From the Departments of Cellular and Molecular
Pharmacology and ¶ Pharmaceutical Chemistry, University of
California, San Francisco, California 94143
Received for publication, August 24, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Although agonists are thought to occupy binding
pockets within the seven-helix core of serpentine receptors, the
topography of these binding pockets and the conformational changes
responsible for receptor activation are poorly understood. To identify
the ligand binding pocket in the receptor for complement factor 5a (C5aR), we assessed binding affinities of hexapeptide ligands, each
mutated at a single position, for seven mutant C5aRs, each mutated at a
single position in the putative ligand binding site. In ChaW (an
antagonist) and W5Cha (an agonist), the side chains at position 5 are
tryptophan and cyclohexylalanine, respectively. Comparisons of binding
affinities indicated that the hexapeptide residue at this position
interacts with two C5aR residues, Ile-116 (helix III) and
Val-286 (helix VII); in a C5aR model these two side chains point
toward one another. Both the I116A and the V286A mutations markedly
increased binding affinity of W5Cha but not that of ChaW. Moreover,
ChaW, the antagonist hexapeptide, acted as a full agonist on the I116A
mutant. These results argue that C5aR residues Ile-116 and Val-286
interact with the side chain at position 5 of the hexapeptide ligand to
form an activation switch. Based on this and previous work, we present
a docking model for the hexapeptide within the C5aR binding pocket. We
propose that agonists induce a small change in the relative
orientations of helices III and VII and that these helices work
together to allow movement of helix VI away from the receptor core,
thereby triggering G protein activation.
Serpentine receptors transmit a diverse array of extracellular
stimuli to heterotrimeric G proteins located on the cytoplasmic face of
the plasma membrane. These receptors promote exchange of GTP for GDP
bound to the The mechanism of receptor activation, which is probably highly
conserved also, resides in the transmembrane helices (8), where most of
the evolutionarily conserved residues are located. Indeed, extra- or
intracellular loops and termini can be exchanged between different
receptors, leaving intact the receptors' capacity to be activated,
while swapping ligand or G protein specificity (9). How is this
conserved activation switch activated by an enormously diverse set of
agonist ligands, widely differing in size and chemical character, which
must occupy similarly diverse binding pockets in the receptors? Small
agonists, including biogenic amines and chromophores, are thought to
bind exclusively to a transmembrane receptor pocket; larger ligands,
such as oligopeptides and proteins, interact in addition with
extracellular domains of their receptors (2, 10, 11). Progress beyond
this generalization has proved difficult; binding interactions have
been studied in biochemical detail only for relatively small ligands,
such as adrenergic amines and retinal, the chromophore of the visual
pigment, rhodopsin (reviewed in Ref. 10).
The receptor for complement factor 5a
(C5a),1 a 74-amino acid
protein, furnishes an instructive experimental model for studying how
larger peptides bind to and activate serpentine receptors (reviewed in
Ref. 12). Deletion mutants of the C5a receptor (C5aR) indicate that C5a
interacts both with the receptor's N terminus and with the
transmembrane bundle; the latter interaction is required for activation
of the C5aR (13, 14). Molecular details of the C5a·C5aR
interaction have been elucidated by pharmacological characterization of
C5a mutants and peptides derived from the C-terminal amino acid
sequence of C5a (13, 15-23). In addition, three-dimensional structures
have been determined for C5a (24) and a hexapeptide antagonist (22)
Me-F-K-P-dCha-W-dR (hereafter termed ChaW; dCha = D-cyclohexylalanine). Taken together, these studies show
that receptor activation is mediated by interaction of C-terminal
residues of C5a with the receptor's transmembrane helix bundle;
hexapeptide ligands analogous to the C-terminal eight residues of C5a
interact exclusively with the transmembrane pocket (13). One residue in
transmembrane helix V of the receptor, Arg-206, is essential for
receptor activation by a hexapeptide agonist; the guanidinium group of
Arg-206 interacts with the terminal carboxylate of this agonist, as
shown by characterization of simultaneously altered ligand and receptor
mutants (21). Unlike its C-terminal residues, remaining regions of C5a
sequence interact with the N terminus of the C5aR (14); this
interaction enhances ligand binding affinity but is not required for
receptor activation.
