines. There are several highly conserved, positively charged residues present in most chemokines, which can be grouped into two sites based on sequence proximity, mutagenesis, and structural proximity. Site 1 comprises residues within the C-terminal helix (which usually begins after a conserved proline, Pro54, in CDF). Site 2 comprises residues from the loop between the second and third β-strands of the main β-sheet (residues 44–47 in CDF). These two sites have been found to be important in binding chemokines to GAGs (32Koopmann W. Krangel M.S. J. Biol. Chem. 1997; 272: 10103-10109Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 33Kuschert G.S. Hoogewerf A.J. Proudfoot A.E. Chung C.W. Cooke R.M. Hubbard R.E. Wells T.N. Sanderson P.N. Biochemistry. 1998; 37: 11193-11201Crossref PubMed Scopus (167) Google Scholar, 34Chakravarty L. Rogers L. Quach T. Breckenridge S. Kolattukudy P.E. J. Biol. Chem. 1998; 273: 29641-29647Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The electric charge distribution on the molecular surface of CDF shows clustered positive patches in three regions (Fig.5). Two of the patches overlap with sites 1 and 2, as described above. It is not known whether CDF can bind to GAGs or whether binding to GAGs is required for CDF to chemoattract lymphocytes or neutrophils, but the free form of fractalkine might require some other type of immobilization to create a haptotactic gradient. To investigate whether CDF binds to GAGs with an affinity similar to other chemokines, we compared CDF with an assortment of chemokines using heparin affinity chromatography (TableII). The binding of CDF to heparin-Sepharose is closest in affinity to MIP-1α. Although the theoretical pI, number of charged residues, and protein sequence of CDF are most similar to those of RANTES and MCP-1, the binding of CDF to heparin-Sepharose is lower. MIP-1α has fewer positive charges on its surface than CDF, RANTES, or MCP-1. Thus, the affinity of CDF for binding to GAGs is not dependent strictly on total net charge but on charge localization on the surface of the protein.Table IIComparison of the concentration of NaCl required to elute various chemokines from heparin-Sepharose and theoretical isoelectric pointsProtein[NaCl], MpI2-aTheoretical.No. K, R2-bNumber of lysine and arginine or aspartate and glutamate residues within the chemokine.No. D, E2-bNumber of lysine and arginine or aspartate and glutamate residues within the chemokine.MIP-1α0.34, 0.392-cFrom Ref. 43.5.1478CDF0.37, 0.222-dFrom Ref. 20.9.56115MIP-1β0.492-eFrom Ref. 6.4.7768Neutrophil-activating peptide-20.562-cFrom Ref. 43.6.911313Interleukin-80.60c,e9.021410MCP-10.609.39137MCP-30.76, 0.802-cFrom Ref. 43.9.74167MCP-40.789.98154Interferon-inducible protein-100.852-cFrom Ref. 43.10.1166Lymphotaxin0.862-cFrom Ref. 43.10.6156RANTES0.959.241052-a Theoretical.2-b Number of lysine and arginine or aspartate and glutamate residues within the chemokine.2-c From Ref. 43Kuschert G.S.V. Coulin F. Power C.A. Proudfoot A.E.I. Hubbard R.E. Hoogewerf A.J. Wells T.N.C. Biochemistry. 1999; 38: 12959-12968Crossref PubMed Scopus (491) Google Scholar.2-d From Ref. 20Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (126) Google Scholar.2-e From Ref. 6Hoogewerf A.J. Kuschert G.S. Proudfoot A.E. Borlat F. Clark-Lewis I. Power C.A. Wells T.N. Biochemistry. 1997; 36: 13570-13578Crossref PubMed Scopus (437) Google Scholar. Open table in a new tab Although it is known CDF does not oligomerize in solution, it does in the crystal. This phenomenon has been seen for the chemokines stromal cell-derived factor-1α (35Dealwis C. Fernandez E.J. Thompson D.A. Simon R.J. Siani M.A. Lolis E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6941-6946Crossref PubMed Scopus (151) Google Scholar) and viral MIP-2 (36Shao W. Fernandez E. Wilken J. Thompson D.A. Siani M.A. West J. Lolis E. Schweitzer B.I. FEBS Lett. 1998; 441: 77-82Crossref PubMed Scopus (15) Google Scholar). The driving forces in forming oligomers within a crystal lattice probably include the high concentrations of protein available in the crystal, as well as the unusual electrostatic environment that would be present under such conditions. Similar conditions might occur in vivo, because chemokines are not fully diffusible in simple buffered solutions but are immobilized within a two-dimensional area on the cell surface. It is unknown what local concentrations of the chemokine could be achieved under such conditions. There are other factors known to drive oligomerization. A key structural determinant for aggregation of CC chemokines has been identified as two key acidic residues, one within the 20s loop (Glu26 in RANTES), the other within the C-terminal helix (Asp67 in RANTES) (29Czaplewski L.G. McKeating J. Craven C.J. Higgins L.D. Appay V. Brown A. Dudgeon T. Howard L.A. Meyers T. Owen J. Palan S.R. Tan P. Wilson G. Woods N.R. Heyworth C.M. Lord B.I. Brotherton D. Christison R. Craig S. Cribbes S. Edwards R.M. Evens S.J. Gilbert R. Morgan P. Randle E. Schofield N. Varley P.G. Fisher J. Waltho J.P. Hunter M.G. J. Biol. Chem. 1999; 274: 16077-16084Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Although there is no acidic residue in CDF