©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis Studies of Interleukin-8
IDENTIFICATION OF A SECOND EPITOPE INVOLVED IN RECEPTOR BINDING (*)

(Received for publication, September 18, 1995; and in revised form, January 2, 1996)

Glyn Williams (§) Neera Borkakoti Gillian A. Bottomley Irene Cowan Amanda G. Fallowfield Philip S. Jones Stephen J. Kirtland Glyn J. Price Lauri Price

From the Roche Research Centre, Welwyn Garden City, Hertfordshire, AL7 3AY United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interleukin-8 (IL-8) is a dimeric, C-X-C chemokine, produced by a variety of cells and which elicits pro-inflammatory responses from the neutrophil. As a prelude to drug design, we have investigated the interactions between IL-8 and its receptor by preparing a number of single-site mutants of IL-8 and determining their activity in receptor-binding and functional assays. In order to define the binding surface as precisely as possible, we have used chemical shifts obtained from nuclear magnetic resonance spectroscopy to screen mutant proteins for structural changes which affect regions of the IL-8 surface remote from the site of mutation. In addition to a previously recognized sequence, Glu^4-Leu^5-Arg^6 in the N-terminal peptide, we have identified a second epitope comprising a contiguous group of non-sequential, solvent-exposed, hydrophobic residues, Phe, Phe, Ile, and Leu. These two receptor-binding regions are separated by over 20 Å in the IL-8 structure and are important both for receptor binding and function. In addition, we have shown through the production of a covalently linked IL-8 dimer, that subunit dissociation is not necessary for biological activity.


INTRODUCTION

Interleukin-8 (IL-8) (^1)is a pro-inflammatory polypeptide produced by a number of cell types (e.g. T-lymphocytes, monocytes, endothelial cells, epithelial cells, and neutrophils) in response to a variety of stimuli (1) . These include lipopolysaccharide, IL-1, and tumor necrosis factor. IL-8 affects the function of several cell types, notably neutrophils, causing chemotaxis, adhesion, and activation and acts on endothelial cells leading to angiogenesis. It mediates shedding of L-selectin and rapid up-regulation of a variety of adhesion molecules (CR1, CD11b/CD18, CD11c/CD18) on the neutrophil surface, facilitating their adherence to endothelial cells(2) . In addition, IL-8 causes remodeling of the neutrophil cytoskeleton and chemotaxis along the IL-8 concentration gradient originating from the site of inflammation(3) . Anti-IL-8 neutralizing monoclonal antibodies have been used in vivo to demonstrate that blocking IL-8 dramatically inhibits neutrophil-mediated inflammation in various animal models, such as lung reperfusion injury(4) .

The three-dimensional structures of IL-8 (5, 6) and three other C-X-C chemokines, neutrophil activating protein-2(7) , platelet factor 4(8) , and melanoma growth stimulating activity(9, 10) , have been determined and are found to be multimeric with a common dimer fold, consisting of two antiparallel alpha-helices lying above a six-stranded, antiparallel beta-sheet (Fig. 1a). The fold of the monomer unit is also shared by the C-C chemokines, RANTES(11, 12) , and macrophage inflammatory protein-1beta (13) although the quaternary arrangement of the monomers is quite different. Site-directed mutagenesis of the charged residues of IL-8 and of the 15 N-terminal amino acids has identified residues Glu^4, Leu^5, Arg^6, and Ile as being important for receptor binding(14) . Syntheses of IL-8 analogues progressively truncated at the N terminus have confirmed the importance of these residues (15) .


Figure 1: Structure of the IL-8 dimer and a putative, covalently-linked mutant. The two chains of the non-covalently linked IL-8 dimer are shown in blue and gray. a, the IL-8 dimer structure is formed by two antiparallel alpha-helices at the C termini of each chain, lying above a six-stranded, antiparallel beta-sheet. Residues Glu and Ala from different chains are well placed to form a disulfide bond after mutation to cysteine. b, a molecular model (INSIGHT, BIOSYM Technologies Inc., San Diego, CA) indicates that the E29C,A69C mutant can form two inter-monomer disulfide bridges with minimal steric effects. The resulting bond tethers the alpha-helix of one monomer to the beta-sheet of the other.



Experiments performed using C-X-C chemokine chimers, suggested by sequence alignments of IL-8, platelet factor 4, -interferon inducible protein-10, and melanoma growth stimulating activity(16) , have indicated that the N-terminal ELR sequence of IL-8 is necessary, but not sufficient for biological activity (monitored in an elastase-release assay or by displacement of the radiolabeled ligand from its receptor). This is supported by the observation that the simultaneous mutation of Glu^4-Leu^5-Arg^6 to Ala^4-Ala^5-Ala^6 is calculated to reduce receptor binding by a factor of approximately 10^7 in total(14) . This represents a contribution to the receptor/ligand binding energy for these three side chains of approximately 10 kcal mol(17) . While a precise figure for the entropic penalty for the bimolecular association of a peptide ligand and a membrane-bound receptor is difficult to determine, estimates for low molecular weight ligand-receptor interactions are available(18) . These estimates indicate that the binding energy provided by the N terminus alone is insufficient to overcome the entropic penalty of bimolecular association. In addition, short peptides having the N-terminal IL-8 sequence are inactive in a receptor-binding assay(19) .

