©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Crystal Structure of Recombinant Human Neutrophil-activating Peptide-2 (M6L) at 1.9- Resolution (*)

(Received for publication, October 13, 1994; and in revised form, December 20, 1994)

Michael G. Malkowski (1) Jean Yang Wu (1) Jerome B. Lazar (2) Paul H. Johnson (2) Brian F. P. Edwards (1)(§)

From the  (1)Department of Biochemistry, Wayne State University, Detroit, Michigan 48201 and the (2)Cell and Molecular Biology Laboratory, Stanford Research Institute International, Menlo Park, California 94025

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Neutrophil-activating peptide-2 (NAP-2) is a 70-residue carboxyl-terminal fragment of platelet basic protein, which is found in the alpha-granules of human platelets. NAP-2, which belongs to the CXC family of chemokines that includes interleukin-8 and platelet factor 4, binds to the interleukin-8 type II receptor and induces a rise in cytosolic calcium, chemotaxis of neutrophils, and exocytosis. Crystals of recombinant NAP-2 in which the single methionine at position 6 was replaced by leucine to facilitate expression belong to space group P1 (unit cell parameters a = 40.8, b = 43.8, and c = 44.7 Å and alpha = 98.4°, beta = 120.3°, and = 92.8°), with 4 molecules of NAP-2 (M(r) = 7600) in the asymmetric unit. The molecular replacement solution calculated with bovine platelet factor 4 as the starting model was refined using rigid body refinement, manual fitting in solvent-leveled electron density maps, simulated annealing, and restrained least squares to an R-factor of 0.188 for 2 data between 7.0- and 1.9-Å resolution. The final refined crystal structure includes 265 solvent molecules. The overall tertiary structure, which is similar to that of platelet factor 4 and interleukin-8, includes an extended amino-terminal loop, three strands of antiparallel beta-sheet arranged in a Greek key fold, and one alpha-helix at the carboxyl terminus. The Glu-Leu-Arg sequence that is critical for receptor binding is fully defined by electron density and exhibits multiple conformations.


INTRODUCTION

Neutrophil-activating peptide-2 (NAP-2) (^1)is a cleavage product of the platelet alpha-granule component, platelet basic protein (PBP) and its derivative, connective tissue-activating peptide III (CTAP-III) (Castor et al., 1983; Walz and Baggiolini, 1990). NAP-2 corresponds to the carboxyl-terminal fragment of PBP, which is cleaved by monocyte-derived proteases, and gives rise to a single peptide of 70 amino acids with a M(r) of 7600 (Walz and Baggiolini, 1989). CTAP-III and beta-thromboglobulin (beta-TG), an additional truncation product of PBP (Begg et al., 1978), can also be cleaved by neutrophil cathepsin G to generate NAP-2 (Cohen et al., 1992).

Members of the chemotactic cytokine family, which are collectively known as chemokines, have four conserved cysteine residues and are divided into two subfamilies according to the position of the first pair of cysteines, which are separated by one amino acid (CXC) or are adjacent (CC) (Baggiolini et al., 1994; Baggiolini and Clark-Lewis, 1992). The members of the two subfamilies differ in their selectivity, with the CXC subfamily targeting neutrophils and the CC subfamily targeting monocytes (Baggiolini et al., 1994). PBP and its successive cleavage products, CTAP-III, beta-TG, and NAP-2, belong to the CXC subfamily. Other members of this CXC subfamily include platelet factor 4 (PF4) (Deuel et al., 1977; Hermodson et al., 1977); interleukin-8 (IL-8) (Sherry and Cerami, 1991; Baggiolini and Clark-Lewis, 1992); the growth-related proteins GROalpha, GRObeta, and GRO (Haskill et al., 1990; Tekamp-Olson et al., 1990); ENA-78, a neutrophil-activating peptide identified in the conditioned medium of stimulated human type II epithelial cell line A549 (Walz et al., 1991); macrophage inflammatory protein-2 (Wolpe et al., 1988); and interferon--inducible peptide-10 (IP-10) (Luster et al., 1985).

Unlike its three natural precursors, PBP, CTAP-III, and beta-TG, NAP-2 has powerful neutrophil-stimulating effects involved in inflammation (Walz et al., 1989). NAP-2 behaves as a typical chemotactic receptor agonist, inducing a rise in cytosolic calcium, chemotaxis, and exocytosis at concentrations between 0.3 and 10 nM. NAP-2, which is approximately half as potent as IL-8 as a neutrophil activator (Walz et al., 1989), is released mainly into the vasculature where platelet activation and aggregation occur, whereas IL-8 is formed within tissues. IL-8, NAP-2, GROalpha, and ENA-78 bind to common receptors on neutrophils (Walz et al., 1989, 1991; Moser et al., 1990, 1991) and share a highly conserved Glu-Leu-Arg (ELR) sequence at their amino-terminal end that has been shown to be critical for receptor binding (Hébert et al., 1991; Clark-Lewis et al., 1991; Moser et al., 1993). Comparable neutrophil responses were not observed with PF4 (Walz et al., 1989; Lenord et al., 1991), although chemotaxis and exocytosis have been reported with concentrations that were 1000-10,000-fold higher than those required for IL-8 (Deuel et al., 1981; Bebawy et al., 1986; Park et al., 1990). No neutrophil-stimulating or other biological activities have been observed with IP-10 (Dewald et al., 1992).

Recent experiments have suggested that post-translationally modified forms of CTAP-III and NAP-2 can function as endoglucosamidases that degrade heparin and heparan sulfate to dimers (Hoogewerf et al., 1993). Chondroitin and dermatan sulfates are not cleaved. This discovery further suggests a possible role for NAP-2 in the breakdown of basement membranes that occurs during metastasis, angiogenesis, and arthritis.

