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Originally published In Press as doi:10.1074/jbc.M410797200 on January 20, 2005

J. Biol. Chem., Vol. 280, Issue 18, 17831-17840, May 6, 2005
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Comparative Agonist/Antagonist Responses in Mutant Human C5a Receptors Define the Ligand Binding Site*

Adrian Higginbottom{ddagger}, Stuart A. Cain{ddagger}§, Trent M. Woodruff¶, Lavinia M. Proctor¶, Praveen K. Madala||, Joel D. A. Tyndall||**, Stephen M. Taylor¶, David P. Fairlie||{ddagger}{ddagger}, and Peter N. Monk{ddagger}{ddagger}{ddagger}§§

From the {ddagger}Academic Neurology Unit, University of Sheffield Medical School, Sheffield S10 2RX, United Kingdom and Department of Physiology and Pharmacology and ||Institute of Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia

Received for publication, September 20, 2004 , and in revised form, January 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites (Ile116, Arg175, Arg206, Glu199, Asp282, and Val286). Agonist peptide 1 (Phe-Lys-Pro-D-cyclohexylalanine-cyclohexylalanine-D-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 (peptide 2). Conversely, mutation of C5aR transmembrane residue Ile116 to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394–3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile116, the latter is not essential for either activation or antagonism. Arg206 and Arg175 both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp282 has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu199 may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
C5a, a 74-residue polypeptide, is involved in several stages of inflammatory processes, causing chemotaxis and degranulation of leukocytes, enhancing vascular permeability, and stimulating cytokine production (2). The C terminus of C5a is rapidly truncated in vivo to C5a des-Arg74 (3), a plasma-stable form that has a different spectrum of bioactivity to that of intact C5a (4). Peptide analogs of the C-terminal domain of C5a (e.g. Phe-Lys-Pro-D-Cha-Cha-D-Arg, peptide 1) (5) are reportedly full agonists at the C5a receptor (C5aR).1 This suggests that the C terminus is solely responsible for receptor activation, the remainder of the molecule conferring high affinity binding (6–8). C5aR is a member of the G protein-coupled receptor superfamily (9, 10), two of the extracellular loops (the second and third) and the N-terminal domain being essential for C5a binding (11). The receptor N terminus is required for high affinity binding of C5a but not for receptor activation by C5a or small peptide agonists (12), which interact with charged residues at the extracellular faces of the transmembrane helical bundle and hydrophobic residues in the core of the receptor (13–15). The nature of the residue at position 5 of 1 has been shown to be crucial for agonist activity (5), with substitution by bulkier, more aromatic molecules such as Trp or 1-naphthylalanine reducing agonist activity (5). Two derivatives of 1, the linear peptide antagonist Phe-Lys-Pro-D-cyclohexylalanine-Trp-D-Arg, 2, (5) and the cyclic peptide antagonist Phe-cyclo-[L-Orn-Pro-D-cyclohexylalanine-Trp-Arg], 3, have been shown to inhibit C5a binding and function at human and rat C5aR (16–18).

A model for the interaction of antagonist 2 with C5aR has recently been proposed using data from a yeast-based system of genetic analysis. Antagonist 2 became a full agonist at the I116A-mutated C5aR. This was interpreted as suggesting that Ile116 in transmembrane helix 3 could be part of an activation switch that is blocked by Trp5 in antagonist 2 (1). An adjacent receptor residue, Val286, in helix 7, was also suggested to contribute to the mechanism. The C-terminal carboxylate has been shown to be involved in the interaction between 2 and C5aR, possibly at Arg175 in helix 4 or Arg206 in helix 5 (6, 13). On the other hand, cyclic compound 3, in which there is no free carboxylate, is an even more potent antagonist than 2, so its antagonist properties are likely dependent upon only its Trp5 residue, the C terminus being blocked. The side chain of the C-terminal Arg74 residue of C5a has been shown to interact with Asp282 at the extracellular side of helix 7 (14). Glu199, near the extracellular face of helix 5, appears to interact with the side chain of Lys68, a residue required for full activity in agonist peptides and C5a des-Arg74 but not intact C5a (19, 20).