A genetic screen of C5aR mutants expressed in Saccharomyces
cerevisiae (25), designed to assess the functional importance of
amino acids in receptor helices III, V, VI, and VII, identified a
cluster of residues situated at extracellular ends of the transmembrane helices that were required for C5aR signaling but are not
evolutionarily conserved. These residues are located at positions
cognate to positions of residues that are thought to interact with
ligands for the Hexapeptide Antagonists and Agonists--
ChaW and its
single site derivatives F1L, dCha4dL, W5Cha, and dR6dH were from the
Daiichi Research Center, University of California at San Francisco, San
Francisco, CA. The peptides were synthesized by standard solid phase
peptide chemistry, purified by high pressure liquid chromatography, and
analyzed by mass spectroscopy.
Construction of Receptor Mutants and Yeast Assays--
Mutated
C5aRs were created as described (25). Analysis of receptor signaling in
yeast was performed by replica plating onto different concentrations of
aminotriazole (AT), as described previously (25). For COS-7 cell
assays, the various C5aR sequences were subcloned into plasmid pDM8
(Invitrogen, Carlsbad, CA).
Mammalian Cell Culture and Transfection--
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium with 10% fetal calf
serum, 100 µg/ml streptomycin sulfate, 100 units/ml penicillin G, and
10 µg/ml gentamicin. Transfections of WT and mutant receptors were
performed using a DEAE-dextran/adenovirus method, as described
(26).
Membrane Preparations--
Membranes of COS-7 cells transfected
with WT or mutant C5aR were prepared by a modification of a previously
described method (26). Cells were harvested and lysed in
phosphate-buffered saline, pH 7.4, with 10 mM EDTA, 2 mM dithiothreitol, and protease inhibitors (phenylmethylsulfonyl fluoride, bacitracin, and leupeptin), and homogenized by passing 10 times through a 27-gauge needle. The supernatant fraction of two successive centrifugations at 900 × g for 10 min was centrifuged at 100,000 × g
for 30 min, and the particulate membrane fractions were resuspended in
lysis solution and stored at Binding Assays--
Membrane preparations expressing WT or
mutant C5aR (1-10 µg of total protein) were incubated at 37 °C
for 1 h with 25 pM (NQ) or 100 pM (all
others) [125I]C5a (2200 Ci/mmol, PerkinElmer Life
Sciences, Boston, MA) in 250 µl of binding buffer (Hanks' balanced
salt solution supplemented with 25 mM HEPES, pH 7.4, and
0.1% (w/v) bovine serum albumin). Recombinant, nonradioactive C5a
(Sigma, St. Louis, MO) was added to the indicated concentrations.
Nonspecific binding was defined as the amount of radioactivity bound in
the presence of 100 nM nonradioactive C5a. Incubations were
terminated by vacuum filtration through presoaked GF/C filters
(Whatman, Clifton, NJ) and rapid washing with 6 ml of ice-cold binding
buffer. Binding data were analyzed by nonlinear regression analysis
using Prism 2.0 (GraphPad Software, San Diego, CA). For competition
binding experiments, 1 nM [125I]C5a (400 Ci/mmol) was incubated with the indicated concentrations of ChaW and analogs.
Structure Determination of ChaW by NMR--
NMR experiments were
performed on a 500-MHz Bruker Avance instrument. All experiments were
carried out at 20 °C with a 1 mM sample of ChaW
dissolved in deuterated Me2SO. Resonance assignments and
structural constraints were obtained from two-dimensional 1H,1H-TOCSY and
1H,1H-NOESY experiments. HNH Activation Assays--
5 × 105 COS-7 cells
cotransfected with 0.25 µg of plasmid encoding G Molecular Modeling--
The rhodopsin crystal structure (7) was
the starting point for our model of the C5aR. Only the transmembrane
helix portions were used; the side chains corresponding to the human
C5aR sequence were substituted for the rhodopsin side chains with the
program SCWRL (31), which uses a backbone-dependent rotamer
library. Only the side chains of positions which differ between bovine rhodopsin and the C5aR were replaced; otherwise, the original coordinates were retained. The NMR structure of ChaW (see above) was
used for manual docking to the C5aR. Consistent with the observed flexibility, torsions of the side chains and backbone (termini only) of
ChaW were adjusted. Side chains of the C5aR model were also rotated
during manual docking: Ile-116, Leu-117, Tyr-121, Arg-206, and Tyr-290.