No other structure of NAP-2 is available, although the crystallization of recombinant NAP-2 was recently reported (Kungl et al., 1994). However, crystal structures of bovine PF4 (St. Charles et al., 1989), IL-8 (Baldwin et al., 1991), and recombinant human PF4 (Stuckey, 1992; Zhang et al., 1994) have been elucidated. The structure of IL-8 has been determined by NMR methods as well (Clore et al., 1990). In all cases, the secondary structure of the monomer consists of an extended loop, three strands of antiparallel beta-sheet folded in a Greek key, and one alpha-helix at the carboxyl terminus of the protein. In solution, IL-8 exists as a dimer in which the two monomers form an extended six-stranded beta-sheet (Clore et al., 1990). In the crystal structure of IL-8, the dimer lies on a crystallographic two fold so there is only a monomer in the asymmetric unit. PF4, which exists primarily as a tetramer in solution, formed orthorhombic crystals with a tetramer in the asymmetric unit that was formed by the back-to-back association of the extended beta-sheets of two dimers (St. Charles et al., 1989). In this paper, we present a refined crystal structure of biologically active, recombinant human NAP-2 {M6L} (^2)in which methionine at position 6 in the NAP-2 sequence has been replaced by leucine to facilitate expression (Castor et al., 1990). The crystal structure contains a PF4-like tetramer in the asymmetric unit of the triclinic unit cell.


MATERIALS AND METHODS

Preparation of Recombinant NAP-2

Escherichia coli cells were transformed with the pBR-CRM-CTAP-Met20,Leu26 {M15,L21} plasmid, which coded for a modified form of CTAP-III that had Met-20 replacing tyrosine and Leu-26 replacing methionine (Waleh et al., 1992). Cleavage at Met-20 in CTAP-III with CNBr generates the amino terminus of native NAP-2, while the M26L substitution prevents the generation of a truncated form of NAP-2. Transformed E. coli cells were grown in shaker flasks in Luria broth containing 50 µg/ml ampicillin at 37 °C. CTAP (Met-20,Leu-26) protein expression was induced at an A of 0.4 by the addition of mitomycin C to a final concentration of 1 µg/ml. Cells were harvested and lysed by suspension in 6 M guanidine HCl, 0.1 M Tris-HCl (pH 8.0), 10 mM EDTA, and 6 mM sodium thiosulfate to protect against oxidation of cysteine by CNBr. The solution was purged with argon to remove oxygen, adjusted to 0.2 M in phosphoric acid and to 0.2 M in cyanogen bromide, and incubated overnight in the dark at room temperature. Besides generating free NAP-2 {M6L}, this treatment also aided in subsequent purification steps by fragmenting the endogenous E. coli proteins. The reaction mixture was dialyzed against deionized water to remove salts, CNBr, and sodium thiosulfate and freeze-dried.

The protein powder was dissolved and incubated for 1 h at 37 °C in 6 M guanidine HCl, 10 mM EDTA, 0.1 M dithiothreitol, 25 mM Tris-HCl (pH 8.6) to reduce disulfide bonds and to remove sulfonate-protecting groups from cysteine sulfhydryls. The solution was prepared for reverse-phase HPLC by acidification with 0.02 volumes of 88% formic acid, addition of 0.43 volumes of acetonitrile in 0.065% (v/v) trifluoroacetic acid, and centrifugation or filtration to remove any precipitate that formed. The reduced NAP-2 was purified on a Vydac 214TP1520 1-inch diameter reverse-phase column using a gradient that increased from 20% of eluant B to 35% at a rate of 0.1%/minute at a flow rate of 10 ml/min. Eluant A was water, and eluant B was acetonitrile (both 0.065% (v/v) in trifluoroacetic acid). The peak of reduced NAP-2 was pooled and lyophilized. NAP-2 was refolded at a concentration of 0.2 mg/ml in 2 M guanidine HCl, 0.1 M Tris-HCl (pH 8.6), 10 mM EDTA, 2 mM oxidized glutathione, 1 mM reduced glutathione at 37 °C for 2 h. The solution was acidified by the addition of 0.02 volumes of 88% formic acid and 0.33 volumes of acetonitrile. The filtered refolded NAP-2 was purified on a Vydac 214TP1022 high-resolution reversed-phase column using the same gradient as described above. The NAP-2 peak was pooled and freeze-dried. Purity was judged to be >98% as assessed by spectral and amino acid composition analyses, amino-terminal sequencing, and analytical HPLC. Typical yields were 20-30 mg/liter of induced culture.

Crystallization

Crystals were grown by the hanging drop method at 22 °C from reservoirs containing 28% polyethylene glycol 4000, 100 mM sodium acetate (pH 4.6), and 200 mM ammonium acetate. The initial concentration of NAP-2 in the drop was 17 mg/ml. The crystals belong to space group P1 with unit cell dimensions of a = 40.8, b = 43.8, and c = 44.7 Å and alpha = 98.4°, beta = 120.3°, and = 92.8°, with four monomers in the asymmetric unit.

Data Collection

Diffraction data were collected at 22 °C on a single crystal (0.40 times 0.20 times 0.20 mm) mounted in a quartz capillary. The Siemens X-1000 area detector was mounted on a Rigaku RU200H rotating anode x-ray generator (CuK radiation) that was operated at 40 kV and 70 mA and equipped with a Supper graphite monochromator. The data collection, which was organized using the program ASTRO (Chambers et al., 1992), was done in eight sweeps of 280 frames/sweep at 0.25°/frame. Each frame was collected for 3 min to ensure that it contained at least one million total counts. The data were merged and scaled using the program XENGEN (Howard et al., 1987), yielding 30,511 reflections, 17,830 of which were unique between 7.0 and 1.9 Å. These data, which represent 86.7% of the possible reflections, had an overall unweighted absolute R of 0.025 based on intensities. For the three shells, 7.0 to 2.1, 2.1 to 2.0, and 2.0 to 1.9 Å, the completeness percentages were 97, 85, and 47%, respectively.