In this paper we report the effects of mutating Ile116, Val286, Arg175, Glu199, Arg206, and Asp282 on the activity of C5a, C5a des-Arg74, and a series of peptide agonists and antagonists. The data suggest that residues other than Ile116 and Val286 may be involved in the mechanism of antagonist action for cyclic antagonist 3 and perhaps also for acyclic ligands. However, the substitution of a more bulky hydrophobic residue for Trp5 in 3 does produce a peptide with very weak agonist activity at I116A-C5aR, suggesting that Ile116 may lie in close proximity to this ligand residue as well. Together with evidence for interactions between the C-terminal Arg6 of the peptides and Arg175, Glu199, Arg206, and Asp282, we have produced a new model of the peptide ligand binding site on C5aR, which accommodates structure/activity relationships that define antagonist function for 2, 3, and analogues (21).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Human C5aR Mutants—The mutant C5aRs, I116A and V286A, were constructed by overlap extension mutagenesis as described previously (15). The C5aR mutant clones were sequenced using ABI Big Dye terminator cycle sequencing kit, and the correct constructs were purified using Nucleobond kit PC500 (Macherey-Na-gel). The production of the C5aR receptor mutants R175A, R175D, E199K, R206A, double mutant E199K/R206A, and D282A have been previously described (14, 19, 22, 23).

Transfection and Cell Culture—RBL-2H3 cells were routinely cultured in Dulbecco's modified Eagle's medium plus 10% (v/v) fetal calf serum, which was supplemented with 400 mg/liter G418 for transfected cells, at 37 °C, 5% CO2. RBL-2H3 cells were transfected by electroporation, as previously described (23). A monoclonal antibody (S5/1; Serotec) that recognizes the N-terminal sequence of the C5aR was used to sort the highest 50% of transfected cells on a BD Biosciences Vantage flow cytometer in two rounds of fluorescence-activated cell sorting.

Production of Peptides and Recombinant Ligands—The ligands used in this study are shown in Table I. C5a, V3, C5a[Ala74], and C5a des-Arg74 were produced in Escherichia coli and purified by the methods described in Crass et al. (23). Agonist and antagonist peptides, synthesized as described previously (21), were supplied by Promics Pty Ltd. (Queensland, Australia). All cyclic peptide antagonists were N-acetylated at the N terminus except 3. An N-acetylated form of 3 had identical properties to the non-acetylated form at the C5aR in RBL cells (data not shown). Polypeptide C5a receptor antagonist C5aRA (24) was a generous gift from Joerg Kohl (Medizinische Hochschule, Hannover, Germany), and antagonist V2[Glu68] was made as described (25).


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  		TABLE I
Peptide sequences

 
Measurement of Receptor Activation—Receptor activation in RBL cells was measured as the release of {beta}-hexosaminidase from intracellular granules as described (26). The percentage of {beta}-hexosaminidase release was calculated as a percentage of the release stimulated by a high dose of C5a (1 µM). Total {beta}-hexosaminidase content was determined after cell lysis with 0.1% Nonidet P-40. Assay of the antagonist activity was performed as described above except that the antagonists were added at varying concentrations for 15 min before the addition of C5a, C5a des-Arg74, or V3 at a final concentration of 50 or 250 nM.IC50, EC50, and S.E. values were obtained by nonlinear regression analysis using GraphPad Prism 3.0.

Chemotaxis Assay—Chemotaxis was measured using a 48-well migration chamber (Neuroprobe), with a 5-µm pore polycarbonate membrane. Cells were harvested by treatment with phosphate-buffered saline plus 5 mm EDTA, resuspended in Dulbecco's modified Eagle's medium plus 0.1% bovine serum albumin at 106/ml, and placed in the top chamber. Dulbecco's modified Eagle's medium plus 0.1% bovine serum albumin containing C5a, C5a des-Arg74, V3, or antagonist peptides was placed in the lower chamber, and the apparatus was incubated at 37 °C for 2 h. Cells were mechanically removed from the upper surface of the membrane, which was then fixed in methanol for 30 s and stained with a 0.1% solution of toluidine blue in 0.1 M sodium phosphate, pH 5.5, for 10 min. The numbers of migrated cells was measured by densitometry using an imaging system (Alpha Innotech Corp.). In some cases the relationship between the observed density of staining and cell number was confirmed by counting stained cells in three fields at a 40x magnification. Migration was calculated as a percentage of migration of positive control (WT-C5aR cells to 1 nM C5a) included in every experiment after subtraction of migration in the absence of ligand. All experiments were performed in triplicate, with a minimum of three repeats.