The C5aR complex was relaxed with the following energy-minimization
protocol: In SYBYL (release 6.5, Tripos, Inc., St. Louis, MO),
essential hydrogens were added to the receptor and all hydrogens were
added to ChaW. Kollman united-atom charges were loaded for the
receptor, and Gasteiger-Marsili charges were computed for ChaW. Energy
minimization of only the ligand and its immediate surroundings was
performed with the "Minimize Subset" option, specifying the entire
ligand as the subset. Other than the charge sets mentioned above and an
increase in the number of iterations to 500, default parameters were
used, including the Tripos force field. Interactive docking and
molecular graphics figures were done in MidasPlus (32) (Computer
Graphics Laboratory, University of California, San Francisco).
Intramolecular Epistasis Points to a Binding Pocket for
C5a--
Residues at 12 positions located near the extracellular ends
of the transmembrane helices were consistently preserved in functioning mutant C5aRs selected in our yeast screen (25); these are colored orange and yellow in Fig.
1. Of these, we chose seven
(orange in the figure) for further analysis, based on two
criteria: (a) a strict requirement for receptor function
(the corresponding single site mutants show no detectable activity in
our yeast assay; see Table I and Ref.
25); and (b) localization on a helical face pointing into
the transmembrane pocket between helices III, V, VI, and VII.
These criteria are compatible with the idea that these seven residues
form part of the C5a binding pocket. If so, amino acid substitutions at
these positions, which block C5a-triggered signals, should not affect
signaling by a constitutively active receptor whose activity is
independent of the agonist. In an intramolecular epistasis experiment,
we therefore combined each of the seven single site substitutions
(L112A, I116A in helix III; A203V, R206H, L207A in helix V; V286A,
S287A in helix VII) with the previously documented (25) activating
double mutation I124N/L127Q (NQ; cyan in Fig. 1). The NQ
double mutation involves residues located in helix III, two helical
turns farther from the extracellular space than residues in the
putative ligand binding pocket. Thus, direct compensatory interactions
between mutated NQ residues and substituted residues in the ligand
pocket are unlikely. The signaling phenotypes of six of the seven
combination mutants were similar to that of NQ itself. Based on this
epistasis analysis of single site substitutions (Table I), we infer
that these six residues are important for ligand-dependent
signaling but not for constitutive signaling, and therefore that they
are likely to comprise a transmembrane binding pocket for C5a. One
combination mutant, NQ/R206H, was inactive; this negative result does
not rule out a role for Arg-206 in binding ligand, however because the
R206H mutation may impair folding or expression of the mutant C5aR.
Mutant Cycle Analysis Identifies a Receptor Switch in the
Ligand-binding Pocket--
Amino acid substitutions for residues that
directly interact with the ligand should alter ligand binding affinity.
Accordingly, we assessed affinities of the seven putative binding
pocket mutants for binding [125I]C5a; binding assays were
conducted with C5aR mutants expressed in COS-7 cells, because in our
hands binding assays were not reproducible in the heterologous yeast
system. To our surprise the binding affinities of the mutant receptors
for C5a were indistinguishable from that of the wild type (WT) C5aR
(Table II). We imagine that the strong
contribution of the receptor's N terminus to its binding affinity for
C5a (13) overshadows more subtle contributions from individual residues
in the transmembrane binding site, which interact with a
different part of C5a (see above). Indeed, replacement of Arg-206 by
alanine (instead of histidine, as in our experiment) similarly failed
to alter affinity for C5a (21).
The high affinity of C5a for all mutant receptors allowed us to
characterize other ligands in competition binding experiments and to
perform a mutant cycle analysis, described below. To do so, we assessed
binding affinities of hexapeptide ligands, including the antagonist
ChaW and four derivatives, each of which differs from ChaW by
substitution of a single amino acid residue (Fig. 2A). Because the hexapeptide
ligands interact exclusively with the transmembrane pocket of the C5aR
(13), their binding affinities, unlike that of C5a, do not depend on
interaction with the receptor's N terminus. A second advantage of
these ligands is that ChaW shows an ordered structure in solution (see
below and Ref. 22), which allowed us to model the putative
ligand-receptor binding complex. Finally, replacement of the tryptophan
at position 5 of ChaW by cyclohexylalanine converts the antagonist into
an agonist (W5Cha) (20). Thus, as described below, a receptor site
found to interact with the side chain at position 5 of the ligand is
likely to comprise part of the elusive ligand-receptor activation
switch.