Molecular Replacement

The structure was solved by the molecular replacement method (Rossman and Blow, 1962) using the XPLOR suite of programs (Brunger, 1988) with a previously determined structure of PF4 as the model (St. Charles et al., 1989). Because the sequence identity between the model and NAP-2 is 50%, residues that were not identical between structures were changed to alanine. The model tetramer was then placed in a 100-Å P1 orthogonal unit cell with its center of mass placed at the origin. The cross-rotation function was calculated for the range (1) = 0-360°, (2) = 0-180°, and (3) = 0-360° (Rao et al., 1980) using the data between 7.0 and 4.0 Å. Patterson correlation refinement (Brunger, 1988) of the 150 most significant solutions identified two rotations, related by 179.5°, whose coefficients were above 0.43; the third best rotation had a correlation coefficient of 0.35. The first rotation ((1) = 297.2°, (2) = 13.9°, and (3) = 40.4°) was used to orient the model in the crystallographic unit cell. For convenience, the model was translated by x = 0.5, y = 0.5, and z = 0.5, so that roughly one tetramer would be contained within the limits of the unit cell. No translation function was calculated because there is only one tetramer in the P1 unit cell. The initial R-factor was 0.539 for data from 7.0- to 3.0-Å resolution.

Structural Analysis

The coordinates for the crystal structure of human IL-8 (entry 3IL8) and for the NMR structure of IL-8 (entry 1IL8) were obtained from the Protein Data Bank (Bernstein et al., 1977). The coordinates for bovine PF4 were obtained from those of St. Charles et al.(1989) by refinement against diffraction data collected to 2.2-Å resolution from crystals of bovine PF4 that were free of nickel tetracyanate ions (Stuckey, 1992). The R-factor is 0.20. The coordinates for human PF4 were obtained by molecular replacement using the bovine coordinates and subsequent refinement to an R-factor of 0.19 at 2.0-Å resolution (Stuckey, 1992). Subunit B in NAP-2 was taken as the reference structure because its amino terminus has the fewest crystal contacts. Hydrogen bonds and surface accessibility values were calculated using the program QUANTA (Polygen Corp., Waltham, MA). The criteria for a hydrogen bond are that the angles C=O . . . H, O . . . H-N, and H-N-C be greater than 90 °C and the distance N . . . O not exceed 3.3 Å for a ``short'' hydrogen bond and 4.0 Å for a ``long'' one (Baker and Hubbard, 1984). Superposition of coordinates between structures was done using the program ALIGN (Satow et al., 1986). Omit maps were calculated in XPLOR.


RESULTS

Structure Solution and Refinement

After 200 cycles of rigid body refinement, allowing the beta-sheet and helical domains to refine independently, and fixing a different monomer in the tetramer every 50 cycles, the R-factor fell to 0.47 for data from 7.0 to 3.0 Å. Omit maps were calculated, in which a monomer was deleted from the tetramer, to see that the density for the deleted monomer was still present. Convinced that the solution gave a reasonable difference map, we proceeded directly to simulated annealing utilizing the slow cool annealing protocol in XPLOR. The dynamics temperature started out at 4000 K and decreased in intervals of 25 K to a final temperature of 0 K with 25 steps (0.5 fs/step) of Verlet dynamics at each temperature. This reduced the R-factor to 0.328 using all the data from 7.0 to 1.9 Å. Side chains in subunit A that had been replaced by alanine in the model were then placed into an electron density map that was calculated with a locally written program to perform noncrystallographic symmetry averaging. (^3)Positional refinement and analysis of electron density maps that were improved through solvent leveling, histogram matching, and Sayre's equations using the program SQUASH (Zhang, 1993) were then used to fit additional residues at the amino and carboxyl termini of the individual subunits and to adjust poorly fit side chains. The R-factor was reduced to 0.234 for all the data from 7.0 to 1.9 Å.

At this point, all the residues were fit to well defined density with the exception of the last four carboxyl-terminal residues in each subunit and the three amino-terminal residues in subunit C, which were disordered. These residues were removed before proceeding with further refinement. Water molecules were then added at positions that were within 2.5-3.5 Å of a hydrogen bonding donor or acceptor and had electron density in both F(o) - F(c) and 2F(o) - F(c) maps. A total of 145 cycles of GPRLSA refinement (Furey et al., 1982), including 265 water molecules, dropped the R-factor to 0.202 for 2 data from 7.0 to 1.9 Å, with root mean square deviations for bonds and angles of 0.020 Å and 5.5°, respectively (Table 1). Finally, the XPLOR program was used to refine only the B values in each subunit. Twenty-five cycles of B refinement dropped the R-factor to 0.188 for data from 7.0 to 1.9 Å and greater than 2 .



The final refined model of the 4 molecules of NAP-2 in the asymmetric unit (Fig. 2) has excellent stereochemistry when evaluated with the program PROCHECK (Morris et al., 1992). On the Ramachandran plot, 90.5% of the residues are in the most favored region, and none lie in the disallowed region. Other well refined structures at an equivalent resolution have 83.8% of their residues, on average, within the most favored region. The nine other parameters evaluated by PROCHECK are also equal to or better than the bounds established from well refined structures at equivalent resolution.


Figure 2: Structure of NAP-2. Top, the alpha-carbon backbones of residues 21(a)-86(a) (thicklines) and of residues 21(b)-86(b) (thinlines) are shown in stereo. The view is approximately down the AB noncrystallographic axis. Bottom, the alpha-carbon backbone of the NAP-2 tetramer is shown in stereo as viewed approximately down the AD noncrystallographic axis. As drawn, subunits A (thicklines) and B (thinlines) are above subunits C (thinlines) and D (thicklines).



Secondary Structure of NAP-2

The secondary structure of NAP-2 is summarized in Fig. 3. The amino terminus, which starts at position 21 in each subunit, is ordered in subunits A, B, and D (Fig. 4), but Ala-21(c), Glu-22(c), and Leu-23(c) are disordered in subunit C. Ala-21 to Ile-34 form a large open loop that is stabilized by the two disulfide bonds Cys-25-Cys-51 and Cys-27-Cys-67. Also, there are bends, as defined by Kabsch and Sander(1983), centered at residues 25 and 27 and one hydrogen bond from Thr-31 to Cys-67. A main chain hydrogen bond exists between the nitrogen of Asp-69 and the carbonyl oxygen of Thr-31, which also contributes to stabilize the loop. His-35 to Asn-38 form a single turn of 3 helix in an external loop that is stabilized by main chain hydrogen bonds between Asn-38 and His-35 and between Ile-39 and Pro-36.