[125I]C5a and ([3H]3CCO)(Phe)-cyclo-[Orn-Pro-D-Cha-Trp-Arg] Binding Assays—Binding assays using 50 pM [125I]C5a or 830 pM ([3H]3CCO)(Phe)-cyclo-[Orn-Pro-D-Cha-Trp-Arg] ([3H]Ac-3) were performed as previously described on either adherent C5aR-transfected RBL cells (23) or isolated human polymorphonuclear granulocytes (PMNs) (18). IC50 and Bmax values were obtained by nonlinear regression and Scatchard analyses, respectively, using GraphPad Prism 3.0.

Ligand Docking to a Homology Model of C5aR—Docking studies were carried out on antagonist peptides 2 and Ac-3 using a homology model of the C5aR,2 based on the crystal structure of bovine rhodopsin (27). The homology model was constructed using the Homology module within InsightII molecular modeling suite (Accelrys Inc., InsightII Modeling Environment, Release 200.1, 2004, Accelrys Inc., San Diego, CA). In this model of C5aR, Arg175 and Glu199 are located just above the top of helices 4, and 5, respectively, but their predicted positions have a high degree of uncertainty since the model is only based on sequence homology with the transmembrane helices of rhodopsin. In contrast, Arg206 is at the extracellular face of helix 5, and its location can be predicted with more confidence.

Docking studies were carried out using the flexible ligand docking program Gold Version 2.1. The most recent and best defined (21) NMR-derived solution structures of 2 (21) and Ac-3 (17) were used as starting points for ligand docking. We have created a series of structure models for C5aR either with a Cys188–Cys109 disulfide bond that closes the loops onto the transmembrane regions or without a disulfide bond and loops directed away from the transmembrane region, which is then exposed for extracellular access. Models were generated using the Homology module within InsightII 2000.1. The receptors were prepared for docking by assigning charges and potentials and adjusting the side chains of specific residues where necessary (Asp282, Arg206). The ligands used were based on the NMR structures that we have previously published and refined (21) for the cyclic and acyclic antagonists (17, 21). Docking studies were carried out using Gold Versions 2.1, and results were viewed using InsightII. All modeling and docking was performed on an SGI R12000 [GenBank] octane work station.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Mutant Human C5aR in RBL-2H3 Cells—Wild type (WT) and mutant (I116A, V286A) C5aR were transfected into RBL-2H3 cells, and stable transfectants were obtained by selection with G418. Homogenous populations of cells were collected by two rounds of fluorescence-activated cell sorting, selecting for the top 50% expressing cells using an anti-C5aR monoclonal antibody. Receptor expression levels were measured by the specific binding of [125I]C5a (Bmax) to detect active receptor and by immunofluorescence to detect total receptor (Fig. 1). By these criteria WT and I116A-C5aR binding levels were similar, but V286A-C5aR showed reduced binding of [125I]C5a despite similar levels of receptor expression as shown by immunofluorescence. Degranulation in response to a high dose of C5a (1 µM) was also assessed and was found to vary widely between cell lines (Fig. 1). I116A-C5aR had a maximal release of only 10% of total {beta}-hexosaminidase, similar to that found previously for R206A-C5aR and E199K/R206A (14). The expression and maximal secretion by cells transfected with R175A and R175D-C5aR have been also been described previously (22). Both of these mutant receptors are activated only weakly by C5a but can be stimulated effectively by a variant of C5a des-Arg74 (V3) isolated by selection from a phage display library (22). R175A secretes ~50% of total {beta}-hexosaminidase in response to 1 µM V3, whereas R175D secretes to a lower maximum level. E199K- and D282A-C5aR have been shown to be expressed at high levels and to secrete substantial amounts of {beta}-hexosaminidase in response to C5a (14, 23). The strength of the degranulatory response is reduced by some mutations of C5aR (results are presented here and elsewhere e.g. Ref. 22), an effect that does not correlate with either C5aR expression or C5a binding and may be due to partial misfolding of mutant receptor. However, the functional response used to measure the activity of ligands depends only on the contribution of properly folded receptors, and there is clearly sufficient active receptor to stimulate measurable degranulation in all cases (14).