Mutant cycle analysis, which has successfully identified amino acids
involved in intermolecular protein-protein interactions (33-36), is
based on the following intuitive principle: If residue a of
the protein ligand (ChaW or its derivatives) interacts with residue
b of the C5aR, the effect of mutating a on the
ligand's binding affinity should depend on whether receptor residue
b is mutated as well. In practice, we simply compared the
relative changes of binding affinities by dividing the respective
inhibition constants (KI; see the legend of
Table II). We assessed binding affinities of ChaW and four singly
substituted derivatives to the WT C5aR and C5aR mutants with
substitutions at each of the seven residues in the putative ligand
binding pocket. Results of these experiments are summarized in Table II
and Fig. 2B and illustrated in Fig.
3, A-F.
Comparing the relative degrees of cooperation between each of four
hexapeptide residues and each of the seven genetically conserved
receptor positions (Fig. 2B) clearly indicates two direct and specific interactions: The side chain at position 5 of the ligand
(tryptophan in ChaW, cyclohexylalanine in the agonist W5Cha) apparently
interacts with two C5aR residues, Ile-116 in helix III and Val-286 in
helix VII. This analysis failed to show interaction of the other three
ChaW positions tested (Phe-1, dCha-4, or dArg-6) with any of the seven
mutated receptor side chains. The apparent specificities of the I116A
and V286A mutations for cooperating with side chains at position 5 of
the ligand make it unlikely that these receptor mutations act
indirectly to alter ligand affinity; otherwise, they would be expected
to alter apparent cooperation with other side chains in the ligand as well.
The I116A C5aR Mutation Converts ChaW into an Agonist--
The
fact that substitutions at position 5 in this series of hexapeptide
ligands determine their capacity to act as agonists (20) suggests that
receptor residues that interact with this side chain of the ligand form
part of the C5aR activation switch, which distinguishes agonists from
antagonists. In keeping with this prediction, replacement of Ile-116 by
alanine in the receptor converted ChaW from an antagonist (or weak
partial agonist) into a very strong agonist (Figs. 2C and 3,
G-I). As assessed by measuring inositol phosphate (IP)
accumulation in response to the hexapeptides, the WT and all the mutant
C5a receptors (Figs. 2C and 3, G-I), expressed
in COS-7 cells, showed robust responses to W5Cha, as well as C5a. In
cells expressing the I116A receptor mutant, ChaW
In contrast to the results in yeast (Table I), in COS-7 cells maximal
IP responses to C5a and the agonist hexapeptide W5Cha were not affected
by any of the seven receptor mutations tested (Figs. 2C and
3, G-I). To account for this discrepancy, we hypothesized that the C5aR in yeast cells is a less robust signal transducer than in
mammalian cells (owing, for example, to differences in protein
expression, folding, stability, functional interactions with G
proteins, etc.). Indeed, enhanced susceptibility of receptors in the
heterologous yeast system to detrimental effects of point mutations
appears to increase the sensitivity with which we can identify residues
that play important functions, even though these functions are not
absolutely required, under some conditions, for signaling of single
site mutants expressed in mammalian cells.
NMR Structure of ChAW--
In combination with previous work (Ref.
21; see below), identification of Ile-116 and Val-286 as sites for
interaction with a specific residue of hexapeptide ligands furnishes
stringent constraints for orienting ChaW relative to the C5a receptor
binding site, provided that the ligand structure is known. We therefore solved the NMR structure of ChaW in Me2SO (see
"Experimental Procedures"). Despite its small size, ChaW shows an
ordered structure in solution, comprising an inverse Docking and Modeling of a ChaW/C5aR Ligand-Receptor
Complex--
With the structure of ChaW in hand, we used
intermolecular contacts as constraints in building a model of the
receptor-antagonist complex. Two of these, detected by mutant cycle
analysis (see above), are interactions of the Trp-5 residue of ChaW
with Ile-116 in helix III and Val-286 in helix VII of the receptor. We
also used constraints identified in a previous study (21) of the receptor's interaction with the C-terminal dArg-6 residue of ChaW. In
this study, DeMartino and coworkers used an R206A receptor mutant and a
series of ChaW derivatives to show that the side chain of Arg-206 acts
as a "gate-keeper," allowing hexapeptide ligands to bind to the
transmembrane receptor core only when the ligands present a C-terminal
carboxylate, which appears to interact with the guanidinium group of
Arg-206. In addition to the carboxylate, the side chain of dArg-6 was
also required for receptor interaction, leading these investigators to
suggest that it interacts with the transmembrane receptor pocket.
These constraints, together with the known backbone structure of ChaW,
orient the hexapeptide in relation to the C5aR model. Fig. 4 shows ChaW
docked in the receptor's putative transmembrane ligand binding pocket
(see "Experimental Procedures"). The orientation of ChaW with
respect to the ligand binding pocket correlates nicely with results of
detailed pharmacological analyses of ChaW and its derivatives (23, 24).