Figure 3: Hydrogen bonds and topology of NAP-2. Main chain hydrogen bonds for the four loops, three strands of beta-sheet, and one alpha-helix found in the NAP-2 monomer are shown as arrows going from the donor to the acceptor. The two disulfide bonds are indicated by thickarrows. Ala-72 to Lys-81 in the helical region are also acceptors in 3-type hydrogen bonds with the residue three positions farther along in the sequence. These hydrogen bonds are generally longer than the alpha-helical type and are not shown. A hydrogen bond was included in the diagram if it was present in all four subunits.




Figure 4: Electron density for the ELR sequence of NAP-2. The electron density in a 2F - F map contoured at 2 is shown in stereo for Glu-22(a), Leu-23(a), and Arg-24(a) in NAP-2. The final refined model is shown in thicklines.



The first strand of the three-stranded beta-sheet is formed by Ile-39 to Gly-46. There are six main chain hydrogen bonds formed with the adjacent subunit that form the extended six-stranded beta-sheet (Fig. 3). Glu-43(a) on the ``interior side'' of the beta-sheet forms an internal salt bridge with Lys-65(c) in the other extended beta-sheet and similarly for the related pair Glu-43(c) to Lys-65(a). The respective Glu-OE2 to Lys-NZ distances are 2.61 and 3.31 Å, respectively. The comparable salt bridges between subunits B and D, namely Glu-43(b)-Lys-65(d) and Glu-43(d)-Lys-65(b), which have Glu-OE2 to Lys-NZ distances of 6.70 and 7.29 Å, respectively, are not made due to the absence of perfect 222 symmetry in the crystal packing.

Lys-47 to Gln-53 form a hairpin loop (Sibanda and Thornton, 1985) between strands I and II of the beta-sheet. There is a 14-atom turn involving main chain hydrogen bonds formed between the nitrogen of Gly-48 and the oxygen of Cys-51 and between the nitrogen of Cys-51 and the oxygen of Gly-48. Strands II and III of the beta-sheet are formed by Val-54 to Thr-59 and by Arg-64 to Leu-68, respectively. They are linked by Leu-60 to Gly-63, which form a hydrogen-bonded type I reverse turn. There are two beta-bulges (Richardson, 1981): a wide type at Ile-39-Gln-40 and a G type at Gly-63-Arg-64.

Asp-69 to Arg-74 loop between strand III and the helix at the end of the molecule. There is a hydrogen bond between Asp-69 and Ala-72 that forms an inverse -turn (Rose et al., 1985). Ile-75 to Asp-86 form an alpha-helix that lies diagonally across the top of the beta-sheet.

Comparison of the 4 NAP2 Molecules

Although the 4 molecules of NAP-2 present in the asymmetric unit of our NAP-2 crystals are crystallographically independent and occupy nonequivalent environments, they all have the 15-residue open loop, three strands of antiparallel beta-sheet, and the 12-residue helix lying across the pleated sheet as described above. The 4 molecules are paired to form two extended beta-pleated sheets of six strands each (Fig. 2), which are then arranged back to back to form a tetramer with approximate dimensions of 35 times 35 times 45 Å^3. The subunits of the tetramer are related by quasi 222 symmetry. The two six-stranded beta-sheets that lie back to back in the tetramer are bisected by a perpendicular rotation axis that relates subunits A to B and subunits C to D with a rotation angle of 178.5° (ALIGN). The two rotation axes parallel to the beta-sheets relate subunits A to C and subunits B to D with a rotation angle of 179.4° and subunits A to D and subunits B to C with a rotation angle of 170.1°.

The individual molecules are similar to one another as evidenced by the average root mean square deviation of 0.47 Å in C-alpha positions when they are overlapped in pairs (Table 2). In these six pairwise comparisons, the C-alpha atoms of 14 residues (Ala-21 to Arg-24, Ile-28, Lys-29, His-35, Gly-48 to His-50, Asn-52, and Ala-84 to Asp-86) were separated by more than twice the average root mean square deviation in at least one of the overlaps. The largest C-alpha deviations between the subunits (2.0-8.58 Å) occur at the amino terminus where Ala-21(a) to Arg-24(a) interact with residues in adjacent asymmetric units and consequently exhibit a markedly different conformation from the same residues in subunits B and D (Fig. 5). Only five of the 14 residues, namely Arg-24, Gly-48, Thr-49, Ala-84, and Asp-86, have C-alpha deviations greater than 0.94 Å that are not also associated with differences in packing contacts with other subunits, either in the same or adjacent asymmetric units. Among these five residues, Thr-49, which is part of the loop between beta-strands I and II, has the largest C-alpha displacement (2.18 Å between subunits B and C) and exceeds 0.94 Å in all pairwise comparisons, except in the overlap of subunits A and C.




Figure 5: Conformation of the ELR sequence in the subunits of NAP-2. The conformations of Ala-21, Glu-22, Leu-23, Arg-24, Cys-25, Leu-26, and Cys-27 are compared for subunits A (thinlines) and B (thick lines). The conformation of Arg-24(b) shown in solidlines is the one most similar to that present in subunit D; the conformation shown in dashedlines is an alternative conformation present in subunit B only. In subunit C, the first three residues are disordered, and the conformation of Arg-24(c) is similar to that of Arg-24(b) shown in solidlines. In subunit D, the four residues have a conformation similar to that shown for subunit B.




DISCUSSION

The secondary structure and main chain hydrogen bonding of NAP-2 as shown in Fig. 3are very similar to those of the other known structures in the CXC chemokine family (St. Charles et al., 1989; Baldwin et al., 1991). Fig. 3includes all of the hydrogen bonds identified in an NMR analysis of the beta-sheet region of NAP-2 (Yang et al., 1994) with the exception of the bond between the nitrogen of Arg-64 and the oxygen of Leu-60, which is a bond between the nitrogen of Gly-63 and the oxygen of Leu-60 in the x-ray structure, as well as three beta-sheet hydrogen bonds that are not detected by NMR, namely the two bonds between Ile-45 and Glu-55 and the bond between the nitrogen of Ile-57 and the oxygen of Glu-43.

