The functions of the C5a
anaphylatoxin are expressed through its interaction with a cell-surface
receptor with seven transmembrane helices. The interaction of C5a with
the receptor has been explained by a two-site model whereby recognition
and effector sites on C5a bind, respectively, to recognition and
effector domains on the receptor, leading to receptor activation
(Chenoweth, D. E., and Hugli, T. E. (1980) Mol.
Immunol. 17, 151-161. In addition, the extracellular N-terminal
region of the C5a receptor has been implicated as the recognition
domain for C5a, responsible for ~50% of the binding energy of the
C5a-receptor complex (Mery, L., and Boulay, F. (1994) J. Biol. Chem. 269, 3457-3463; DeMartino, J. A., Van Riper,
G., Siciliano, S. J., Molineaux, C. J., Konteatis, Z. D., Rosen, H., and Springer, M. S. (1994) J. Biol.
Chem. 269, 14446-14450). In this work, the interactions of C5a
with the N-terminal domain of the C5a receptor were examined by use of
recombinant human C5a molecules and peptide fragments
M1NSFN5YTTPD10YGHYD15DKDTL20DLNTP25VDKTS30NTLR(hC5aRF-1-34), acetyl-HYD15DKDTL20DLNTP25VDKTS30NTLR
(hC5aRF-13-34), and
acetyl-TL20DLNTP25VDKTS30N-amide (hC5aRF-19-31) derived from human C5a receptor. Binding induced resonance perturbations in the NMR spectra of the receptor fragments and the C5a molecules indicated that the isolated Nterminal domain or residues 1-34 of the C5a receptor retain specific binding to C5a
and to a C5a analog devoid of the agonistic C-terminal tail in the
intact C5a. Residues of C5a perturbed by the binding of the receptor
peptides are localized within the helical core of the C5a structure, in
agreement with the results from functional studies employing mutated
C5a and intact receptor molecules. All three receptor peptides,
hC5aRF-1-34, hC5aRF-13-34, and hC5aRF-19-31, responded to the
binding of C5a through the 21-30 region containing either hydrophobic,
polar, or positively charged residues such as Thr24,
Pro25, Val26, Lys28,
Thr29, and Ser30. The 21-30 segment of all
three receptor fragments was found to have a partially folded
conformation in solution, independent of residues 1-18. These results
indicate that a short peptide sequence, or residues 21-30, of the C5a
receptor N terminus may constitute the binding domain for the
recognition site on C5a.
 |
INTRODUCTION |
The C5a anaphylatoxin is a 74-residue glycoprotein derived from
the fifth component (C5) of the complement system upon proteolytic activation (1). The C5a molecule plays an important role in host
defense against invading microorganisms or tumor cells. Inappropriately accumulated C5a, on the other hand, stimulates smooth muscle
contraction, causes vasodilation, increases vascular permeability, and
can even recruit and stimulate granulocytes leading to the release of
inflammatory molecules (2-4). The implication of C5a in various immune
and inflammatory diseases prompted extensive structure-and-function studies (5-16). In particular, NMR analysis defined C5a as being composed of a 4-helix core structure (residues 1-64) followed by a
10-residue C terminus with conformational variability (7-9, 16).
Determination of the three-dimensional structure of C5a followed by
mutagenesis allowed the identification of many C5a residues required
for receptor activation and others that may be more important for the
structural integrity of the C5a protein (10-15).
There appear to be two major structural elements within C5a required
for an effective activation of the C5a receptor (4). The 4-helix region
of C5a or residues 1-64 encodes a recognition site for receptor
binding (5, 6). The flexible C-terminal residues, on the other hand,
comprise the predominant effector site for receptor activation (5,
17-19). Accordingly, peptides derived from the C terminus of C5a were
found to be specific agonists of the C5a receptor. These C5a peptides
have been useful for mapping the effector sites and led to the
development of moderately potent receptor agonists (17-19). A C5a
analog truncated at the C terminus, or C5a-1-69, was found to be a C5a
receptor antagonist (5). Mutagenesis studies within this portion of C5a
identified some residues important for receptor binding (10-15).
However, the recognition site has not been defined conclusively because
the important residues identified by these studies were distributed
almost throughout C5a. Recently, a new C5a antagonist was synthesized,
and its solution structure was determined (20). The structural
difference between the C5a semi-synthetic antagonist and intact C5a
(7-9) provided further insights into the nature of the receptor
recognition site on C5a (20).