Thus Lys-2 and Pro-3 of the hexapeptide, sites at which
substitutions do not affect binding affinity or antagonistic potential,
point away from the putative ligand binding pocket of the C5aR model
(Fig. 4). In contrast, substitutions for Phe-1 greatly decreased
binding affinity of ChaW (23). The location of the Phe-1 side chain
close to the extreme extracellular end of helix V may also favor
contacts with the N-terminal end of the receptor's second
extracellular loop, a region that appears to mediate high affinity
binding of C5a: Replacement of this region in a C5a/formyl peptide
receptor chimera abolished C5a binding (37). Although the dCha-4 side
chain is close to that of Leu-112 (helix III) in the receptor binding
pocket (Fig. 4), mutant cycle analysis did not indicate an
energetically important interaction between the two side chains.
In our docking model the long arginine side chain at position 6 of ChaW
penetrates deeply into the receptor's helix bundle, between helices
III, V, and VI (Fig. 4C). Its guanidinium group, located one
helical turn lower than any of the genetically conserved residues in
the putative ligand binding pocket, is cradled in a pocket of aromatic,
mostly conserved residues in helices V and VI (green in Fig.
4C). Interaction of a positively charged side chain
(arginine or lysine) with aromatic residues is not unlikely in protein
structures; indeed, a recent analysis (38) of interactions between
cationic groups and delocalized Trigger Zone for Receptor Activation--
Our results indicate
that the side chain at position 5 of ligand hexapeptides is clasped by
hydrophobic side chains of amino acids in helix III (Ile-116) and helix
VII (Val-286). The role of position 5 in determining whether a
hexapeptide acts as an agonist or an antagonist, combined with
conversion of ChaW into an agonist by the I116A mutation, points to the
cleft between helices III and VII as a potentially pivotal site at
which the ligand may trigger C5aR activation of the C5aR.
Unfortunately, we do not know why cyclohexylalanine at ligand position
5 induces conformational changes required for receptor activation, but
tryptophan at the same position does not. In our model complex (Fig.
4), the tryptophan and cyclohexylalanine side chains appear to fit
equally well into the cleft between Ile-116 and Val-286. A previous
pharmacological analysis of ChaW and derivatives altered at position 5 (20) provides a tantalizing clue, however, suggesting that contact
between residues Ile-116 and Val-286 and the agonist's side chain at
position 5 may pull helices III and VII closer to one another. These
experiments (20) showed that agonist activity decreased with changes in
the side chain at position 5 in the following order: Leu, Cha > Phe > naphthylalanine > Trp. Only the transition from a
naphthyl to an indole side chain completely prevented C5aR activation,
however. The principal chemical difference between the condensed
aromatic rings of these groups, which are of similar size, is the
presence of a hetero-aromatic nitrogen in the indole.
Thus, we speculate that the receptor is activated by insertion of a
hydrophobic group (e.g. cyclohexylalanine) between helices III and VII but that robust activation is not compatible with the
greater separation of these helices that would be induced by insertion
of the bulky indole group with its nitrogen, which could further
perturb an otherwise purely hydrophobic helix interface. Although this
explanation is severely limited by our lack of knowledge of the
structure of the receptor's active state, it is compatible with the
reverent phenotype of the I116A mutation, in which replacement of
isoleucine by alanine could compensate for the indole to allow agonism
by ChaW, whereas the smaller size difference between valine and alanine
in the V286A mutant furnishes too little extra room.
Even though we cannot provide a satisfying explanation of
how ligand interaction with Ile-116 and Val-286 activates
the C5aR, it is likely that the same site in other receptors plays a
role in triggering receptor activation, as suggested by studies of the
Perspective--
Our model of the ChaW·C5aR complex (Fig. 4) is
based on an NMR structure of the ligand, a homology model of the
receptor helices, and interactions between specific side chains
predicted from genetic analysis. The general location and orientation
of ChaW in the predicted binding pocket within the receptor's helix
bundle are probably correct, because they explain a large array of
pharmacological evidence from studies of mutant peptides and mutant
C5aRs (see above). This docking model sets the stage for further
experiments, including analysis of the interactions of the C5aR and C5a
itself, as well as approaches to designing small-molecule antagonists for the C5aR.