Tertiary and Quaternary Interactions

In addition to the disulfide and hydrogen bonds shown in Fig. 3, several clusters of hydrophobic residues contribute to the integrity of the subunits. For example, Ile-34(a) and Pro-36(a), which reside in the loop structure, cluster with Ile-39(a), Leu-42(a), Ile-66(a), and Leu-68(a) in the beta-sheet and with Ile-75(a) and Ile-78(a) in the alpha-helix. At the helix-sheet interface, Leu-42(a) and Val-56(a) in the beta-sheet contact Val-79(a) and the C-, C-, and C- atoms of Lys-82(a). Similarly, the dimer interface is strengthened by hydrophobic contacts between residues in each helix, for instance Val-79(a), Lys-82(a) (C-, C-, and C-), and Leu-83(a), and between residues in the beta-sheet of the partner, Leu-42(b), Val-44(b), and Val-56(b). The overlap of the inner hydrophobic surfaces of the alpha-helices with the beta-sheet strands from the adjacent monomer creates a shallow hydrophobic groove between the helices that has a floor defined by Leu-42(a), Val-44(a), Val-54(a), and Val-56(a) along with the equivalent residues in subunit B and walls defined by Pro-70(a), Ile-75(a), Lys-76(a) (side chain C-, C-, and C- atoms), Val-79(a), and Leu-83 along with the same residues from subunit B. The CD dimer has a similar groove.

The last four residues, Glu-87, Ser-88, Ala-89, and Asp-90, are disordered in the electron density maps of NAP-2 {M6L}. They are not part of the binding site for receptors (Clark-Lewis et al., 1994), but when one to three of these residues are removed, the activity of NAP-2 increases 4-fold (Brandt et al., 1993). Possibly, these residues inhibit the activity of NAP-2, which is most active as a monomer (Schnitzel et al., 1994), by extending the alpha-helices farther across the adjacent beta-sheets and thereby stabilizing the dimer interface.

The interface of the AB and CD dimers also is stabilized through hydrophobic interactions. Leu-26(a) clusters with its counterpart, Leu-26(c), and also with Ile-57(c) and the side chain C-, C-, and C- atoms of Lys-65(c). Ile-45(a) in the beta-sheet also interacts with the C-, C-, and C- atoms of Lys-65(c). Similarly, Leu-26(b) contacts Leu-26(d). However, the interface between the two dimers, which contains the four buried salt links discussed earlier, appears to be predominantly hydrophilic in that 18 of the 22 residues in the CD dimer that are within 3.5 Å of a residue in the AB dimer are hydrophilic.

Additional evidence for structural significance of the residues mentioned above comes from the similarity between the specific residues in NAP-2 and residues in homologous proteins within the chemokine family (Fig. 1). The potential for salt links between Glu-43(a) and Lys-65(c) and between Lys-65(a) and Glu-43(c) is conserved in bovine PF4, human PF4, and IP-10, while in IL-8, the acidic and basic residues have switched positions, with lysine replaced by arginine at position 43. Also, almost all of the hydrophobic residues mentioned above as having important structural roles in NAP-2 occupy the same positions throughout the chemokine family and are highly conserved.


Figure 1: Sequence homology within the chemokine CXC subfamily. The sequences have been aligned to show the conservation of the four cysteine residues. The sequence of NAP-2 is compared to other known proteins belonging to the CXC family, namely human IL-8 (Sherry and Cerami, 1991; Baggiolini and Clark-Lewis, 1992), ENA-78 (Walz et al., 1991), GROalpha (Haskill et al., 1990; Tekamp-Olson et al., 1990), macrophage inflammatory protein-2 (Wolpe et al., 1988), bovine (Ciaglowski et al., 1986) and human (Poncz et al., 1987) PF4, and IP-10 (Luster et al., 1985). The conserved ELR sequence and the 12 structurally conserved hydrophobic residues are boxed. The residue positions in PF4 are in boldface above the sequences, and the corresponding numbers in IL-8 are given below.



One exception occurs at position 26, where wild-type NAP-2 has methionine and other chemokines have valine, leucine, threonine, or glutamine (Fig. 1). The hydrophobic contacts described above for Leu-26 in our NAP-2 structure, which replaces the wild-type methionine, suggest that a medium-sized hydrophobic residue at this position shifts the dimer/tetramer equilibrium toward tetramers. Human and bovine PF4, which have Leu-26 and Val-26, respectively, form tetramers in the crystal similar to those of our NAP-2 mutant and form mostly tetramers in solution (Bock et al., 1980; Mayo and Chen, 1989), whereas NAP-2 and IL-8, which have Met-26 and Gln-26, respectively, exhibit a monomer/dimer/tetramer equilibrium in solution (Schnitzel et al., 1994; Yang et al., 1994). At physiological concentrations of 10M and lower, both IL-8 and NAP-2 exist and act primarily as monomers (Rajarathnam et al., 1994; Schnitzel et al., 1994; Yang et al., 1994), although protein cross-linking experiments indicate that IL-8 can also bind as an oligomer to its receptor (Schnitzel et al., 1994). When Gln-26 in IL-8 is replaced by leucine, it remains active (Clark-Lewis et al., 1994), as does NAP-2 when Met-26 is replaced by leucine (Castor et al., 1990).