Since the cloning of the C5a receptor (21, 22), work has been focused
on defining the structural features of the receptor molecule required
for the C5a-receptor interaction (23-31). The cell-surface receptor
for C5a is a member of the G-protein-coupled receptor superfamily, with
an extracellular N-terminal region, an integral membrane helical
domain, and a C-terminal tail extending into the cytoplasmic space
(32). The extracellular and intracellular faces of the C5a receptor
have three peptide loops, respectively, each connecting the C and the N
termini of the seven individual transmembrane helices. The agonist
functions of C5a are thought to be expressed by the binding of its
C-terminal tail M70QLGR74 (or the effector
site) to an activation domain on the receptor formed by the
transmembrane helices (21, 22, 29-31). The 4-helix core region or
recognition site of C5a may serve to assist receptor activation through
interactions with a recognition domain on the extracellular face of the
receptor molecule (4, 6, 28, 33). However, there is a lack of detailed
structural information about the C5a/C5a-receptor complex (4), or any
ligand-receptor complex involving G-protein-coupled receptors (32), for
an adequate understanding of the molecular mechanism of receptor
activation.
Unfortunately, it is not yet feasible to define in atomic resolution
the structural details of the entire C5a/C5a-receptor complex. On the
other hand, structural information may be derived through a study of
the binding interactions of C5a with soluble fragments of the receptor.
Several lines of investigation suggest that the recognition domain for
the C5a-receptor interaction is localized in the extracellular
N-terminal region of the C5a receptor (4). First, antibodies
recognizing the receptor N-terminal fragments interfere with C5a
binding (23, 24). Second, truncation of the N terminus of the C5a
receptor results in reduced binding of the receptor to intact C5a, but
not to the agonist C5a tail peptides (27, 28). Third, partial or
complete replacement of the receptor N terminus diminished cell
responses to C5a binding (26). Fourth, replacement of some of the
N-terminal Asp residues of the receptor with either Ala or Asn results
in dramatically decreased affinity for C5a (26, 27). There is therefore
a possibility that the receptor N terminus may represent an autonomous binding domain showing specific interactions with C5a. In this paper,
we report an NMR characterization of the interactions of the N-terminal
fragments of the C5a receptor with C5a and with an antagonist analog,
CGS-28805, of C5a. NMR work was facilitated by the finding that the
isolated receptor N terminus, residues 1-34 of the human C5a receptor,
retained binding to C5a. The NMR results provide additional evidence
for specific contacts between C5a and the N-terminal region of the C5a
receptor. Receptor residues responsible for these contacts are further
localized to residues Asp21 to Ser30 containing
only two out of the five aspartic acids identified by mutagenesis as
important for binding to C5a.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Peptides and Proteins--
Peptide fragments
(Table I) derived from residues 1-34 of
the human C5a receptor were synthesized using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry on a
multiple-channel peptide synthesizer (Symphony Protein Technology Co.).
The synthetic peptides were purified by high pressure liquid
chromatography using a Vydac C18 reverse-phase column (Vydac, Hesperia,
CA) and with a gradient of 10-70% acetonitrile in a flow phase of
99.9% of H2O and 0.1% of trifluoroacetic acid. The
identities of the purified peptides were verified by use of electrospray mass spectrometry, which showed that peptide hC5aRF-1-34 had a molecular mass of 3954 (expected: 3950.79), hC5aRF-13-34 of 2603 (expected: 2602.24), and hC5aRF-19-31 of 1459 (expected: 1458.55).
Sequential assignments of the proton NMR resonances also confirmed the
expected sequences of all the peptides.
Recombinant C5a (Thr1-Met) and a C-terminally truncated C5a
analog, C5a-1-71 (Thr1-Met, Cys27-Ser,
Gln71-Cys), were obtained from expressing bacterial cell
lines as described elsewhere (12). C5a was renatured as the glutathione
adduct and CGS-28805 as the cysteine adduct, C5a-1-71
(Thr1-Met, Cys27-Ser,
Gln71-Cys-S-S-Cys), of the C5a-1-71 analog (20). To
prepare CGS-28805, cysteine/cystine was used as the redox couple to
replace reduced and oxidized glutathione in the renaturation buffer.
Electrospray mass spectrometry was used to verify the molecular weight
of both proteins. C5a had a molecular weight of 8601 (expected: 8603) and CGS-28805 of 8052 (expected: 8050). The C5a preparation had a
binding affinity (IC50) of 0.007 nM whereas
that of the CGS-28805 was 0.1 nM based on in
vitro competitive binding assays against 125I-labeled
C5a (12, 14). The CGS-28805 molecule had an antagonist potency
(IC50) of better than 400 nM, a value close to
those of C5a-1-69 (5) and another C5a antagonist, C5a-1-71
(Thr1-Met, Cys27-Ser,
Gln71-Cys-S-S-Cys-Leu-Gly-D-Arg) (20).