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  		FIG. 1.
C5a receptor expression by transfected RBL cells. Receptor expression levels on untransfected RBL cells (NX) or RBL cells transfected with WT-, I116A-, or V286A-C5aR were measured by immunofluorescence (open bars) and are shown as log median channel numbers; means are from a single experiment performed in duplicate. Expression levels were also assessed by Scatchard analysis of [125I]C5a binding (hatched bars), shown as Bmax values in dpm/well to adherent cells in a 96-well microtiter plate from n separate experiments (see Table I) performed in triplicate. The maximal degranulation levels of these cell lines (filled bars) in response to 1 mM C5a are shown as the percentage of total cellular {beta}-hexosaminidase mean ± S.E. released into the supernatant from at least three separate experiments performed in triplicate.

 
The Effects of Mutation of C5aR Residues on Receptor Affinity for C5aR Ligands—The affinities of WT and mutant C5aR for the full agonist C5a and the partial agonist C5a des-Arg74 were measured by pretreating transfected RBL cells with these agents, then incubating with 50 pM [125I]C5a (Table IIA). Only the mutation of Arg175 caused a substantial loss of affinity for C5a, with 30- and 152-fold decreases observed for R175A and R175D, respectively. In contrast, mutation of either Ile116 or Val286 increased affinity for C5a des-Arg74, whereas other mutations had no effect or caused only a small decrease (R175D). The binding of peptide ligands was increased in most cases measured, with only R175D-C5aR showing substantially decreased affinities for the ligands relative to WT-C5aR. These data suggest that mutation of these residues, with the exception of R175D, have relatively small effects on the overall energy of interaction of these ligands for the receptor.


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  		TABLE II
Affinities of C5a and C-terminal analogues for C5a receptor and selected receptor mutants

 
Peptide Ligand Binding Sites on C5aR—To determine whether linear and cyclic peptide antagonists bind to the same site on C5aR, competition binding studies using radiolabeled cyclic antagonist [3H]Ac-3 were performed on human PMNs (Table IIB). C5a competed for [3H]Ac-3 binding with a 6-fold higher IC50 than for [125I]C5a, confirming the high affinity of cyclic antagonist for C5aR. The linear antagonist 2 also appeared to have a higher affinity when competing for [3H]Ac-3 binding relative to [125I]C5a. Two forms of cyclic antagonist, Ac-3 and 3, had comparable affinities when competing with [125I]C5a or [3H]Ac-3 for C5aR binding (Table IIB). These data support the idea that C5a and the peptide ligands (agonist 2 and antagonist 3) bind to the same (or a very similar) site on C5aR.

The Effects of Mutation of C5aR Residues on C5aR Agonists—The responses of mutated receptors to C5a largely correlated with binding affinity. In contrast, the response to the partial agonist C5a, des-Arg74 was completely inhibited in several cases (R206A-, R175A- or R175D-, E199K-C5aR), suggesting a selective loss of responsiveness in the absence of the Arg74 side chain (Table IIC). The agonist form of the linear peptide, 1, was a superagonist at several mutant receptors (I116A-, V286A-, D282A-, R175A- or R175D-C5aR) stimulating a greater degree of activation than 1 µM C5a/V3, but it failed to activate R206A-C5aR. Two other peptide agonists of C5aR, 4 and 5, also stimulated supramaximal levels of release from the receptor mutants I116A-, V286A-, D282A-C5aR. R175A- or R175D-C5aR did not respond to these peptides, whereas R206A, not activated by 1, showed a substantial response to 4 and 5. In contrast, E199K-C5aR responded well to peptide 1 but very poorly to 4 and 5. The presence of an L-Arg at the C terminus of peptides 4 and 5 may have been responsible for this mixed pattern of receptor activation. However, a variant of peptide 1 with L-Arg instead of D-Arg (1-L-Arg) failed to activate either R206A or E199K-C5aR, suggesting that additional factors must be involved. The C-terminal amide analog of 1 (1-amide) is a substantially weaker agonist at WT-C5aR than 1, suggesting that a C-terminal carboxylate might be important for agonist activity. Interestingly, receptors R206A- and R175A- or R175D-C5aR showed a reverse response, indicating that 1-amide was a stronger agonist at these receptors than 1.