We report here the preparation, structural characterization, and activity of 29 single-site mutants and one double-mutant of human IL-8 which were designed to identify receptor-binding regions in addition to the N-terminal peptide. It has been suggested that a reduction in receptor binding activity greater than 10-fold on mutation is sufficient to identify that residue as being within the contact surface for the interaction(20) . However, such differences arise from relatively small changes in binding energy and, in order to locate the binding surface as precisely as possible, the structural effects of mutation on the conformations of local residues should also be considered. While the structural characterization of each mutant at high resolution by nuclear magnetic resonance (NMR) or x-ray crystallography is impractical, a method based on NMR chemical shifts was developed, by which IL-8 mutants could be screened rapidly to assess the likely degree of structural change. Those mutants in which significant structural change was indicated were then either subjected to a more detailed analysis or excluded from the receptor-binding analysis.

In a previous study, Hébert and co-workers (14) have systematically mutated the charged residues of IL-8, singly or in groups, to alanine. Charged residues were selected on the grounds that they are more likely than neutral or hydrophobic residues to be solvent-exposed and therefore available for receptor binding. We set out to extend this approach to the remaining surface-exposed residues while using structural data from NMR to check the effects of mutation on the protein structure wherever possible. The residues mutated in this study and by Hébert et al.(14) are shown in Fig. 2; together they account for 86% of the solvent-exposed surface of the dimer.


Figure 2: Coverage of the IL-8 dimer surface by mutagenesis. Mutations made by Hébert et al.(14) , principally of charged residues, are shown in blue. Additional residues which were mutated in this study are shown in yellow. The combined sets cover approximately 86% of the solvent-accessible dimer surface. The residues which have not been mutated are expected to play an important role in the protein structure and include four cysteine and two glycine residues, as well as a number of residues whose side chains form hydrophobic contacts within the dimer. Ribbon and space-filling representations are shown, and the view given is in the plane of the beta sheet, perpendicular to the mean helix axis.



While IL-8 is a dimer under the conditions of the x-ray and NMR structural studies, there is some disagreement as to the association state under physiological conditions(21, 22) . Experiments indicate that all necessary receptor-binding epitopes are contained within a single monomer unit of IL-8(23) . If dissociation of the dimer occurs on receptor binding, further surfaces of IL-8 will be exposed which may interact with the receptor. In order to investigate the importance of the dimer interface in receptor activation, we have constructed a covalently-linked IL-8 dimer in which residues Glu and Ala have been mutated simultaneously to cysteine, leading to the formation of two inter-monomer disulfide bridges (Fig. 1b).


EXPERIMENTAL PROCEDURES

Mutagenesis

The cloning and expression strategy took into account the finding of Hébert et al.(24) , that monocyte-derived IL-8 can be generated from endothelial-derived IL-8 by thrombin digestion. A synthetic gene encoding the 77 amino acids of endothelial-derived IL-8, with 6 additional histidine residues at the N terminus, was constructed from 6 overlapping oligonucleotides and cloned into the expression vector pDS56/RBSII(25) . The ligation reaction was used to transform Escherichia coli strain M15, containing the LacI-producing plasmid pDM1.1. Transformants were selected on L-agar containing 100 µg/ml ampicillin and 25 µg/ml kanamycin. For the induction of gene expression, cultures were grown in antibiotic-supplemented L-broth to an OD of 0.7, and isopropyl-1-thio-beta-D-galactopyranoside added to a final concentration of 1 mM. Cultures were harvested 3 h post-induction. (His)(6)-labeled endothelial-derived IL-8 was extracted with 6 M guanidine hydrochloride, containing 3% mercaptoethanol, from the E. coli cell paste and loaded directly on to a Ni-nitrilotriacetic acid column(26) . The column was washed sequentially with 6 M guanidine hydrochloride, 8 M urea, 2 M urea, and thrombin cleavage buffer (50 mM Tris-HCl, 200 mM NaCl, 2.5 mM CaCl(2), pH 7.5). The recombinant protein was eluted in thrombin cleavage buffer containing 0.5 M imidazole. Monocyte-derived IL-8 was generated from (His)(6)-labeled endothelial-derived IL-8 by incubating with human thrombin (Sigma) for 1 h at 37 °C (1 unit of thrombin/10 µg of recombinant protein) and the products of the digest chromatographed on a wide-pore reverse-phase high performance liquid chromatography column (21.4 times 50-mm Dynamax C8) in 0.1% trifluoroacetic acid and a 20-55% acetonitrile gradient at a flow rate of 20 ml/min.

Site-directed mutants were produced by single-stranded mutagenesis (27) using synthetic oligonucleotides containing the desired change(s) in codons and verified by DNA sequencing. All mutants were expressed and purified as described above.

Human Neutrophil Isolation

Heparinized blood from volunteers was layered onto Lymphocyte Separation Medium (ICN Biomedicals) and centrifuged (300 times g, 30 min) to yield a fraction enriched in granulocytes and red cells. This fraction was removed and mixed with an equal volume of 1.8% (w/v) dextran in phosphate-buffered saline, pH 7.4, and stood (1 h, room temperature) to sediment the red cells. The neutrophil-rich fraction was centrifuged (300 times g, 10 min) and residual red cells were disrupted by hypotonic shock(28) . The neutrophils were isolated and washed by centrifugation (300 times g, 10 min) in phosphate-buffered saline. Cell viability (assessed by trypan blue exclusion) was >96%.