Samples for NMR measurements were prepared by dissolving 1-2 mg of the
purified peptides in 450 µl of an aqueous solution containing 20 mM sodium acetate and 0.2 mM EDTA. A volume of
50 µl D2O was added to the peptide solutions to provide
the deuterium lock signal for the NMR spectrometers. The pH values of
the peptide samples were adjusted to 5 or 6.8 with dilute NaOH or HCl
solutions. The concentrations of the peptides were approximately 0.5 mM for one-dimensional and 1 mM for
two-dimensional NMR experiments. Stock solutions of C5a and the C5a
antagonist, CGS-28805, were prepared by dissolving desired amounts of
the proteins into the same buffers used for the peptide samples. The
concentrations of C5a and CGS-28805 were approximately 0.6 mM for two-dimensional NMR measurements.
NMR Measurements--
All NMR experiments were carried out on a
Bruker AMX2-500 MHz and/or a DRX-500 MHz NMR spectrometer at sample
temperatures of 5, 15, and 30 °C. Phase-sensitive detection by
time-proportional phase incrementation was employed for both
two-dimensional nuclear Overhauser effects,
NOESY1 (34) or ROESY (35,
36), and total correlation, TOCSY (37, 38), experiments. The intense
water signal was suppressed either by selective irradiation during the
relaxation delay and during the NOE mixing time or by a WATERGATE
sequence (39, 40) combined with the flipback of water signals (41, 42).
The NOESY spectra were acquired with mixing times of 200 and 350 ms.
The ROESY spectra were acquired with a mixing time of 250 ms. TOCSY
spectra were obtained with mixing times of 30-60 ms using the TOWNY-16
isotropic mixing sequence (43). All free induction decays were acquired with a size of 2048 complex data points. The free induction decays data
matrices were accumulated with sine modulation along the t1 dimension. The initial
t1 delays were chosen such that the zero-order
and first-order phase corrections along the F1 spectral dimension were 90 and 0, respectively (44). The number of increments along the t1 dimension was 350-400 for NOESY
and ROESY and 300 for TOCSY data sets. The NMR data were processed
using an in-house program, nmrDSP, incorporating fast cosine and sine
transformation, linear predication, and optimized base-line correction
procedures. Prior to spectral transformation, the free induction decays
data were multiplied by cosine-squared window functions along both the
t1 and t2 time dimensions
and zero-filled to 1024 real points along the t1
dimension. Spectral display, analysis, and comparisons were achieved by
use of the FELIX software and/or through the graphics interface of the
Sybyl NMR module, TRIAD.
Proton Resonance Assignments--
Sequence-specific assignments
of the proton resonances of the receptor peptides
(Tables
II-IV)
were achieved by use of a combination of two-dimensional TOCSY and
NOESY experiments. Intraresidue spin systems were identified based on
TOCSY cross-peak patterns. The identified spin systems were assigned to
the corresponding residues in the primary sequence through sequential
NOE connectivities in the NOESY spectra (45). The
CH protons of
Pro9 and Pro25 were used in place of the normal
NH for the tracing of the sequential NOE connectivities. The backbone
amide proton resonance for Asn2 was not observed in the
TOCSY spectra. The assignment for this residue was made on the basis of
sequential NOE connectivities. The two-dimensional NMR spectra allowed
the assignment of most of the proton resonances (Tables II-IV),
despite that peptide hC5aRF-1-34 contains many repeats of a few
residues, for example, 6 Asp and 6 Thr out of its 34 amino acid
residues. The assignment of the proton resonances of two shorter
peptides, hC5aRF-13-34 and hC5aRF-19-31 (Tables I-IV), was
straightforward following similar TOCSY patterns of the common amino
acid residues with peptide hC5aRF-1-34.
Assignments of the proton resonances of C5a have been described
previously (16, 20). In short, the intraresidue spin systems were
identified using through-bond connectivities observed in COSY or TOCSY
type experiments. The sequential assignments were achieved by use of
the sequential NOE contacts observed in the NOESY spectra of C5a (45).
Similar approaches were applied to the assignments of the proton
resonances for the C5a antagonist, CGS-28805.
 |
RESULTS AND DISCUSSION |
The Isolated N-terminal Domain of the C5a Receptor Retains Binding
to C5a--
Interactions between the receptor peptide hC5aRF-1-34 and
C5a were followed by NMR spectroscopy through perturbations of the proton resonances of the receptor peptide accompanying complex formation. Fig. 1 shows the amide NH
region of the proton NMR spectra of peptide hC5aRF-1-34 (Fig.