We infer from our observations that C5a agonists activate the receptor
by interacting with a trigger zone for activation, located between
neighboring residues in helices III and VII. As summarized above, quite
different experimental approaches in other laboratories have identified
pivotal sites for receptor activation at the same helix-helix contact
site in two other serpentine receptors, the
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subunit of the heterotrimer, allowing the
and
subunits to disengage from one another and activate intracellular effectors (1). Of several serpentine receptor families
(2), the rhodopsin-like family is the largest (3). Low resolution
models of the three-dimensional structure of the seven-helix bundle in
the serpentine receptor core were based on patterns of conserved
primary structure, biochemical observations with many receptors, and a
low resolution electron cryomicroscopy structure of rhodopsin (4).
Three such models, constructed independently (3, 5, 6), predict
three-dimensional structures of the transmembrane helices that are
remarkably similar to one another and to a recent three-dimensional
crystal structure of rhodopsin at atomic resolution (7). These
similarities make the crystal structure a promising platform for
designing and interpreting experiments aimed at elucidating structure
and molecular mechanisms of other members of the rhodopsin-like family
of serpentine receptors.
2-adrenoreceptor, rhodopsin, and other serpentine
receptors, suggesting that structurally different agonists activate
their receptors by a common mechanism, which involves similarly located elements of the receptors' ligand binding pockets. To address this
hypothesis, we characterized interactions of C5a and other ligands with
C5aR mutants in which alanine or other residues replace natural
residues at sites in the putative ligand binding pocket. These
experiments identified a transmembrane binding pocket for the
antagonist ChaW, as well as an apparent activation switch that involves
ligand interaction with adjacent residues in helices III and VII. On
this basis we propose a model of the complex of the C5aR with the
synthetic antagonist, ChaW. The model and our biochemical observations
point to a site for ligand-dependent activation that is
probably conserved in many serpentine receptors.
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70 °C.
coupling
constants were obtained from one-dimensional slices along the
acquisition dimension of the NOESY experiment with the help of the
program INFIT (27). NMR data processing and analysis were carried out
using the programs PROSA (28) and XEASY (29). Structure calculations
were performed with the software package DYANA (30) using 84 NOE
distance constraints and 2 HNH
coupling constants for the Trp and
dCha residues.
16 and with 0.25 µg of plasmid encoding WT or mutant C5a receptors were incubated
overnight with 2 µCi of [3H]inositol (21 Ci/mmol,
PerkinElmer Life Sciences, Boston, MA), washed with assay medium (RPMI
1640 supplemented with 20 mM HEPES, pH 7.4, and 5 mM LiCl), incubated with or without 10 nM C5a
for 1 h at 37 °C, aspirated and incubated with 750 µl of 20 mM cold formic acid at 4 °C for 30 min, and adjusted
with 100 µl of solution I (6 ml of concentrated ammonium
hydroxide/liter). Poly-Prep chromatography columns (Bio-Rad, Hercules,
CA) with 1 ml of AG 1-X8 resin (100-200 mesh, Bio-Rad) were
equilibrated with 10 ml of solution II (4 M ammonium
formate, 0.2 M formic acid) followed by 5 ml of solution III (10-fold dilution of solution II). Samples were loaded, columns were eluted with 1 ml of solution III, and the resulting inositol fractions were collected. Columns were washed with 4 ml of solution IV
(40 mM ammonium formate, 0.1 M formic acid),
eluted with 1 ml of solution II, and the resulting inositol phosphate
(IP) fractions were collected. Inositol phosphate accumulation was
reported as the fraction of total inositol used ([IP]/([IP] + [total inositol]) × 100%).
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Fig. 1.
The transmembrane ligand binding pocket of
the C5aR. Genetically conserved residues (25) presumed to
form the transmembrane ligand binding pocket of the C5aR
(yellow and orange), viewed from the
extracellular medium. The orange residues were subjected to
mutant cycle analysis, as described in the text. Ile-124 and Leu-127,
which are substituted in the constitutively activating I124NL127Q
(NQ) double mutant (see text), are cyan. Helices
are designated by roman numerals, and key amino acid side
chains are designated by amino acid (one-letter code) and by
number in the C5aR sequence.
Signaling strength of WT and mutant C5aR constructs in yeast
C5a) or an a-factor prepro/C5a ligand (+C5a). Receptor
signaling was assayed by growth of histidine-deficient media in the
presence of AT: +++++, growth on 10 mM AT; ++++, growth on
5 mM AT; +++, growth on 2 mM AT; ++, growth on
1 mM AT; +, growth on 0.5 mM AT; 0, no growth
on 0.5 mM AT.