ELR Region

The amino-terminal residues Glu-22, Leu-23, and especially Arg-24 have been identified in IL-8 as being important for receptor binding (Hébert et al., 1991; Clark-Lewis et al., 1991, 1993; Moser et al., 1993). This ELR sequence, which is conserved in the chemokines NAP-2, GROalpha, and murine inflammatory protein-2 (Fig. 1), which compete with IL-8, is necessary but not sufficient for binding to the two classes of IL-8 receptors on neutrophils (Oppenheim et al., 1991). For example, IL-8 binds tightly to both receptors, whereas the other chemokines bind tightly to only the type II receptor (Moser et al., 1991; Holmes et al., 1991; Murphy and Tiffany, 1991). When the ELR sequence is introduced into PF4 and IP-10, the former acquires activity, but the latter does not (Clark-Lewis et al., 1993). Besides the ELR motif, a compact tertiary structure with intact disulfide bonds is needed (Peveri et al., 1988; Van Damme et al., 1989) in addition to appropriate residues at positions 22-43, 47-52, and 66 (IL-8 residues 4-22, 30-35, and 49) (Clark-Lewis et al., 1994).

The conformation of the amino-terminal ELR region is well defined in every subunit of NAP-2 except subunit C (Fig. 4), which lacks density for Ala-21(3) to Leu-23(c). However, the particular structure adopted by an ELR region depends strongly upon the local environment. The ELR regions of subunits A and B have very different structures (Fig. 5) because they are stabilized by different interactions: the former with a symmetry-related NAP-2 tetramer and the latter with subunit D in its own tetramer. Conversely, the ELR region of subunit D reciprocally interacts with subunit B in the same tetramer and adopts a conformation similar to that seen in subunit B. Because of crystal packing and asymmetry in the NAP-2 tetramer, neither interaction is accessible to the ELR region in subunit C, which is consequently disordered. Clearly, the main chains of the ELR regions exhibit unusual flexibility despite being tethered to the core of the protein through disulfide bridges at Cys-25 and Cys-27. Moreover, the side chain of Arg-24(b), which has no intra- or intermolecular contacts, exhibits two conformations, indicating that this critical residue in receptor binding also has intrinsic conformational flexibility (Fig. 5). At physiological concentrations, where NAP-2 probably functions as a monomer (Rajarathnam et al., 1994; Schnitzel et al., 1994; Yang et al., 1994), the ELR region would be unconstrained by intermolecular interactions, much like the case with subunit C, and could adopt an extended conformation on the receptor if necessary.

The positions of the ELR residues in the IL-8 crystal structure (Baldwin et al., 1991) match those in subunit B of NAP-2 better than in subunit A or D. The C-alpha atoms of the two leucine residues and the two arginine residues are separated by 3.65 and 1.11 Å, respectively (Glu-22 is not defined in the IL-8 crystal structure). Although Leu-23(b) and Arg-24(b) in NAP-2 and Leu-23 in the IL-8 crystal structure have no intermolecular contacts, the side chain of Arg-24 in IL-8 forms three hydrogen bonds with a symmetry-related molecule in the crystal. The first five amino-terminal residues in the IL-8 NMR structure are considered as being partially disordered due to a standard deviation of 5 Å in the atomic positions of these residues (Clore et al., 1990). In the bovine PF4 crystal structure, the amino-terminal residues are disordered, and position 24, which is the first residue defined by electron density in the structure, is a Gln instead of Arg (St. Charles et al., 1989).

It has also been suggested that Glu-22 {E4} in IL-8 may form a salt bridge with either Lys-40 {K23} or Lys-59 {K42} in the adjacent monomer of IL-8, but since neither lysine is critical for receptor binding, it is more likely that Glu-22 interacts with the receptor and not with the other subunit in the dimer (Hébert et al., 1991). The equivalent residues in NAP-2 are Gln-40 and Thr-59, so no ion pair is possible in our structure. Ile-28 in IL-8 has also been shown to be sensitive to mutagenesis and important for receptor binding (Hébert et al., 1991). The side chain of Ile-28, which occupies excellent electron density in our structure, has only one contact within 4.0 Å (Lys-29 C-) and is sufficiently exposed to interact directly with the receptor.

Activation of NAP-2

beta-TG, CTAP-III, and PBP, which differ from NAP-2 only in having an additional 11, 15, and 24 residues, respectively, added to the amino terminus, are all inactive toward the IL-8 receptors at physiological concentrations (Walz et al., 1989). The longer isoforms, which predominate in quiescent platelets, are converted to NAP-2 by cathepsin G, which cleaves the Tyr-21-Ala-22 peptide bond and releases the activation peptide. The system has strong positive feedback in that NAP-2 activates neutrophils, which release cathepsin G (Cohen et al., 1992), which in turn activates platelets. Since a longer amino terminus does not induce a detectable change in the secondary structure of the NAP-2 portion (Yang et al., 1994), it is likely that the extended amino-terminal chain inhibits by blocking access to one or more of the sites on NAP-2 that bind the receptor. The neural network method of Holley and Karplus (1989) predicts a turn of alpha-helix in the activation peptide consisting of Glu-12 to Asp-16. This predicted structure places two acidic groups on the same side of the helix, where they could interact with a positive residue such as Arg-24 (Fig. 6).


Figure 6: A model for the activation peptide of NAP-2. A helix is predicted by the method of Holley and Karplus (1989) for Glu-12 to Asp-16 in the activation peptide of beta-TG (Gly-10 to Tyr-20). The activation peptide, which presents two negatively charged side chains on one side of the helix, is shown bending back to interact with the ELR region of NAP-2. The activation peptide is released from beta-TG to form NAP-2 by cleavage between Tyr-20 and Ala-21 (dashedarrow).



The activation peptide present in beta-thromboglobulin (residues 10-20) has a provocative similarity to the amino-terminal extracellular domain of the IL-8 receptor in having a high concentration of acidic residues. Clubb et al.(1994) have proposed from NMR data that the receptor peptide, which has nine acidic residues among 40 positions for the type I receptor and 12 acidic residues among 49 for the type II receptor, binds to IL-8 in the cleft between residues 30-38 {residues 12-21} and the third strand of beta-sheet, where it could interact with a cluster of basic residues. NAP-2 has three conserved residues whose basic side chains project into this region, namely Lys-29, Lys-37, and Arg-64.