1a) and the same peptide in the presence of a less than
stoichiometric amount of C5a (Fig. 1b). Comparison of the
two spectra shows that only a subset of the proton resonances from the
receptor peptide (Fig. 1a) were selectively perturbed upon
the addition of C5a (Fig. 1b). The perturbed resonances
exhibited changes in chemical shifts and some broadening in line
widths, indicating a fast association and dissociation of the
C5a-peptide complex (46). Most of the resonance perturbations are
relatively small, presumably as a result of low affinity of the
isolated receptor N terminus for C5a and/or a minimal change of the
conformations of the receptor peptide between the free and the
C5a-bound states (46). The binding experiment was also carried out
(Fig. 1c) with an antagonist analog of C5a, CGS-28805, a
molecule lacking 3 residues from the C-terminal tail of native C5a (see
the "Experimental Procedures"). Addition of the C5a antagonist had
larger effects on the proton NMR spectra of the receptor peptide as
more resonance signals appear to be shifted or broadened (Fig.
1c). The pattern of resonance perturbations remained the
same when the experiments were repeated at a higher temperature of
30 °C or at pH 6.8 (spectra not shown) for both complexes. The
isolated N-terminal domain, hC5aRF-1-34, of the C5a receptor therefore
retains binding to C5a even in the absence of the C-terminal agonist
tail of intact C5a (as in CGS-28805).

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Fig. 1.
Amide region of the proton NMR spectra of
peptide hCRF-1-34 at pH 5 and 15 °C. a, free hCRF-1-34;
b, in the presence of human C5a; and c, in the
presence of CGS-28805, an antagonist of C5a. The concentration of the
receptor peptide is 0.6 mM and those of the C5a ligands are
0.075 and 0.09 mM, respectively. Proton resonances were
perturbed upon the addition of the C5a proteins to the peptide
solution. The most affected resonances are indicated by
arrows, and the unperturbed resonances are labeled with
asterisks.
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The perturbed proton resonances were assigned to the specific residues
of the receptor peptide based on a comparison of the two-dimensional
TOCSY spectra of the receptor peptide in the presence and absence of
C5a. Fig. 2A shows the
superposition of the NH side chain connectivities of peptide
hC5aRF-1-34 before (in blue) and after (in pink)
the addition of C5a at ~0.13 molar ratio to the peptide. Clearly,
most of the peptide peaks are not affected by C5a binding although
slight perturbations can be seen along the NH resonance frequencies of
residues Thr24, Val26, Asp27,
Lys28, and Thr29. These same residues of the
receptor peptide are perturbed in the presence of ~0.15 molar ratio
of the C5a antagonist, CGS-28805 (Fig. 2B), but with more
dramatic change of resonances than by C5a (Fig. 2A). Binding
of the C5a antagonist also perturbed the NH signals of residues
Asp21 and Ser30. Residues Val26,
Asp27, and Lys28 displayed significant
broadenings in their NH resonances in addition to shifts of the
resonance frequencies (Fig. 2B). The amide proton resonances
of Val26 and Asp27 were shifted downfield,
whereas the NH signals of Thr24, Lys28, and
Thr29 moved upfield in the presence of both C5a and
CGS-28805. Resonance perturbations were also observed for the
CH and
CH3 protons of Val26 and for the
CH and
one of
CH protons of Pro25, despite the lack of
significant effects on the side chain proton resonances of other
residues in the same peptide. Differential broadening of the amide
proton resonances of Val26, Asp27, and
Lys28 of the receptor peptide indicates that these protons
must have significantly different resonance frequencies and chemical
environment upon binding to C5a, given that the C5a and the antagonist
were present in less than 15% of the molar concentration of the
receptor peptide (Figs. 1 and 2).

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Fig. 2.
The NH region of the TOCSY spectra for
hC5aRF-1-34 in the presence and absence of C5a and the C5a
antagonist. The TOCSY spectra of the free peptide hC5aRF-1-34 are
shown in blue and in pink are the spectra in the
presence of C5a (A) or the C5a antagonist (B).
The concentration of the receptor peptide is 1 mM, and
those of the proteins are 0.13 mM for C5a and 0.15 mM for the C5a antagonist, CGS-28805. Only a few resonances
were perturbed by C5a binding (see also Fig. 1), whereas most amide
protons remained at their original positions. The perturbed resonances
are labeled by their corresponding residues (see also Tables II-IV).