Analysis of saturation and inhibition binding for C5aR constructs
) for an intermolecular
interaction between two residues (one on the receptor and one on the
ligand, respectively) is defined as
= (Ki wtwt × Ki mtmt)/(Ki wtmt × Ki mtwt), where Ki wtwt is the
inhibition constant of the WT C5aR with ChaW,
Ki mtwt the inhibition constant of an individual
C5aR mutant with ChaW, Ki wtmt the inhibition
constant of the WT C5aR with an individual ChaW mutant, and
Ki mtmt the inhibition constant of a mutant
receptor with a ChaW mutant. For ease of comparison, the cooperation
value of each pairing has been normalized by setting as = 1.0 the
relative affinity change of ChaW versus a substituted ligand
for the WT C5aR. In addition, where the
for an interaction is less
than 1.0, the value is reported as its reciprocal.
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Fig. 2.
Identification of intermolecular contacts
between ChaW and specific residues of the C5aR. A,
amino acid sequences of the C-terminal octapeptide of C5a, a deleted
form of C5a that acts as a related antagonist (17), the hexapeptide
ChaW, and ChaW derivatives used in these experiments. Mutated residues
are shown in open and shaded representation. The
two agonists (C5a and W5Cha) are marked by asterisks.
B, summary of the mutant cycle analysis of binding
affinities of all combinations of ChaW and its derivatives with the WT
and all mutant C5a receptors. Bars indicating the
interaction of W5 in ChaW with Ile-116 and Val-286 in the C5aR are
colored black. Refer to Table II for calculations of
cooperation values ( ). C, agonist-induced (C5a, W5Cha) or
antagonist-induced (ChaW) inositol phosphate production mediated by the
WT or mutant C5a receptors. The front row indicates
background (bck) levels in the absence of ligand. Activity
is given as the percentage fraction of total inositol converted to
inositol phosphate. The bar indicating ChaW-mediated
activation of the I116A mutant is colored black.
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Fig. 3.
Selected results illustrating or extending
the mutant cycle analysis of the interaction of ChaW and its
derivatives with the WT and mutant C5a receptors. Results are
shown for WT C5aR and the I116A and V286A mutant receptors and the C5a,
ChaW, and W5Cha ligands only. A-C, saturation binding
experiments performed to determine the affinity of C5a ( ) for the WT
and the I116A and V286A mutant receptors. D-F, competition
binding experiments performed to determine the affinities of ChaW (
)
and W5Cha (
) for the WT and the I116A and V286A mutant receptors.
G-I, stimulation of inositol phosphate accumulation by C5a
(
), ChaW (
), and W5Cha (
). The ligand concentration (nM)
required for half-maximal effect is indicated next to each respective
curve. Panels show one of three or more independent experiments
performed with duplicate or triplicate determinations (A-C)
or the means ± S.E. of three independent experiments, each
performed with duplicate determinations
(D-I).
at best a weak
partial agonist for the WT C5aR
induced IP accumulation comparable to
responses observed with W5Cha acting on the WT or any mutant C5aR
(Figs. 2C and 3, G-I). This surprisingly robust functional cooperation, in combination with the highly cooperative effects of the I116A mutation on ligand binding affinity, argues strongly for a direct interaction between the side chain at this position and position 5 of the hexapeptide ligand. The V286A receptor mutant C5aR, carrying a substitution for the other side chain that
appears to interact with the residue at position 5 of the hexapeptide,
did not recognize ChaW as an agonist; with this receptor mutant the
molar potencies of both C5a and W5Cha were slightly lower than those
observed with the WT and I116A receptors (Fig. 3, G-I).
Similar to ChaW, the three other ChaW derivatives, F1L, dCha4dL, and
dR6dH, induced a response with the I116A mutant only (data not shown).
-turn formed by
the Lys-Pro-dCha residues and a distorted type II
-turn that
includes the residues of the
-turn and the tryptophan at position 5. This structure (Fig. 4) appears to agree
with an earlier NMR study that identified a well-defined backbone
conformation composed of the same structural motifs (22). Because the
authors of this analysis chose not to make their ChaW coordinates
available to us, we cannot directly compare our structure with
theirs.
View larger version (39K):
[in a new window]
Fig. 4.
Model of the C5aR in complex with the
antagonist ChaW. A, top (extracellular) and
B, side views of the docking of ChaW to the C5aR.