Preparation of Neutrophil Membranes

Neutrophils (10^8 cells/ml in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl(2), 0.5 mM EDTA, 100 kalikrein inhibitor units/ml aprotinin, and 2 µM phenylmethylsulfony fluoride at 4 °C) were disrupted by ultrasound (3 times 15 s at 23 kHz and with 12 µm peak-to-peak amplitude). The membranes were isolated by differential centrifugation at 4 °C (400 times g for 20 min and 95,000 times g for 40 min) and resuspended in 50 mM Tris-HCl, pH 7.5, containing 0.5 mM EDTA. The protein content was estimated by the method of Lowry et al.(29) and aliquots of the membrane suspension were stored at -70 °C.

Ligand Binding Experiments

Competition experiments with the mutants and radiolabeled IL-8 for binding to neutrophil membranes were carried out in a 96-well microtiter filtration system (Millipore MultiScreen) using 0.22-µm pore size, low protein-binding filters. The reaction mixture (100 µl, final volume) contained 1.0% (w/v) bovine serum albumin, 0.1% sodium azide, 0.25 nMI-labeled IL-8 (2,000 Ci/mmol, Amersham), mutant protein, 0-6 µM, and 5 µg of membrane preparation in phosphate-buffered saline. Quadruplicate estimations were performed for each concentration of mutant. Nonspecific binding was defined as that determined in the presence of a 1,000-fold excess of native IL-8. The reaction was carried out at 4 °C for 90 min and terminated by vacuum filtration (Eppendorf, event 4160). The filters were washed 3 times (200 µl per well) with ice-cold phosphate-buffered saline, dried, and punched out using a MultiScreen multiple punch assembly. Radioactive material on the filters was determined in a -counter (Wallac). Binding data was analyzed and K(i) values calculated using the EBDA/LIGAND programs(30) .

Calcium Transient Assay

Isolated neutrophils were resuspended at 10^7/ml in Hanks' balanced salt solution (Life Technologies, Inc.) containing 0.1% (w/v) bovine serum albumin. The cells were incubated with 1 µM Fura 2/AM (Fluka) at 37 °C for 30 min. The cells were washed with Hanks' balanced salt solution prior to resuspension in the same medium at 2 times 10^6 neutrophils/ml. The cells were kept at room temperature until required for testing. Fluorescence changes on addition of mutant protein to stirred cell suspensions at 37 °C were monitored in a fluorescence spectrophotometer (Perkin Elmer LS50) at an emission wavelength of 510 nm and using dual excitation wavelengths of 340 and 380 nm. The cytosolic calcium ion concentrations were calculated by the ratio method of Grynkiewicz et al.(31) using 0.1% Triton X-100 to release all the intracellular dye and saturate Fura 2 with Ca (R(max)), and with addition of EGTA to determine Ca-insensitive fluorescence (R(min)).

Design and Preparation of Covalently Linked Dimeric IL-8

In order to investigate the importance of the IL-8 dimer interface in receptor binding, the simultaneous mutation of two residues to cysteine was used to produce a covalently-tethered dimer. The mutated residues should, in the first instance, be capable of forming an intermonomer disulfide bond, but should also be arranged so that intramonomer disulfide bond formation is not possible. A recent survey of the relative orientation of pairs of cysteines in protein x-ray structures has shown that, for disulfide bond formation, the Cbeta carbon atoms of the bonded cysteines should be separated by 3.0 to 4.5 Å(32) . In addition, the study suggests that a Cbeta-Cbeta separation of greater than 5 Å is sufficient to ensure that disulfide bond formation is not feasible.

Examination of the x-ray structure of IL-8 shows that three pairs of residues have intermonomer Cbeta-Cbeta separations between 3.0 and 4.5 Å and intramonomer separations of greater than 5 Å; these are Leu-Val, Glu-Ala, and Pro-Glu. The side chains of Leu and Val form important hydrophobic interactions in the core of IL-8, while Pro participates in a turn between the outermost strand of beta-sheet and the C-terminal helix. In contrast, Glu and Ala are solvent-exposed residues whose side chains make no significant structural contacts and consequently these residues were selected for mutation. A model showing the putative disulfide bridge between Cys and Cys is given in Fig. 1b. By tethering each alpha-helix to the beta-sheet, the new disulfide bond formed should restrict their motion apart, thus also preventing receptor binding in the interhelix cleft as originally proposed for IL-8(5, 33) .

The double mutant E29C,A69C was produced using the procedure described above. After metal chelate chromatography and thrombin cleavage, the linked dimer preparation was centrifuged (3000 times g for 6 h) in a microconcentrator (Microsep, 3000 M(r) cutoff, Filtron Corp.) in order to concentrate the sample and effect buffer exchange before chromatography. The filtered sample was chromatographed on a Superdex 16/60 column, equilibrated with 50 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl, at a flow rate of 0.8 ml/min. The correctly folded dimeric material was detected via its reaction with an anti-IL-8 monoclonal antibody (Amersham). This fraction eluted with a K of 0.39, which corresponded from the calibration curve to a molecular mass of 16,800 Da. Fractions were pooled, dialyzed, and freeze-dried.

Electrospray mass spectrometry of the dimer preparation gave a molecular mass of 16,776 ± 4 Da, corresponding closely to the calculated mass of 16,771.6 Da. After reduction with 2-mercaptoethanol, material corresponding to the monomer was observed (molecular mass 8391 ± 2Da).

The purity of the product was further examined by ion exchange chromatography (Poros cation exchange column, Perseptive Biosystems) using a 50 mM sodium formate buffer at pH 4.0, 4.6, or 5.5 with an elution gradient from 0.15 to 2.0 M NaCl. At each pH, the dimer was eluted as a single peak of UV-absorbing material.