Both frequency shifts and line broadening were observed for the
perturbed resonances. Note that resonance perturbations are for the
same residues of hC5aRF-1-34 in the presence of either C5a
(A) or the C5a antagonist, CGS-28805 (B).
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Receptor binding assays showed that the C5a receptor has high affinity
interactions with intact C5a with a Kd (or IC50) in the subnanomolar range, between 4 pM
and 5 nM depending on the types of assays used (4, 6, 9,
12, 26, 33). Mutagenesis experiments indicated that the receptor N
terminus contributed to almost half of the binding energy between the
C5a ligand and the surface-expressed intact receptor molecules (27). A
two-site binding mode between C5a and the receptor (4, 6, 28) predicts
that the isolated receptor N terminus can have a binding affinity for
C5a with a Kd value as low as ~1 µM.
Indeed, NMR results here indicate that there are interactions between
C5a and the isolated N-terminal receptor domain, hC5aRF-1-34, involving specific residues (Figs. 1 and 2). These interactions appear
to be enhanced when C5a was replaced by a C5a antagonist, CGS-28805,
with three residues removed from the C terminus of C5a. Consistent
patterns of resonance perturbations on the receptor peptide
hC5aRF-1-34 by both C5a and the C5a antagonist suggest that
interactions detected by NMR (Figs. 1 and 2) are very likely consequences of specific binding between the isolated receptor N
terminus and the C5a molecules.
The N-terminal Receptor Fragment Binds to the Recognition Site
within C5a--
Interactions of the isolated receptor N terminus with
C5a were also examined by following the changes in the proton NMR
spectra of C5a induced by the binding of the receptor peptide. Again, there were localized changes in the one-dimensional proton spectra of
C5a upon the addition of peptide hC5aRF-1-34. It was difficult to
analyze the binding effects using these one-dimensional NMR data since
C5a with 74 residues had a significantly more complex proton spectrum
(not shown) than that for the 34-residue receptor peptide (Fig.
1A). On the other hand, the structured C5a produces well
resolved two-dimensional NOESY spectra (7-9) with which perturbed
resonances can be analyzed and assigned. In the presence of the
receptor peptide, C5a and CGS-28805 exhibited a large number of the NOE
connectivities characteristic of the three-dimensional structure of C5a
(16, 20). Except for shifts in the positions of a small number of
cross-peaks, the NOESY spectra remain essentially unchanged (spectra
not shown) without the intensity distortions or grossly broadened peaks
seen with the spectrum of the receptor peptide (Fig. 2).
The shifted proton resonances of C5a in the presence of the
hC5aRF-1-34 peptide were assigned to the corresponding residues in
C5a. Fig. 3 shows the chemical shift
differences for the C5a resonances in the absence and presence of the
receptor peptide at 0.3 molar ratio to C5a. Clear resonance
perturbations (>0.01 ppm) by peptide binding were observed for the NH
protons of Val17p,2 Lys19p, and
Ser42p. In addition, detectable peak shifts (0.005-0.01
ppm) were found for the NH proton resonances of Val18p,
Lys20p, Cys21p, Tyr23p,
Ala38p, and Gly44p. Compared with free C5a, the
backbone amide proton resonances of Val17p,
Lys20p, Tyr23p, and Ser42p were
shifted downfield, whereas those of Val18p,
Lys19p, Cys21p, Ala38p, and
Gly44p were moved to upfield (Fig. 3). Furthermore,
resonance shifts were also detected for the
CH,
CH, and
CH
protons of His15p with the result for the
CH proton
shown in Fig. 3. More pronounced effects were observed when the
experiments were carried out with the C5a antagonist, CGS-28805, as
shown in Fig. 3. It is also seen that binding of the receptor peptide
perturbed the same set of residues in both the native C5a and the C5a
antagonist.

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Fig. 3.
Resonance perturbations in C5a (open
circles) and in CGS-28805 (filled circles) induced by
the binding of the hC5aRF-1-34 peptide. The values of the changes
were the chemical shift differences between the resonances of the
proteins before and after the addition of the peptide. All illustrated
changes were for backbone amide protons except for residue 15 (His15p) for which the side chain CH proton was used.
Only residues 15-44 were shown since no detectable resonance shifts
were observed for the residues beyond this region of the C5a sequence.
The concentrations of C5a and CGS-28805 were approximately 0.6 mM in acetate buffer at pH 5 and 30 °C in the absence or
presence of ~0.2 mM of the hC5aRF-1-34 peptide.