Another similarity between the activation peptide in beta-TG and the amino-terminal extracellular domain of the IL-8 type II receptor is the prediction that residues 43-46 (ESLE) of the receptor have the same helical structure, with two acidic groups on the same side of the helix, as predicted for the activation peptide. Moreover, residues 43-46 are in the one stretch of the receptor sequence that exhibits significant similarity to the sequence of the activation peptide. The predicted alpha-helical region in the receptor peptide lies near the first transmembrane alpha-helix in the proposed receptor structure (LaRosa et al., 1992). Therefore, cleavage of beta-TG or a longer precursor into NAP-2 not only would remove the residues blocking the ELR sequence or other subsite, but would also allow the newly exposed amino terminus of NAP-2 to approach the receptor membrane closely and to interact with the putative alpha-helix in the receptor sequence. The type I receptor, for which NAP-2 has a much lower affinity, is less similar in sequence to the activation peptide and cannot have a helix with two acidic residues on the same side. These predictions raise the intriguing possibility that the activation peptide mimics, at least in part, the receptor interactions with NAP-2.

However, extensive alanine scanning of the IL-8 type I receptor sequence did not find any critical acidic residues among the 40 residues of the amino-terminal extracellular domain (Hébert et al., 1993; Leong et al., 1994). Replacement of Thr-18, Pro-21, or Tyr-27 reduced significantly the binding constant for IL-8 but not the mobilization of calcium, while replacement of Cys-30, which is near the predicted helical region in the type II receptor (as the conserved Cys-40), abolished both binding and transduction. An additional 19 residues in the second, third, and fourth extracellular domains were also shown to be involved in IL-8 binding.

Comparison with Other Chemokine Structures

Together, the crystal structures of NAP-2 and IL-8 identify some of the nuances of the interaction between the chemokines and their receptors. Two types of human neutrophil receptors for IL-8, whose sequences are 77% identical, have been separately cloned and expressed in mammalian cells (Moser et al., 1991; Holmes et al., 1991; Murphy and Tiffany, 1991; Cerretti et al., 1993). IL-8 activates both type I and II receptors while NAP-2, which has 44% identity to IL-8, activates only the type II receptors at equivalent concentrations, although at high concentration, it can completely displace bound IL-8 from the surface of neutrophils (Petersen et al., 1994). In contrast, PF4 and IP-10 activate neither receptor (Clark-Lewis et al., 1993, 1994). However, when residues 22-43, 47-52, and 66 in IP-10 are replaced by the equivalent residues in IL-8 {residues 4-22, 30-35, and 49}, the hybrid IP-10 is as potent as IL-8 in activating neutrophils (Clark-Lewis et al., 1994), which suggests that positions 22-43, 47-52, and 66 in the CXC chemokine sequence define the binding site(s) for both types of receptor. Of course, it is still possible that other residues, which by chance are identical in IL-8 and IP-10, are also critical parts of an extended binding site. However, outside of the three subsites defined above, IP-10 has only nine residues that are identical to those in IL-8, and of these, only one is on the surface in the IL-8 structure and accessible to a receptor molecule.

Consequently, the explanation for IL-8 binding strongly to both receptors but NAP-2 only binding tightly to the type II receptor probably lies in the structural or chemical differences in these three subsites. An upper limit for structural differences attributable to error and thermal motion can be calculated from the differences among the independently determined structures of the four NAP-2 subunits. The maximum average C-alpha displacement relative to subunit B is 0.58 ± 0.28 Å for subsite I (residues 25-39 in subunit C), 1.09 ± 0.58 Å for subsite II (residues 47-52 in subunit C), and 0.42 Å for subsite III (residue 66 in subunit A). The amino-terminal ELR residues, which have large differences among some subunits as discussed above, were omitted from these calculations.

By these criteria, the structure of NAP-2, as measured by C-alpha positions relative to those of IL-8, exceeds the average displacement by more than two standard deviations at positions 28 and 32-37 ( Fig. 7and Fig. 8) in subsite I, but at no positions in subsite II or III. Although four of the seven residues at positions 32-37 are identical between IL-8 and NAP-2, the structural differences are not surprising given the insertion of Pro-33A in the sequence of IL-8. This site is the strongest candidate for a structural difference excluding NAP-2 from binding to the type I receptor.


Figure 7: Comparison of NAP-2 with IL-8 and PF4. The alpha-carbon backbone of subunit B of NAP-2 (thicklines) is compared with that of IL-8 (A, thinlines; x-ray structure) and human PF4 (B, thinlines). The superpositions were calculated with residues 23-86 and 24-85, respectively.




Figure 8: Deviations in C-alpha atom positions. The deviations between C-alpha (Ca) positions are shown for NAP-2 overlapped with the IL-8 x-ray structure (top) and the human PF4 structure (bottom).



Gly-48 and Pro-49 are critical for maximal binding of IL-8 to its receptors (Clark-Lewis et al., 1994). If these two residues in IL-8 are switched or replaced by the equivalent residues in IP-10 (Ser-48 and Gln-49), binding is decreased 170-fold (Clark-Lewis et al., 1994). Although residues 48 and 49 in NAP-2 are shifted by over 1.8 Å from those in IL-8, shifts of similar magnitude are seen among the NAP-2 subunits themselves. Because the chemokine structure is unusually flexible in this region, any effects of structural differences must be secondary to those caused by differences in sequence, namely the presence of Thr-49 in NAP-2 instead of the proline found in IL-8.

Surprisingly, mutagenesis of His-50 in IL-8 {H33} does not affect activity, in contrast to its immediate neighbors, Gly-48 {G31} and Pro-49 {P32} (Clark-Lewis et al., 1994), although its side chain is near the ELR region in the IL-8 NMR structure, where it forms a hydrogen bond with Gln-26. A possible explanation lies in the NAP-2 structure, which shows that the His-50 side chain has two conformations available, one of which is buried. The side chains of His-50(a) ((1) = -54°) and His-50(c) ((1) = -48°) are solvent-accessible (side chain-accessible surface areas of 56.7 and 64.6 Å^2, respectively) and near the ELR region. His-50(a) forms two hydrogen bonds within the ELR segment (between the nitrogen of Arg-24(a) and the NE2 of His-50(a) and between the ND1 of His-50(a) and the oxygen of Glu-22(a)). The only other residue in the NAP-2 tetramer, excluding contacts due to crystal packing, with side chain atoms within 4.0 Å of the ELR region is Thr-49(a), which contacts the side chain of Leu-23(a). The side chain of His-50(c) is in a similar conformation and within 3.5 Å of Arg-24(c), but the angles of the possible hydrogen bonds exceed the limits.