After desalting, NMR spectra of the linked dimer were obtained which indicated that the protein was homogeneous and had the expected cross-linking. Disulfide bonds between residues Cys^9-Cys and Cys^7-Cys were maintained and signals corresponding to two new cysteine residues were apparent.

NMR Methods

Freeze-dried samples, containing 0.5-3.5 mg of protein, were dissolved in 450 µl of 99.6% D(2)O (Aldrich) and the pH adjusted to 5.2 ± 0.1 using small aliquots of DCl and NaOD. NMR spectra were recorded at 40 °C, using a Bruker AM400 spectrometer. Two-dimensional TOCSY spectra were acquired using a 30 ms, MLEV-17 spin lock(34, 35) , and with 200 and 2048 complex points in 1 and 2, respectively. The spectral width was typically 4500 Hz, giving a digital resolution of 1.1 Hz per point (0.003 ppm per point) in 2 after zero filling. All chemical shifts were measured in the directly acquired dimension.

Proton assignments were taken from the literature(36) . TOCSY spectra were used to obtain the chemical shift information from complete spin systems comprising a single amino acid side chain. Most signals of a mutant protein could then be assigned by inspection since chemical shift changes were, in general, small and, where significant changes did occur, these affected one or two signals only and did not encompass the entire amino acid spin system.

Ring-current shift calculations were performed using the program VNMR (37) with IL-8 coordinates taken from a 1.6-Å refinement of the x-ray structure (6) or from the NMR structure(5) . Slightly better agreement between the observed NMR shift changes on mutation of an aromatic ring to alanine and the calculated shifts due to that ring, was obtained from calculations incorporating the x-ray coordinates and these coordinates are used in the work presented here.


RESULTS

Structural Studies of IL-8 Mutants

Given the large number of mutants studied here, a full structure determination of each, by NMR or x-ray diffraction, was impractical. For this reason, a method was sought whereby the extent and degree of structural differences between wild-type IL-8 and a single-site mutant could be estimated quickly and with reasonable confidence(38) .

Values of proton chemical shifts from NMR spectra can be determined rapidly and reliably and are dependent on three-dimensional molecular structure(39, 40, 41) . For a protein of the size of IL-8 (molecular mass = 8.4 kDa per monomer), TOCSY experiments can be performed on 1-2 mg of protein and data can be collected, processed, and interpreted within 48 h giving chemical shifts for at least two protons from the majority of amino acid residues. TOCSY spectra are preferred over other chemical shift correlation experiments (e.g. COSY) since they contain redundancies which allow the assignment of chemical shifts to be checked. Since a change in the chemical shift of a proton will be taken as a potential indicator of structural change, it is important that the assignment of signals is consistent between wild-type and mutant protein spectra. While nuclear Overhauser enhancements (NOE) are more directly related to molecular structure than chemical shifts(42) , NOE spectra generally require more sample, contain more correlation peaks and have no redundancies. Thus NOE spectra are considerably more crowded than TOCSY spectra and, without prior analysis of a TOCSY spectrum, can be interpreted only in those cases where chemical shift changes between the wild-type and mutant proteins are minimal. It should be stressed that the NMR method described here does not require isotope-labeled material and the samples are unaffected by the analysis.

The structure of IL-8 has been determined using NMR (5) and a nearly complete list of proton assignments is available(36) . For those mutants prepared in sufficient quantities, a one-dimensional NMR spectrum was first obtained and compared with that of the wild-type. In many cases, direct inspection of these one-dimensional spectra clearly showed that the chemical shift changes on mutation were negligible (<0.04 ppm) except in those regions which contained signals from the mutated residue itself. For the remaining mutants, where the one-dimensional NMR spectrum indicated significant changes in chemical shifts, two-dimensional TOCSY data were obtained.

When TOCSY spectra were compared, it was found that approximately 76 (±8) chemical shifts from 24 (±1) amino acids could be assigned unambiguously, by inspection, in all spectra. The signals from which these shifts are taken are in uncrowded regions of the spectra or give readily identifiable patterns of cross-peaks in a TOCSY spectrum. For these peaks, there is no ambiguity in assignment, despite the variation in shifts between the mutants. Inspection of the IL-8 structure shows that these 24 residues are distributed randomly throughout the protein and are thus suitable for use as ``reporter groups'' to investigate the occurrence of conformation-dependent changes. All shifts were measured with a precision of ±0.01 ppm. This implies that two independent measurements of a large number of shifts from two identical protein spectra would have an RMS error of 0.012 ppm. It is difficult to estimate the reproducibility of these measurements which would be caused by changes in ionic strength, pH, and temperature. However, experience indicates that for most signals this is likely to be less than ±0.02 ppm.

When two proteins differing by a single point mutation are compared (and the mutated residue is excluded from the comparison), the RMS shift difference for the representative set of 76 (±8) chemical shifts is in the range 0.018 to 0.041 ppm, or 1.5 to 3.5 times greater than the expected RMS for identical proteins. The increase in RMS must reflect either a structural change or a change in local magnetic susceptibilty around the site of mutation, or both. Susceptibility changes will be particularly severe if the mutation involves the loss or gain of an aromatic ring but otherwise are expected to be significant only for those residues in van der Waals contact with the mutated side chain. Two of the mutations studied here involve the loss of an aromatic ring (F21A and F17L) and one involves its gain (L43H); in these cases, the observed shift changes were corrected for the effects of the ring susceptibility using the program VNMR.