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Residues of C5a contributing to receptor binding have been identified
through chemical modification and mutagenesis studies. Table V is a list of the C5a residues
implicated in receptor binding by mutagenesis and those whose proton
resonances were perturbed significantly by the receptor peptide
hC5aRF-1-34. Residues with resonance perturbations are localized
within the 15-44 region of C5a, whereas the isolated receptor N
terminus did not perturb any residues from the agonist C-terminal tail
of C5a. The binding interactions between C5a and the isolated receptor
peptide detected by NMR are specific effects since the NMR
signals of C5a perturbed by the receptor peptide correspond closely to
those C5a residues identified by mutagenesis as important for
binding to intact receptor molecules (Table V). The NMR results
therefore provided additional evidence for the interaction between C5a
and its receptor involving a contact between a recognition site on C5a
and the N-terminal region of the C5a receptor (6, 28). The receptor
recognition site on C5a is most likely organized by residues 15-44
within the helical core structure of C5a. In addition, the C5a
antagonist, CGS-28805, binds to the isolated receptor N terminus
peptide, hC5aRF-1-34, using the same contact surface as in the agonist C5a. Interestingly, the antagonist CGS-28805 appears to have enhanced interactions with the receptor peptide despite that it had a somewhat decreased binding affinity (~0.1 nM) compared with the
native C5a molecule (~0.007 nM) in receptor binding
assays (see "Experimental Procedures").
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Table V
C5a residues contributing to receptor binding and with perturbations of
the proton resonances by the receptor fragment, hC5aRF-1-34
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Residues 21-30 of the C5a Receptor Constitute a Binding Domain for
C5a--
Fig. 2 showed that only 7 out of a total of 34 residues of
the receptor peptide have specific resonance shifts and/or line broadening in the presence of C5a. Both C5a and CGS-28805 perturbed the
receptor N-terminal fragment in the region of residues 21-30, whereas
no significant changes were found for residues 1-18. These results
indicate that C5a may contact the C-terminal portion of the receptor
peptide hC5aRF-1-34 independent of the N-terminal residues 1-18 of
the same peptide. Two shorter receptor peptides (hC5aRF-13-34 and
hC5aRF-19-31 in Table I) were synthesized, and their interactions with
CGS-28805 were examined. These two subfragments were found to not only
bind to the C5a antagonist but also the perturbed resonances by the
antagonist in the shorter peptides belong to the same set of residues
as observed for hC5aRF-1-34 (Fig. 4).
Frequency shifts were observed for the amide proton resonances of
Thr24, Val26, Asp27,
Lys28, and Ser30 of hC5aRF-13-34. Obvious
perturbations were found for the amide protons of residues
Thr24, Val26, Asp27, and
Lys28 of hC5aRF-19-31.

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Fig. 4.
Summary of the NOE (or ROESY) connectivities
observed for the three receptor peptides, hC5aRF-1-34, hC5aRF-13-34,
and hC5aRF-19-31. Most of the NOEs were those observed at a
temperature of 15 °C with NOE mixing times of 350 ms for NOESY and
250 ms for ROESY (for peptide hC5aRF-19-31). Three
d N(i,i + 2) NOE contacts between
Asp21 and Asn23, between Leu22 and
Thr24, and between Val26 and Lys28
were found at 5 °C and are shown by hatched lines. The
residues with resonance perturbations upon the binding of CGS-28805
(Fig. 2) are shown by the underlined and bold
letters.
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Binding induced perturbations of proton resonances identified with the
three receptor peptides, hC5aRF-1-34, hC5aRF-13-34, and hC5aRF-19-31
(Fig. 4), suggest that the contact region for C5a may be localized
within the segment of residues 21-30 in the N terminus of the C5a
receptor. For the shorter receptor fragments, there appear to be fewer
residues with resonance perturbations that are mostly centered around
the hydrophobic residue Val26 (Fig. 4). This result is not
surprising in light of the fact that residues Val26,
Asp27, and Lys28 have the most severely
perturbed proton resonances with both resonance shifts and line
broadening effects in the full-length peptide hC5aRF-1-34 (Fig.
2C). It is also possible that the truncated receptor
fragments may have a binding mode slightly different from the longer
peptide hC5aRF-1-34. The surface area on C5a contacted by the shortest
receptor fragment, hC5aRF-19-31, was then examined by following the
resonance perturbations in CGS-28805 induced by the binding of this
shorter receptor fragment. Clear peak shifts were observed for residues
His15p, Val17p, Val18p,
Lys19p, Lys20p, Ser42p, and
Gly44p of CGS-28805 as seen for the binding of peptide
hC5aRF-1-34 (Fig. 3). However, the shorter receptor peptide did not
perturb the resonances of residues Cys21p,
Tyr23p, and Ala38p. On the other hand, residues
Ala50p and Phe51p were slightly perturbed
(spectra not shown) which were not affected by the binding of the
longer peptide hC5aRF-1-34. Regardless, all the C5a residues with
resonance perturbations are still localized in the same structural
region of C5a molecules (16, 20). The shortest receptor peptide
hC5aRF-19-31 therefore retains specific binding to the C5a antagonist,
suggesting that residues in this region of the receptor represent a
binding site for C5a.
