(Received for publication, September 18, 1995; and in revised form, January 2, 1996)
From the
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-Leu
-Arg
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.
Interleukin-8 (IL-8) ()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 -helices lying above a
six-stranded, antiparallel
-sheet (Fig. 1a). The
fold of the monomer unit is also shared by the C-C chemokines,
RANTES(11, 12) , and macrophage inflammatory
protein-1
(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
, Leu
, Arg
, 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 -helices at the C termini of each chain, lying above
a six-stranded, antiparallel
-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
-helix of
one monomer to the
-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
-Leu
-Arg
to
Ala
-Ala
-Ala
is calculated to reduce
receptor binding by a factor of approximately 10
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 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).
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.
Examination of
the x-ray structure of IL-8 shows that three pairs of residues have
intermonomer C-C
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
-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
-helix to the
-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 g for 6 h) in a microconcentrator
(Microsep, 3000 M
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-Cys
and
Cys
-Cys
were maintained and signals
corresponding to two new cysteine residues were apparent.
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.
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
. (
)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
- and
-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
-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
-protons than for other
classes of CH protons in diamagnetic proteins. However, plotting the
chemical shift changes of
- and
-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 - and
-proton chemical shift changes. Chemical shift changes, defined as
=
-
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
(representative
set RMS = 0.040 ppm, Table 1). The Ile
side
chain is packed against the disulfide bridge formed between Cys
and Cys
. 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).
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, Arg
,
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
)Leu
-Arg
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
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. (
)
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-Leu
-Arg
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.
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
of greater than 10
-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
-Leu
-Arg
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 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.