RMS shift differences between a mutant IL-8 and the wild type are given in Table 1for nine mutant proteins, including the double-mutant E29C,A69C. A tenth mutant, F17A, was found by NMR to be misfolded and aggregated and so the shifts of its representative set of signals could not be determined. RMS differences are given for the whole representative set of signals as well as for two subsets. The tendency for the structural effects of mutation to be limited in space is clearly shown by calculating separate RMS shift differences for the five most-shifted signals of the representative set and for the remainder. Excluding the double mutant, the RMS shift for the five most strongly shifted signals is in the range 0.141 to 0.040 ppm and, in four cases (A35K, P32A, F21A, and I10A), the RMS for the remainder has a value which is indistiguishable from 0.012 ppm (i.e. no structural change). In all cases, the five most strongly shifted signals arise from residues close to the site of mutation, although not necessarily in van der Waals contact with the mutated side chain. Significantly, after correction for ring-current effects, the four mutants which show signs of more extensive conformational change (F17L, I40A, V41A, and L43H) also have the largest local shift changes. The data therefore suggest that, in all the mutants studied, there is a local conformation change involving amino acid side chains around the site of mutation. The size of this conformational change varies from a minimum for the mutants I10A and A35K to a maximum for the mutants F17L and I40A. In the latter cases and those of V41A and L43H, the conformation change may extend a significant distance from the site of mutation.



The double-mutation E29C,A69C causes RMS shift changes approximately twice as large as the single mutation L43H and is thus placed in the same structural class. When examined in detail, the changes in the double mutant are found to be confined to those residues at the C terminus (Glu and Asn) and to a localized group of residues which are in contact with Glu or with each other: Ile, Leu, Val, Val, Phe, and Leu.

In order to assess the degree of structural change that such RMS shift changes represent, the mutants F21A, F17L, and L43H were selected for more detailed structural study using NOE data. For these mutants, few or no differences in NOE patterns were found when compared to the wild-type protein. For example, the imidazole side chain of His in the L43H mutant makes some intramolecular contacts which are not made by Leu of wild-type IL-8. However, modeling studies show that these contacts are readily made by the new side chain and that most of the observed shifts can be accounted for by the presence of the new aromatic ring with some small (<20°) changes in the side chain torsion angles of Phe and Leu. (^2)Thus, even in the most significant cases, the observed chemical shift changes either represent movements of non-hydrogen atoms of less than 1 Å or must occur in mobile, less tightly packed regions of the protein structure where NOE effects are small. Inspection of the shifted residues in conjunction with the IL-8 structure suggests that both factors contribute to the relative insensitivity of the NOE compared with the chemical shift in detecting structural changes.

In a second approach, which concentrated on the effects around the protein backbone, RMS shift differences were calculated for all measurable alpha- and beta-protons in the protein sequence. Although NH proton shifts are available, previous work has shown that, for two proteins of high structural homology, but lower sequence homology, NH chemical shifts are extremely sensitive to changes in hydrogen bond lengths and angles and can give a misleading impression of the extent of torsion angle changes(43) . Inclusion of NH shifts in an RMS calculation thus biases the result toward these protons. Calculation of the RMS shift changes for all measurable alpha-protons (typically 60 out of a possible 74 signals) gave values for the different mutants which agreed qualitatively with the values obtained using the representative set of Table 1, but which covered a narrower range. This is in contrast to the greater range of conformation-dependent shifts observed for alpha-protons than for other classes of CH protons in diamagnetic proteins. However, plotting the chemical shift changes of alpha- and beta-protons against residue number (Fig. 3) showed that the backbone changes were localized to a small number of residues in the sequence and that the RMS actually represented larger shift changes, but from fewer residues.


Figure 3: Sequence variation of alpha- and beta-proton chemical shift changes. Chemical shift changes, defined as Delta = - are shown for two representative IL-8 mutants, I10A and I40A. Residues for which no assignment has been made in the mutant NMR spectrum, and the mutated residue itself, are omitted in each case. For the I10A mutation, the changes are small except for those residues which are adjacent in the sequence (representative set RMS = 0.018 ppm, see Table 1). For the I40A mutant, the shift changes are larger and are dominated by two regions of the sequence, around Cys and Cys^9 (representative set RMS = 0.040 ppm, Table 1). The Ile side chain is packed against the disulfide bridge formed between Cys and Cys^9. Smaller effects are also evident for residues around Leu and Val whose side chains also make contact with Ile.



In summary, with the exception of F17A, the mutations studied here lead to a few, very specific, changes in the conformation of the polypeptide backbone and more widespread, small readjustments of amino acid side chains. Even when RMS shift changes are comparatively large (e.g. F17L and L43H), NOE studies indicate that the corresponding structural changes are minor (<1 Å for all atoms giving measurable NOEs).

Bioactivities of IL-8 Mutants

Table 2shows the activities of the 29 single-site mutants and one double-mutant, as well as a classification of their structural homology with the wild-type. This classification is based on the NMR chemical shift method described above and, for those mutants with significant shift changes (F17L, I40A, V41A, L43H, and E29C,A69C), on additional NOE and modeling studies. Fifteen mutations among 11 residues led to greater than 10-fold reduction in the receptor binding activity of IL-8 in one or both bioassays.