Mutagenesis studies showed that residues throughout the extracellular
N-terminal domain of the C5a receptor, especially aspartic acid
residues, are important for the interaction of the intact receptor
molecule with C5a (25-27). Furthermore, a mutant receptor missing the
first 22 residues from the N terminus had reduced binding to C5a by
600-fold, and a receptor without residues 1-30 had the affinity for
C5a reduced by almost 50,000 (27, 28). The C5a receptor lost the
binding for C5a after Asp10, Asp15, and
Asp16 or all the 5 Asp residues, Asp10,
Asp15, Asp16, Asp21, and
Asp27, were replaced by Asn residues (26). Interestingly,
little effects on affinity were found when either Asp10,
Asp27, or both Asp21 and Asp27 were
replaced by Asn (26). On the other hand, the receptor has a decreased
binding affinity of more than 40-fold after Asp10,
Asp15, Asp16, Asp18, and
Asp21 were substituted with Ala residues (27). Another
15-fold reduction of binding affinity was found when Asp27
was replaced with Ala within a receptor missing the first 22 residues
(27). Taken together, the mutagenesis results demonstrate that all the
Asp residues play some role in determining the binding of the intact
receptor to C5a. Only two (Asp21 and Asp27) out
of the six Asp residues in the receptor peptide are perturbed by C5a
binding based on the analysis of the proton NMR signals in the isolated
receptor peptides. The other residues in the 21-30 region of the
receptor are either positively charged or with hydrophobic side chains,
such as Leu21, Pro25, Val26, and
Lys27 (Fig. 4). Residues 21-30 of the receptor N terminus
may therefore be involved in direct contacts with C5a, whereas other
important residues within the region of 1-18 of the receptor may
contribute to the C5a binding through indirect interactions.
The 21-30 Region of the Receptor N Terminus Has a Partially Folded
Conformation in Solution--
Potential interactions within the
receptor N terminus were examined through an analysis of nuclear
Overhauser effects (NOEs) between all the protons of the receptor
peptide hC5aRF-1-34. Two-dimensional NOE (NOESY) experiments were
performed at temperatures of 5 and 15 °C. At 15 °C, many NOE
connectivities were observed between residues next to each other in the
peptide sequence (spectra not shown). In particular, sequential NOEs
were observed between the amide protons of the stretch of residues from
His13 to Thr32 but not for residues
Met1 to Gly12. In addition, existence of NOE
contacts between the
CH proton of the preceding residue to the
CH
protons of Pro9 and Pro25 indicates both
proline residues assume the trans conformation in peptide
hC5aRF-1-34. On the other hand, there were no medium and long range
NOE contacts, indicating that the free hC5aRF-1-34 peptide is in a
state of dynamic averaging of many conformations with no particularly
well defined secondary structures. Upon a lowering of the temperature
to 5 °C, long range NOE contacts were still absent. Only three
medium range d
N(i,i + 2) NOEs between residues Asp21 and Asn23, Leu22 and
Thr24, and between Val26 and Lys28
were observed in the NOESY spectra of hC5aRF-1-34 acquired at this
temperature (Fig. 4). Therefore, there appear to be no contacts between
the N-terminal residues 1-18 and residues 21-34 in the receptor
peptide hC5aRF-1-34.
The existence of non-sequential NOEs, along with many sequential NH-NH
NOE contacts, indicates that the free hC5aRF-1-34 peptide may have
locally folded conformations within residues 20-30 or the C-terminal
region of the peptide. The consecutive NH-NH NOE connectivities suggest
the existence of some population of
or 310 helices.
However, the absence of characteristic (i,i + 3) NOE
contacts and presence of three medium range NOEs suggest that some
turn-like local structure may exist in the free hC5aRF-1-34 peptide.
One possible conformation within residues 20-30 of the hC5aRF-1-34
peptide could be a nascent helix involving residues from
Leu20 to Thr24 followed by a
turn starting
from Pro25 to Lys28. It should be noted that
this folded conformation is located within the same region where most
residues have perturbed proton resonances upon the binding of C5a (Fig.
4).