Roman numerals designate transmembrane helices. Ligand
backbone and side chains are cyan, except that the
C-terminal carboxylate is red, the C-terminal arginine side
chain dark blue, and the indole side chain at position 5 pink. Receptor residues identified as interacting with
specific parts of the ligand (Ile-116 in helix III, Arg-206 in helix V,
and Val-286 in helix VII) are orange; other receptor
residues studied in our experiments, including Leu-112 (helix III),
Ala-203 (helix V), Leu-207 (helix V), and Ser-287 (helix VII), are
yellow. Aromatic side chains of the green
receptor residues (Tyr-121 in helix III, Phe-211 in helix V, and
Phe-251, Trp-255 and Tyr-258 in helix VI) in C are
postulated by the docking model to interact with the dArg-6 side chain
of ChaW.
-electron systems in proteins found
such interactions to be much more frequent and energy-rich than
previously assumed. This predicted location for the dArg-6 side chain
of the ligand is especially intriguing in light of previous evidence
(21) that this side chain is necessary for receptor activation.
2-adrenoreceptor and retinal rhodopsin. A mutant
2-adrenoreceptor is activated by formation of a Zn(II)
bridge between receptor residues at positions precisely cognate to
Ile-116 and Val-286 of the C5aR (39). In rhodopsin, the chromophore
11-cis-retinal forms a Schiff base with the side chain of
Lys-296 in helix VII (one turn below the position of Val-286 in the
C5aR), which in turn participates in a salt bridge with Glu-113 of
helix III (one turn above Ile-116 in the C5aR); rhodopsin is activated
when this salt bridge is broken by light-triggered conversion of the
chromophore to the all-trans conformation (reviewed in Ref.
40). Moreover, constitutive activity of rhodopsin results from
mutations that prevent formation of this helix III/VII salt bridge
(that is, substitutions for either Glu-113 or Lys-296). We are thus
left with strong hints that receptors share a common activation trigger located at about the same level of the helix III/VII interface, although none of the available evidence tells us how the trigger works.
2-adrenoreceptor (39) and rhodopsin (40). Similarly
placed activation triggers for three structurally different agonists, a
74-residue polypeptide, a biogenic amine, and a hydrophobic chromophore, suggest that this region plays a similar role in other
serpentine receptors. This suggestion is in accord with other evidence
suggesting that helix VII may move when receptors are activated. In
rhodopsin, light activation exposes an epitope for monoclonal antibody
located at the cytoplasmic end of helix VII, suggesting that activation
induces a movement of this helix (41). In addition, truncating
mutations that completely removed helix VII were found in C5aR mutants
that signaled constitutively in yeast (25), suggesting that movement of
this helix in the course of normal activation by ligands may relieve
constraints that hold the receptor in an inactive conformation.
Experiments in several laboratories (42, 43), including ours (44, 45), indicate that a movement of helix VI relative to helix III is necessary
for activation of G proteins. We therefore speculate that a
ligand-induced movement of helix VII relieves a constraint on helix VI,
allowing helix VI to move away from helix III.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM-27800 (to H. R. B.). The Computer Graphics Laboratory, University of California, San Francisco was supported by National Institutes of Health Grant P41-RR-01081.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ An Advanced Career Postdoctoral Fellow of the Swiss National Science Foundation. Present address: Institute for Research in Biomedicine, Via Vincenco Vela 6, CH-6501 Bellinzona, Switzerland.
A Howard Hughes Medical Institute Physician Postdoctoral
Fellow. Present address: Depts. of Medicine and Molecular Biology and
Pharmacology, Washington University School of Medicine, Box 8103, 660 S. Euclid Ave., St. Louis, MO 63110.
** To whom correspondence should be addressed: Dept. of Cellular and Molecular Pharmacology, University of California, Box 0450, 513 Parnassus Ave., San Francisco, CA 94143. Tel.: 415-476-8161; Fax: 415-476-5292; E-mail: bourne@cmp.ucsf.edu.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M007748200
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ABBREVIATIONS |
---|
The abbreviations used are: C5a, complement factor 5a; C5aR, complement factor 5a receptor; ChaW, Me-F-K-P-dCha-W-dR; dCha, D-cyclohexylalanine; NQ, double mutation I124N/L127Q; 1H, 1H-TOCSY, proton-proton total correlation spectroscopy; 1H, 1H-NOESY, proton-proton nuclear Overhauser enhancement spectroscopy; IP, inositol phosphate; WT, wild type; AT, aminotriazole.
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