corresponding to Glu26, CDF residue Asp67 does correspond to the second acidic residue in RANTES. Because the aggregation of MIP-1α, MIP-1β, and RANTES is pH-dependent, the oligomers are likely to be held primarily by electrostatic forces. Also, it was found that only one mutation is required to abolish all oligomerization in MIP-1α, whereas single mutations lower the amount of oligomerization in RANTES from large, nonspecific aggregates to dimers and tetramers (29Czaplewski L.G. McKeating J. Craven C.J. Higgins L.D. Appay V. Brown A. Dudgeon T. Howard L.A. Meyers T. Owen J. Palan S.R. Tan P. Wilson G. Woods N.R. Heyworth C.M. Lord B.I. Brotherton D. Christison R. Craig S. Cribbes S. Edwards R.M. Evens S.J. Gilbert R. Morgan P. Randle E. Schofield N. Varley P.G. Fisher J. Waltho J.P. Hunter M.G. J. Biol. Chem. 1999; 274: 16077-16084Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Because CDF has a more disperse positive charge on its surface than RANTES, similar to MIP-1α, as well as only a single acidic residue, it seems to be in agreement with data for MIP-1α and RANTES that CDF does not oligomerize in solution (20Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (126) Google Scholar, 28Hoover, D. M., Shaw, J. P., Gryczynski, I., Proudfoot, A. E. I., Wells, T. N., and Lubkowski, J. (2000)Prot. Pept. Lett., in pressGoogle Scholar). Because the CDF dimer formed within the crystal is both asymmetric and distinct from CC chemokines in form, a comparison may not totally explain the forces behind oligomerization. Although it is known that CDF does not oligomerize in solution, it cannot be ruled out that fractalkine might dimerize while tethered to a membrane. An analysis of the electrostatic surface maps and structure shows that this dimerization is possible. The dimeric interface is relatively devoid of charges, whereas the side opposite the interface is highly charged (data not shown). Also, both C termini for the two monomers within the dimer are on the same side (Fig. 1), unlike the dimers seen in CC chemokine structures (37Clore G.M. Gronenborn A.M. FASEB J. 1995; 9: 57-62Crossref PubMed Scopus (118) Google Scholar). The C-terminal helix is likely to extend farther from the main β-sheet, as seen in monomer D. Thus, while bound to the membrane by a flexible mucin-like tether, CDFs might form dimers. CDF binds to heparin with roughly the same affinity as MIP-1α (20Mizoue L.S. Bazan J.F. Johnson E.C. Handel T.M. Biochemistry. 1999; 38: 1402-1414Crossref PubMed Scopus (126) Google Scholar,32Koopmann W. Krangel M.S. J. Biol. Chem. 1997; 272: 10103-10109Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The electrostatic surface of CDF is most similar to MIP-1α. We expect that the binding of CDF to heparin in vivo would be similar to that of MIP-1α. Mutation studies have shown that a cationic cleft composed of residues Arg18, Arg46, and Arg48 (Lys19, Arg45, and Arg48 in CDF) is required for binding MIP-1α to heparin; the neutralization of any one of these positive charges eliminates binding to heparin (32Koopmann W. Krangel M.S. J. Biol. Chem. 1997; 272: 10103-10109Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The same is probably true for CDF. Although disruption of MIP-1α-GAG interactions has little or no effect on binding to and signaling through CCR1 on Chinese hamster ovary cells, it impairs the ability of MIP-1α to induce both a change in the shape of monocytes and chemotaxis, presumably through disruption of interactions with CCR5 (32Koopmann W. Krangel M.S. J. Biol. Chem. 1997; 272: 10103-10109Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 38Graham G.J. Wilkinson P.C. Nibbs R.J. Lowe S. Kolset S.O. Parker A. Freshney M.G. Tsang M.L. Pragnell I.B. EMBO J. 1996; 15: 6506-6515Crossref PubMed Scopus (72) Google Scholar). MIP-1α, as opposed to interleukin-8, MCP-1, or RANTES, does not oligomerize on the surface of human umbilical vein endothelial cells, and its activation of CXCR1-, CCR1-, and CCR2-transfected Chinese hamster ovary cells is only slightly weakened by deglycosylation (6Hoogewerf A.J. Kuschert G.S. Proudfoot A.E. Borlat F. Clark-Lewis I. Power C.A. Wells T.N. Biochemistry. 1997; 36: 13570-13578Crossref PubMed Scopus (437) Google Scholar). Thus, although CDF might bind to GAGs, this binding probably only affects the chemoattractant and activation activities of CDF with specific receptors. In summary, we have shown that CDF forms a novel quaternary structure relative to other chemokines. CDF binds to heparin and is most similar to MIP-1α in terms of electrostatic properties, although it is most similar to MCP-1 in terms of sequence homology. The coordinates for CDF were deposited at the Protein Data Bank, accession code 1f2l. We thank Dr. Alexander Wlodawer for support of the work presented here, Dr. Zbigniew Dauter for suggestions on data collection, and Dr. Amanda Proudfoot for the gift of RANTES. We also thank Anne Arthur for editorial assistance." @default.
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• W1978532711 title "The Crystal Structure of the Chemokine Domain of Fractalkine Shows a Novel Quaternary Arrangement" @default.
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