Similar conformations in peptide hC5aRF-1-34 may also exist in the two
shorter receptor peptides hC5aRF-13-34 and hC5aRF-19-31. In the case
of hC5aRF-13-34, sequential NOE connectivities were observed
throughout the peptide sequence, including many NOEs between the
sequential amide protons (Fig. 4). The three medium range NOEs observed
with peptide hC5aRF-1-34 were also present in this truncated peptide.
Almost identical chemical shifts were found for the proton resonances
of residues 14-34 with the exception of His13 which became
the N terminus in the peptide (Table III). The further truncated
peptide hC5aRF-19-31 exhibited very few NOEs in the NOESY spectra,
apparently as a result of its small size. Rotating-frame NOE spectra
(ROESY) were instead acquired which helped the identification of these
missing NOEs. Several sequential NH-NH ROESY contacts were observed in
addition to the sequential
CH-NH and
CH-NH ROESY connectivities.
Most importantly, the three non-sequential d
N(i,i + 2) NOE contacts present in two
longer peptides were also observed in the shortest peptide (Fig. 4). In
addition, the peptide hC5aRF-19-31 still has proton chemical shifts
similar to those of the corresponding residues in hC5aRF-1-34 (Table
IV). These results indicate that the folding of residues 21-30 in the isolated C5a receptor N terminus is independent of the presence of
residues 1-18 in hC5aRF-1-34. The folded conformations in the receptor peptides may be important for the specific interaction with
C5a since the same residues are strongly affected by C5a binding.
A Model for the C5a-Receptor Complex--
Identification of
residues 21-30 of the isolated receptor N terminus as a contact site
for C5a provides an explanation for the apparent interspecies
variations within the amino acid sequence of the receptor N-terminal
domain (47). In fact, human, dog, and mouse C5a receptors share a
stronger sequence similarity in the region of residues
Thr19 to Ser30 of the human sequence, as noted
previously (25). Human C5a binds to both dog and mouse C5a receptors
(21, 47), presumably because a similar conformation of the C5a binding
region, or residues 21-30 of the human receptor, is stabilized through
interactions of the different receptor N terminus (residues 1-18) with
the corresponding extracellular regions of the receptor variants. The
N-terminal region for intramolecular interactions within the C5a
receptor may be localized within residues
D10YGHYD15DKD of the human receptor, since a
chimeric C5a receptor with the first 8 residues replaced with those
from a formylpeptide receptor functioned normally, whereas replacement
of the first 13 residues resulted in loss of binding affinity for C5a
(26). The target binding site for residues Asp10 to
Asp18 in the receptor N terminus may be the second and/or
the third extracellular loops, since additional loop-swap experiments
suggested that, besides the N terminus, both the second and the third
extracellular loops of the C5a receptor are important for C5a binding
(48).
The results discussed so far can be represented by a model describing
potential interactions within the C5a-receptor complex (Fig.
5). The spatial arrangement of the seven
transmembrane helices is based on a computer-generated model for the
C5a receptor (49, 50). In this model, residues 10-18 of the receptor N
terminus make intramolecular contacts with the extracellular loops
(most likely the second extracellular loop) of the C5a receptor,
thereby stabilizing a conformation of residues 21-30 of the receptor N terminus required for the "recognition site" binding to C5a. Upon binding, C5a is positioned by the N terminus of the receptor to permit
a proper interaction of its agonistic C terminus with the cell-surface
expressed receptor, leading to receptor activation. Further work is in
progress to elucidate the detailed intermolecular interactions within
the C5a-receptor peptide complex and the potential conformational
changes in C5a upon binding of the N-terminal domain of the C5a
receptor.

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Fig. 5.
A model for the interaction of C5a with its
receptor. Seven transmembrane helices are shown by
cylinders. Their arrangement is based on a model of the C5a
receptor given by Grotzinger et al. (49). A disulfide bond
between Cys109 in the first extracellular and
Cys189 in the second extracellular loops is shown by a
red line. The C5a protein core only contacts residues 21-30
of the receptor N terminus. Residues 1-18 (in particular residues
10-18) of the receptor N terminus may interact with the positively
charged and hydrophobic stretch of residues within the extracellular
loops (the second extracellular loop shown here) of the C5a receptor.
The C5a protein molecule is held in a proper position by the N terminus
of the receptor, allowing its C-terminal tail to bind to and activate
the receptor.
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We thank Wayne C. Guida and Janine LePage of
Ciba-Geigy for their continuous support and encouragement and
Willem Stevens for useful discussions. Betty Zhu is acknowledged
for help with NMR data processing and computer graphics and Hui Xiang
for peptide purification.