The side chain of His-50(b) ((1) = 63°) adopts a different conformation than that of His-50(a) and His-50(c) in that it points away from the ELR region and is buried within the core of the protein (solvent-accessible surface area of 3.9 Å^2). Specifically, the ND1 of His-50(b) forms an intersubunit hydrogen bond with the carbonyl oxygen of Gly-63(d). The His-50 side chains in the IL-8 x-ray structure and in three subunits of the PF4 tetramer have an orientation similar to that of His-50(b) in the NAP-2 structure. The side chain conformation of His-50(d) lies between that of His-50(a), His-50(c), and His-50(b), with a (1) angle of -47° and a solvent-accessible surface area of 41.9 Å^2. His-50(d) lies within 5.7 Å of the ELR region, but does not have contacts with it. However, the NE2 of His-50(d) does form an intersubunit hydrogen bond with the carbonyl oxygen of Asp-62(b).

The third region with significant structural differences among the CXC chemokines involves the alpha-helices at the carboxyl terminus of each subunit. In the crystal structures of NAP-2 and IL-8 and the NMR structure of IL-8, the pair of antiparallel helices in the AB dimers (and CD dimers in NAP-2) traverse the extended beta-sheets at an angle of approximately 58°. The distance between the helices in each pair is 8.5 Å for the crystal structures of NAP-2 and IL-8, but 10 and 11 Å for the crystal structure of bovine PF4 and the NMR structure of IL-8, respectively. Moreover, the helices of bovine and human PF4 are translated approximately 1.8 Å with respect to the helices in NAP-2 and IL-8 (Fig. 7B), which accounts for the larger than average root mean square deviation between the helical regions (Table 2).

Mutagenesis studies have shown that residues 52-72 in IL-8, which include the carboxyl-terminal alpha-helix, are not directly involved in receptor binding or activation (Hébert et al., 1991; Clark-Lewis et al., 1991), but play a structural role instead. The helix has also been implicated in the binding of heparin to PF4 (Hardin and Cohen, 1976) and of heparin and heparan sulfate to IL-8 (Webb et al., 1993). The binding of heparan sulfate to IL-8 increases the rate of chemotaxis by 3-4-fold and enhances Ca release. Heparin had a similar effect on Ca response, but did not enhance chemotaxis. The binding of IL-8 to heparan sulfate is abolished with the removal of residues 69-90 (Webb et al., 1993). Although the heparinbulletIL-8 complex has a different overall charge and perhaps a slightly different conformation, the increased activity of IL-8 could also be due to the stabilization of IL-8 oligomers when the anionic polysaccharide binds across or between the pairs of helices.

Although no crystal structure exists of the complex between heparin and any chemokine, Stuckey et al.(1992) have proposed a model of heparin binding to bovine PF4 that predicts that seven basic residues, namely His-35, Lys-37, Lys-61, Lys-76, Lys-77, Lys-80, and Lys-81, interact with the negatively charged heparin chain that runs perpendicular to a pair of alpha-helices in a dimer. NAP-2 has Lys-80 replaced by glutamine and a new lysine at position 82, which dramatically changes the arrangement of positive charges in the helices (Fig. 9) as proposed by Lawler(1981). When NAP-2 is overlapped with bovine PF4 in the model of Stuckey et al. (1992), Lys-76 and Lys-82 do not interact with the heparin acidic groups, which would explain the weaker heparin binding of NAP-2 relative to PF4. Platelet basic protein, the precursor of NAP-2, elutes from a heparin-Sepharose column with 0.6-0.7 M NaCl (Holt and Niewiaroski, 1985), which is comparable to IL-8, which elutes from heparin columns at 0.5 M NaCl (Van Damme et al., 1989).


Figure 9: Comparison of the positively charged side chains in the carboxyl-terminal alpha-helix of NAP-2 and recombinant human PF4. The main chain atoms for residues 75-85 and the side chain atoms for lysines 76, 77, 81, and 82 in the carboxyl-terminal alpha-helix of subunit B of NAP-2 (thicklines) are shown in stereo overlapped with the equivalent main chain atoms and the side chain atoms for residues 76, 77, 80, and 81 of recombinant human PF4 (thinlines).




FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM33192 (to B. F. P. E.) and National Institutes of Health Training Grant T32 HL07602 (to M. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1NAP) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Wayne State University, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-1506; Fax: 313-577-2765.

(^1)
The abbreviations used are: NAP-2, neutrophil-activating peptide-2; PBP, platelet basic protein; CTAP, connective tissue-activating peptide; beta-TG, beta-thromboglobulin; PF4, platelet factor 4; IL-8, interleukin-8; GRO, growth-related protein; IP-10, interferon--inducible peptide-10; HPLC, high pressure liquid chromatography.

(^2)
The residues of NAP-2 and the other chemokines are identified in the text by the three-letter code for amino acids and the equivalent position of the residue in the sequence of bovine platelet factor 4 (Fig. 1). Where appropriate, residues are also identified between braces by the one-letter code and the position of the residue in its own sequence. By this convention, the 70 residues in NAP-2 extend from Ala-21 {A1} to Asp-90 {D70}. We use residue positions 21-90 when speaking generally of all four subunits. Specific residues in the separate subunits of the NAP-2 tetramer are distinguished by subscripts, for example, sequence numbers 21^a-90(a) for subunit A.

(^3)
P. Martin, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Philip Martin for valuable advice, especially during data reduction, and for computer programs to average electron density maps using noncrystallographic symmetry.


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