(Received for publication, October 13, 1994; and in revised form, December 20, 1994)
From the
Neutrophil-activating peptide-2 (NAP-2) is a 70-residue
carboxyl-terminal fragment of platelet basic protein, which is found in
the -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
= 98.4°,
= 120.3°,
and
= 92.8°), with 4 molecules of NAP-2 (M
= 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
-sheet arranged in a Greek key fold, and one
-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.
Neutrophil-activating peptide-2 (NAP-2) ()is a
cleavage product of the platelet
-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
of
7600 (Walz and Baggiolini, 1989). CTAP-III and
-thromboglobulin
(
-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, -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 GRO
, GRO
, 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 -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,
GRO
, 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 -sheet
folded in a Greek key, and one
-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
-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
-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} (
)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.
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.
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 - F
and 2F
- F
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
-carbon backbones of residues 21
-86
(thicklines) and of residues
21
-86
(thinlines) are shown
in stereo. The view is approximately down the AB noncrystallographic
axis. Bottom, the
-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).
Figure 3:
Hydrogen bonds and topology of NAP-2. Main
chain hydrogen bonds for the four loops, three strands of -sheet,
and one
-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
-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
, Leu-23
,
and Arg-24
in NAP-2. The final refined model is shown in thicklines.
The first strand of the three-stranded
-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
-sheet (Fig. 3). Glu-43
on the
``interior side'' of the
-sheet forms an internal salt
bridge with Lys-65
in the other extended
-sheet and
similarly for the related pair Glu-43
to
Lys-65
. 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
-Lys-65
and Glu-43
-Lys-65
, 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 -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
-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
-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
-helix that lies diagonally across the top of the
-sheet.
The individual molecules are similar to one
another as evidenced by the average root mean square deviation of 0.47
Å in C- positions when they are overlapped in pairs (Table 2). In these six pairwise comparisons, the C-
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-
deviations between the subunits (2.0-8.58
Å) occur at the amino terminus where Ala-21
to
Arg-24
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-
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
-strands I and
II, has the largest C-
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 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
is similar to
that of Arg-24
shown in solidlines. In
subunit D, the four residues have a conformation similar to that shown
for subunit B.
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 -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
-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.
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
-helices farther across the adjacent
-sheets and thereby
stabilizing the dimer interface.
The interface of the AB and CD
dimers also is stabilized through hydrophobic interactions.
Leu-26 clusters with its counterpart, Leu-26
,
and also with Ile-57
and the side chain C-
, C-
,
and C-
atoms of Lys-65
. Ile-45
in the
-sheet also interacts with the C-
, C-
, and C-
atoms
of Lys-65
. Similarly, Leu-26
contacts
Leu-26
. 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 and Lys-65
and between Lys-65
and Glu-43
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), GRO (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).
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 to Leu-23
. 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
, 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- 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
and Arg-24
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.
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 -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
-TG to form NAP-2 by cleavage between Tyr-20 and Ala-21 (dashedarrow).
The activation peptide present in
-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
-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 -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
-helical
region in the receptor peptide lies near the first transmembrane
-helix in the proposed receptor structure (LaRosa et al.,
1992). Therefore, cleavage of
-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
-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.
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- 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-
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
-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- atom positions. The
deviations between C-
(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 (
= -54°) and His-50
(
= -48°) are
solvent-accessible (side chain-accessible surface areas of 56.7 and
64.6 Å
, respectively) and near the ELR region.
His-50
forms two hydrogen bonds within the ELR segment
(between the nitrogen of Arg-24
and the NE2 of His-50
and between the ND1 of His-50
and the oxygen of
Glu-22
). 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
, which contacts the
side chain of Leu-23
. The side chain of His-50
is in a similar conformation and within 3.5 Å of
Arg-24
, but the angles of the possible hydrogen bonds
exceed the limits.
The side chain of His-50 (
= 63°) adopts a different
conformation than that of His-50
and His-50
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
). Specifically, the ND1 of His-50
forms
an intersubunit hydrogen bond with the carbonyl oxygen of
Gly-63
. 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
in the NAP-2 structure. The side chain
conformation of His-50
lies between that of
His-50
, His-50
, and His-50
, with a
angle of -47° and a solvent-accessible
surface area of 41.9 Å
. His-50
lies
within 5.7 Å of the ELR region, but does not have contacts with
it. However, the NE2 of His-50
does form an intersubunit
hydrogen bond with the carbonyl oxygen of Asp-62
.
The
third region with significant structural differences among the
CXC chemokines involves the -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
-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 -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
heparin
IL-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 -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 -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
-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).
The atomic coordinates and structure factors (code 1NAP) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.