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J. Biol. Chem., Vol. 275, Issue 30, 23187-23193, July 28, 2000

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The Crystal Structure of the Chemokine Domain of Fractalkine Shows a Novel Quaternary Arrangement^*

David M. Hoover, Laura S. Mizoue^§, Tracy M. Handel^§, and Jacek Lubkowski^¶

From the  Macromolecular Crystallography Laboratory, Program in Structural Biology, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 and the ^§ Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720

Received for publication, March 27, 2000, and in revised form, April 14, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 -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 vivo is not well understood.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemokines, or chemoattractant cytokines, are small (5-20 kDa), basic, heparin-binding proteins that show 20-70% identity in their amino acid sequences (1). 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 (2-4). Chemokines are secreted onto the cell surface and immobilized by binding to GAGs¹ (5). This immobilization is accompanied by oligomerization, which seems to enhance the affinity of the chemokines for their receptors (6, 7).

To date, more than 40 different human chemokines have been identified (8). 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 (9).

Recently, a new chemokine was discovered that contains the cysteine motif CX₃C. This 372-residue protein, known as fractalkine (10, 11) or neurotactin (12), contains an extracellular 76-residue chemokine domain (CDF or chemokine domain of fractalkine) 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 (13, 14). 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 (10, 12, 15). The fractalkine receptor, CX₃CR1, is expressed in leukocytes and binds tightly (K_d ~1-4 nM) to CDF (15-18); the complex seems to play an anti-inflammatory role (19).

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-1. 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sample Preparation and Crystallization-- 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 (20). 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 (6 M 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 dimensions a = 110.47 Å, c = 123.99 Å.

Structure Solution-- 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 (21) and were refined using SHARP (22). The phases were further improved by electron density modification and solvent flattening using DM (23) and PHASES (24). 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 -strands could be identified only when the symmetry for space group P6₁22 or P6₂22 was applied.

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 (20), Met⁶² was identified as the ordered selenium site. A relatively good fit to the electron density was achieved for the regions corresponding to the -strands and C-terminal -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 (25). The model was refined using X-PLOR (26) and SHELXL (21), giving a final R-factor of 0.238 and a free R-factor of 0.321. See Table I for further details.

Heparin-Sepharose Chromatography-- The chemokines RANTES (regulated on activation normal T cell expressed), MCP-1, MCP-3, MCP-4, MIP-1, and MIP-1 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 M NaCl 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 (27). MCP-1, MCP-3, and MCP-4 were overexpressed in E. coli and purified using standard protocols. MIP-1 and MIP-1 were purchased from PeproTech (Princeton, NJ).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Crystal Packing and Space Group Dependences-- 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.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Stereo ribbon drawing of the CDF asymmetric unit. Note the extension of the -helix for monomer D. -Strands are shown as arrows; - and 310-helices are shown as coils. This figure was made using the program RIBBONS (39).

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 approximately c = 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 -helix of which is ordered for an extra 11/2 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.

Dimerization-- 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 -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 (20, 28). 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 (29, 30)) but remain monomeric at lower concentrations in solution (31). 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 -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 (30) 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-Lys¹⁴ to form the intramolecular -sheet. The first disulfide also causes a bulge in the -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).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Stereo drawing showing the superposition of CDF monomers. The monomers are colored as in Fig. 1. The monomers were superimposed using the program ALIGN (40), with all atoms included, and the figure was made using the program MOLSCRIPT (41).

Description of the Structure-- 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 -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 higher B-factor than Met⁶², which might be the reason why only one selenium site was determined by the multiple anomalous diffraction experiment.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Ribbon drawing of the CDF monomers. Shown here is a representation of monomer D. Monomer B is nearly identical to monomer D, whereas monomers A and D differ within residues 5-15. Also, residues 68-74 are ordered in monomer D producing a C-terminal extension of the -helix by 1.5 turns (see "Results"). This drawing was made using the program MOLSCRIPT (41).

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 (20) 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.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Stereo drawings showing the superposition of CDF monomer D on RANTES, MCP-1, and the NMR model of CDF. A, RANTES (28) (green) and MCP-1 (30) (blue) superimposed onto CDF (gray). B, the NMR model of CDF (20) (red) superimposed onto CDF (gray). Disulfide bonds are shown in the same color as the models, with sulfurs colored yellow. C, 2 Fo  Fc  electron density (1.5 ) contoured around residues Cys8-Cys12 and Ala32-Lys36 of the D monomer of CDF. Superpositions were done using the program ALIGN (40), with only backbone atoms included. This figure was made using the program MOLSCRIPT (41).

Charge Distribution and Heparin Binding-- 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 (5). 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 (12). 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 -strands of the main -sheet (residues 44-47 in CDF). These two sites have been found to be important in binding chemokines to GAGs (32-34).

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 (Table II). The binding of CDF to heparin-Sepharose is closest in affinity to MIP-1. 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-1 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.

View larger version (77K): [in this window] [in a new window]   Fig. 5.   Charge distribution of chemokines showing potential GAG binding sites. A, CDF; B, RANTES; C, MCP-1; D, MIP-1; and E, interleukin-8. Site 1 contains Arg44 and Arg67, and site 2 contains Lys14, Lys18, and Arg47. A third patch is present, encompassing residues Lys36, Arg37 (not shown), Lys54, and Lys59. Arg74 is only ordered in monomer D and is isolated from the three sites. Charges were calculated using GRASP (42), and surface maps were calculated and displayed using INSIGHT (Molecular Simulations, Inc.).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-1 (35) and viral MIP-2 (36). 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) (29). 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-1, MIP-1, 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-1, whereas single mutations lower the amount of oligomerization in RANTES from large, nonspecific aggregates to dimers and tetramers (29). Because CDF has a more disperse positive charge on its surface than RANTES, similar to MIP-1, as well as only a single acidic residue, it seems to be in agreement with data for MIP-1 and RANTES that CDF does not oligomerize in solution (20, 28).

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 (37). The C-terminal helix is likely to extend farther from the main -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-1 (20, 32). The electrostatic surface of CDF is most similar to MIP-1. We expect that the binding of CDF to heparin in vivo would be similar to that of MIP-1. 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-1 to heparin; the neutralization of any one of these positive charges eliminates binding to heparin (32). The same is probably true for CDF. Although disruption of MIP-1-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-1 to induce both a change in the shape of monocytes and chemotaxis, presumably through disruption of interactions with CCR5 (32, 38). MIP-1, 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 (6). 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-1 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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported in part the AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health, and by the NCI, National Institutes of Health HIV Drug Resistance Program (to J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1f2l) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^¶ To whom correspondence should be addressed. Tel.: 301-846-5494; Fax: 301-846-5991; E-mail: jacek@ncifcrf.gov.

Published, JBC Papers in Press, April 17, 2000, DOI 10.1074/jbc.M002584200

	ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; CDF, chemokine domain of fractalkine; MIP, macrophage inflammatory protein; MCP, macrophage chemoattractant protein; NMR, nuclear magnetic resonance.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/30/23187    most recent M002584200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoover, D. M. Articles by Lubkowski, J. Search for Related Content PubMed PubMed Citation Articles by Hoover, D. M. Articles by Lubkowski, J. Social Bookmarking                      What's this?