Of these 15 mutants, one, F17A, has been shown to be aggregated and misfolded and the result cannot be interpreted simply. In addition, three mutants, I40A, L43A, and L49A, have NMR spectra indicative of small structural changes which extend beyond the immediate environment of the mutated side chain. Of the remaining mutants, eight, L5A, R6A, I10A, F17L, F21A, I22A, L43H, and E29C,A69C have NMR spectra which indicate minor conformation changes around the site of mutation only. The effects of these mutations can therefore safely be interpreted in terms of a direct role for these residues in receptor binding. No structural studies were carried out on the remaining mutants, E4D, R6K, F17W, and L51A.

Fig. 4shows the locations of the seven residues, Leu^5, Arg^6, Ile, Phe, Phe, Ile, and Leu for which mutation leads to significant receptor-binding effects with negligible structural consequences. These residues define two distinct regions on the IL-8 surface. One is the previously identified N-terminal peptide sequence, (Glu^4)Leu^5-Arg^6 and includes Ile, which lies at the edge of this region. The other is a ``hydrophobic pocket'' comprising Phe, Phe, Ile, and Leu which form a shallow depression on the protein surface. The sequence between Cys^7 and Cys has previously been shown to be required for IL-8 binding to its receptor (16) and this region has, in part, been implicated in the binding to IL-8 of peptides derived from the high affinity IL-8 receptor A (IL-8RA)(44) . In addition, the proximate residues Tyr and Lys have been shown to be responsible for the specificity difference between rabbit and human IL-8 in their interaction with the human A receptor (45) . Residues in this region of a monomeric variant of IL-8 namely Tyr, Phe, Leu, and Ser, have also been implicated in receptor binding by Lowman et al. (^3)


Figure 4: Effects of mutations on IL-8 activity. Residues whose mutation leads to 10-fold or greater loss of activity in receptor-binding or calcium-transient assays are highlighted. Where mutation led to negligible structural changes the residue is colored red. Residues whose mutation may cause significant structural changes remote from the site of mutation, or for which no structural data are available, are shown in magenta. A single monomeric subunit of IL-8 is illustrated and the orientation is identical to that of Fig. 2. Two distinct epitopes are evident: the N-terminal sequence Glu^4-Leu^5-Arg^6 and Ile, and a cluster of solvent-exposed hydrophobic residues comprising Phe, Phe, Ile, and Leu. This latter pocket is completed by Leu, shown in magenta. Two mutations, I40A and L51A, probably cause conformational changes around Cys on mutation (see Fig. 3and text) and the side chains (shown in magenta) may not themselves interact directly with the receptor.



Table 2also indicates that the covalently-linked dimer mutant E29C,A69C is equipotent with wild-type IL-8 in the calcium-transient assay. Thus, dissociation of the IL-8 monomers and exposure of the dimer interface is not obligatory for receptor activation.


DISCUSSION

The results described above have led to the identification of a number of residues of IL-8 which are involved in receptor binding. These residues lie in two separate well defined regions of the protein surface.

Mapping of the water-accessible surface of the IL-8 dimer shows that the pocket formed by Phe, Phe, Ile, and Leu is uniquely hydrophobic when compared to the remainder of the IL-8 surface. Hydrophobic surfaces are often found at the interface between non-covalently associated proteins and the entropic effects of hiding such surfaces from the solvent may be a major driving force for association(46) .

The available structures of IL-8 show that the ELR segment is separated by over 20 Å from the hydrophobic pocket in both the same and in the second monomer. Although the position of the N-terminal peptide is not well defined in these structures, it is clear that, in the absence of large conformational changes, these two receptor binding regions must interact with distinct sites on the IL-8 receptor. The combined effect of the loss of the side chains of Phe, Phe, Ile, and Leu is predicted to be a reduction in K(d) of greater than 10^5-fold. This represents a lower limit since the mutation F17L is semi-conservative and there may well be additional contributions to binding from Ile, Leu, and Leu which are not included here due to the structural change concomitant on their mutation (Ile and Leu) or the absence of structural data (Leu). The relative contribution of this region to receptor binding would therefore appear to be comparable to that of the Glu^4-Leu^5-Arg^6 epitope.

Given the good coverage of the surface of IL-8 by the other mutations described here and the absence of any requirement for subunit dissociation, it is thus likely that the two regions described above, the N-terminal peptide and the hydrophobic pocket around Phe and Phe, are the major epitopes on IL-8 for interaction with its receptors. Since all mutations for which data are available lead to comparable changes in the activities of IL-8 in both the receptor-binding and receptor-function assays, receptor activation appears to depend simply on IL-8 binding.

The mutations at position 43 were designed to test the hypothesis that this region made a hydrophobic contact with the IL-8 receptor based on the reduced activity of L43A. The mutation L43H was expected to occupy some increased space in the hydrophobic pocket and perhaps to introduce a positive charge at this site, while the L43N and L43D mutants were introduced to investigate the effects of hydophilic and negatively charged groups. Attempts to introduce more bulky side chains (L43W) gave poor yields of protein. While the L43A and L43H mutants have lowered affinity for the receptor, the L43D and L43N mutants are equipotent with the wild-type. Detailed NOE and ring current shift analysis of the L43H mutant indicates that the histidine side chain is accommodated with small adjustments to the surrounding residues but occupies more space than the leucine side chain of the wild-type protein. The NMR data also indicate that, in the hydrophobic environment of the pocket, the histidine pK(a) is lowered by more than 2 log units and the side chain is uncharged. Although there was insufficient material for a detailed study of the L43D and L43N mutants, this result suggests that the aspartic acid side chain will also be uncharged in this environment. This would account for the similarity of the activities of the L43D and L43N mutants and highlights the need for biophysical data when constructing charge-modified mutants. Since these mutants are equipotent with the wild-type, the Leu -methyl groups do not appear to make a significant contribution to receptor binding. The reduced affinity of the L43H mutant must therefore be ascribed to an unfavorable steric interaction between the histidine side chain and the receptor while the reduced affinity of the L43A mutant is probably a result of the conformational changes occurring elsewhere.

In contrast, some mutants show indications of an appreciable structural change but do not exhibit significant changes in receptor-binding affinity (P32A, V41A). In these cases, it is unlikely that the mutated residues, and their immediate neighbors, make important contacts with the receptor in the IL-8 receptor complex.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed. Tel.: 1707-366548; Fax: 1707-373504; glyn.williams{at}roche.com.

(^1)
The abbreviations used are: IL-8, interleukin-8; COSY, correlated spectroscopy; IL-1, interleukin-1; NOE, nuclear Overhauser effect; RANTES, regulated upon activation, normal T-cell expressed and secreted; TOCSY, total correlation spectroscopy; RMS, root mean square.

(^2)
G. Williams, G. J. Price, and L. Price, manuscript in preparation.

(^3)
H. B. Lowman, P. H. Slagle, L. DeForge, C. Wirth, B. L. Gilece-Castro, J. Bourell, and W. J. Fairbrother, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. B. K. Handa for synthesis of oligonucleotides, Dr. B. Sherborne for performing the solvent-accessibility determination and for assistance with the ring-current shift calculations, Dr. W. Vetter (Roche, Basel) for mass spectrometric analysis of the covalently-linked IL-8 dimer, and Dr. H. Lowman and co-workers for providing a copy of their manuscript prior to publication. We also acknowledge the contributions of Dr. M. Attwood, Dr. W. A. Thomas, and Z. Hussain in many helpful discussions of the work and C. Nunns in purification of the covalently-linked dimer. Fig. 1, Fig. 2, and Fig. 4were produced using INSIGHT (BIOSYM Technologies Inc., San Diego, CA); Fig. 3was produced with the assistance of J. Nix.


REFERENCES

  1. Oppenheim, J. J., Zachariae, C. O. C., Mukaida, N., and Matsushima, K. (1991) Annu. Rev. Immunol. 9, 617-648 [CrossRef][Medline] [Order article via Infotrieve]
  2. Detmers, P. A., Lo, S. K., Olsen-Egbert, E., Walz, A., Baggiolini, M., and Cohn, Z. A. (1990) J. Exp. Med. 171, 1155-1162 [Abstract]
  3. Westlin, W. F., and Gimbrone, M. A., Jr. (1991) FASEB 5, A1624
  4. Mulligan, M. S., Jones, M. L., Bolanski, M. A., Baganoff, M. P., Deppeler, C. L., Meyers, D. M., Ryan, U. S., and Ward, P. A. (1993) J. Immunol. 150, 5585-5595 [Abstract/Free Full Text]
  5. Clore, G. M., Appella, E., Yamada, M., Matsushima, K., and Gronenborn, A. M. (1990) Biochemistry 29, 1689-1696 [Medline] [Order article via Infotrieve]
  6. Baldwin, E. T., Weber, I. T., St. Charles, R., Xuan, J-C., Appella, E., Yamada, M., Matsushima, K., Edwards, B. F. P., Clore, G. M., Gronenborn, A. M., and Wlodawer, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 502-506 [Abstract]
  7. Malkowski, M. G., Wu, J. Y., Lazar, J. B., Johnson, P. H., and Edwards, B. F. P. (1995) J. Biol. Chem. 270, 7077-7087 [Abstract/Free Full Text]
  8. Zhang, X., Chen, L., Bancroft, D. P., Lai, C. K., and Maione, T. E. (1994) Biochemistry 33, 8361-8366 [Medline] [Order article via Infotrieve]
  9. Kim, K.-S., Clark-Lewis, I., and Sykes, B. D. (1994) J. Biol. Chem. 269, 32909-32915 [Abstract/Free Full Text]
  10. Fairbrother, W. J., Reilly, D., Colby, T. J., Hesselgesser, J., and Horuk, R. (1994) J. Mol. Biol. 242, 252-270 [CrossRef][Medline] [Order article via Infotrieve]
  11. Skelton, N. J., Aspiras, F., Ogez, J., and Schall, T. J. (1995) Biochemistry 34, 5329-5342 [Medline] [Order article via Infotrieve]
  12. Chung, C., Cooke, R. M., Proudfoot, A. E. I., and Wells, T. N. C. (1995) Biochemistry 34, 9307-9314 [Medline] [Order article via Infotrieve]
  13. Lodi, P. J., Garrett, D. S., Kuszewski, J., Tsang, M., L.-S., Wartherbee, J. A., Leonard, W. J., Gronenborn, A. M., and Clore, G. M. (1994) Science 263, 1762-1767 [Medline] [Order article via Infotrieve]
  14. Hébert, C. A., Vitangcol, R. V., and Baker, J. B. (1991) J. Biol. Chem. 266, 18989-18994 [Abstract/Free Full Text]
  15. Clark-Lewis, I., Schumacher, C., Baggiolini, M., and Moser, B. (1991) J. Biol. Chem. 266, 23128-23134 [Abstract/Free Full Text]
  16. Clark-Lewis, I., Dewald, B., Geiser, T., Moser, B., and Baggiolini, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3574-3577 [Abstract]
  17. Cunningham, B. C., and Wells, J. A. (1993) J. Mol. Biol. 234, 554-563 [CrossRef][Medline] [Order article via Infotrieve]
  18. Williams, D. H., Cox, J. P. L., Doig, A. J., Gardner, M., Gerhard, U., Kaye, P. T., Lal, A. R., Nicholls, I. A., Salter, C. J., and Mitchell, R. V. (1991) J. Am. Chem. Soc., 113, 7020-7030
  19. Moser, B., Dewald, B., Barella, L., Schumacher, C., Baggiolini, M., and Clark-Lewis, I. (1993) J. Biol. Chem. 268, 7125-7128 [Abstract/Free Full Text]
  20. Prasad, L., Sharma, S., Vandonselaar, M., Quail, J. W., Lee, J. S., Waygood, E. B., Wilson, K. S., Dauter, Z., and Delbaere, L. T. J. (1993) J. Biol. Chem. 268, 10705-10708 [Abstract/Free Full Text]
  21. Schnitzel, W., Monschein, U., and Besemer, J. (1994) J. Leukocyte Biol. 55, 763-770 [Abstract]
  22. Burrows, S. D., Doyle, M. L., Murphy, K. P., Franklin, S. G., White, J. R., Brooks, I., McNulty, D. E., Scott, M. O., Knutson, J. R., Porter, D, Young, P. R., and Hensley, P. (1994) Biochemistry 33, 12741-12745 [Medline] [Order article via Infotrieve]
  23. Rajarathnam, K., Sykes, B. D., Kay, C. M., Dewald, B., Geiser, T., Baggiolini, M., and Clark-Lewis, I. (1994) Science 264, 90-92 [Medline] [Order article via Infotrieve]
  24. Hébert, C. A., Luscinskas, F. W., Kiely, J-M., Luis, E. A., Darbonne, W. C., Bennett, G. L., Liu, C. C., Obin, M. S., Gimbrone, M. A., Jr., and Baker, J. B. (1990) J. Immunol. 145, 3033-3040 [Abstract/Free Full Text]
  25. Stuber, D., Ibrahimi, I., Cutler, D., Dobberstein, B., and Bujard, H. (1984) EMBO J. 3, 3143-3248 [Abstract]
  26. Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R., and Stuber, D. (1988) Bio/Technology 6, 1321-1325
  27. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492 [Abstract]
  28. Leonard, E. J., Yoshimura, T., Rot, A., Noer, K., Walz, A., Baggiolini, M., Walz, D. A., Goetzl, E. J., and Castor, C.W. (1991) J. Leukocyte Biol. 49, 258-265 [Abstract]
  29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  30. McPherson, G. A. (1985) Kinetic, EBDA, Ligand, Lowry: A Collection of Radioligand Binding Analysis Programs, Biosoft, Cambridge, United Kingdom
  31. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450 [Abstract]
  32. Klaus, W., Broger, C., Gerber, P., and Senn, H. (1993) J. Mol. Biol 232, 897-890 [CrossRef][Medline] [Order article via Infotrieve]
  33. Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620 [Medline] [Order article via Infotrieve]
  34. Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528
  35. Bax, A., and Davis, D. G. (1986) J. Magn. Reson. 65, 355-360
  36. Clore, G. M., Appella, E., Yamada, M., Matsushima, K., and Gronenborn, A. M. (1989) J. Biol. Chem. 264, 18907-18911 [Abstract/Free Full Text]
  37. Hoch, J. C. (1983) The Influence of Protein Structure and Dynamics on NMR Parameters (Overhauser). Ph.D. thesis, Harvard University
  38. Rajarathnam, K., Clark-Lewis, I., and Sykes, B. D. (1994) Biochemistry 33, 6623-6630 [Medline] [Order article via Infotrieve]
  39. Wishart, D. S., Sykes, B. D., and Richards, F. M. (1992) Biochemistry 31, 1647-1651 [Medline] [Order article via Infotrieve]
  40. Williamson, M. P., Kikuchi, J., and Asakura, T. (1995) J. Mol. Biol. 247, 541-546 [CrossRef][Medline] [Order article via Infotrieve]
  41. Bartik, K., Dobson, C. M., and Redfield, C. (1993) Eur. J. Biochem. 215, 255-266 [Abstract]
  42. Barsukov, I. L., and Lian, L-Y. (1993) in NMR of Macromolecules: A Practical Approach (Roberts, G. C. K., ed) pp. 315-337, Oxford University Press Oxford
  43. Gao, Y., Lee, A. D. J., Williams, R. J. P., and Williams, G. (1989) Eur. J. Biochem. 182, 57-65 [Abstract]
  44. Clubb, R. T., Omichinski, J. G., Clore, G. M., and Gronenborn, A. M. (1994) FEBS Lett. 338, 93-97 [CrossRef][Medline] [Order article via Infotrieve]
  45. Schraufstatter, I. U., Ma, M., Oades, Z. G., Barritt, D. S., and Cochrane, C. G. (1995) J. Biol. Chem. 270, 10428-10431 [Abstract/Free Full Text]
  46. Fersht, A.R. (1985) Enzyme Structure and Mechanism, Second edition, p. 299, W. H. Freeman and Co., New York

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