The linear antagonist, 2, did not stimulate secretion from cells transfected with WT or mutant C5aR at concentrations up to 100 µM, with the exception of I116A-C5aR. In these cells, the response to 2 had an EC50 of 622 nM, similar to the agonist peptide 1 (Table IIC). Antagonist 2 was, however, only a partial agonist, stimulating a maximal release of 62% of the response to 1 µM C5a. In contrast, the cyclic antagonist 3 (and Ac-3) had no stimulatory effect at concentrations up to 100 µM (Table IIC) but did act as a partial agonist at R175D-C5aR, stimulating cells to the same level as C5a, with an EC50 of 2410 nM (Table III). Neither of two polypeptide C5aR antagonists, derived by mutagenesis of C5a, namely C5aRA and V2[E68], had any agonist activity on any mutant C5aR at concentrations of up to 3 and 10 µM, respectively (data not shown).


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  		TABLE III
Comparative antagonist potencies on mutant C5a receptors in transfected RBL cells

 
These data were confirmed by studies using chemotaxis as an even more sensitive read-out of receptor activation (Fig. 2). WT-C5aR cells migrated very efficiently to C5a (maximum migration at 1 nM) but were less sensitive to C5a des-Arg74 (maximum at 100 nM). In contrast, D282A-C5aR cells migrated equally well to both stimuli (maxima at 100 nM). E199K- and R206A-C5aR cells responded more strongly to C5a (maxima at 10 nM and 1 µM, respectively) than to C5a des-Arg74 (maxima >10 µM in both cases). R175A (and R175D, data not shown) displayed only very weak responses to C5a, but V3 stimulated higher levels of migration (maxima at 1 nM). As expected from the degranulation data, I116A-C5aR responded quite poorly to C5a, with <50% of the migratory response of WT-C5aR cells, although the maximum response occurred at 1 nM C5a. The linear antagonist peptide 2 stimulated migration in I116A but not WT-C5aR cells, whereas the cyclic antagonist peptide 3 had no effect on either type of receptor.



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  		FIG. 2.
The stimulation of migration of transfected RBL cells. RBL cells transfected with WT or mutant C5aR were stimulated to migrate through 5-µm pore size polycarbonate filters in response to C5a (•), C5a des-Arg74 ({circ}), V3 ({blacksquare}), peptide 2 ({triangledown}), or peptide 3 ({triangleup}). Migration was measured by staining filters with toluidine blue and expressed as a percentage of the migration of WT-C5aR cells to 1 nM C5a. The results are the means ± S.E. of at least three separate experiments performed in triplicate.

 
The Effects of Mutation of C5aR Residues on Antagonist Activity—The antagonist activities were initially measured by preincubating cells with antagonist, then adding C5a (or V3 for R175D-C5aR) to a final concentration of 250 nM (Fig. 3, Table III). Under these conditions, 3 acted as a complete antagonist in all cases except R175D, where only 88% of the degranulation response to V3 was inhibited, with similar IC50 values for WT and all mutant receptors (Fig. 3, Table III). In contrast, 2 was a full antagonist at V286A-, E199K-, R206A-, and E199K/R206A-C5aR and a partial antagonist for both WT and I116A-C5aR (64% inhibition at 100 µM antagonist) but had no significant effect on R175D-C5aR (<50% inhibition at 100 µM). In separate experiments at a lower dose of C5a (50 nM), 100 µM 2 completely inhibited activation of WT-C5aR but inhibited only 40% of the response of I116A-C5aR, whereas 3 remained a full antagonist at both receptors (data not shown).



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  		FIG. 3.
Inhibition of C5a-stimulated degranulation by hexapeptide antagonists in RBL cells transfected with wild type and mutant C5aR. RBL cells transfected with WT or mutant C5aR were incubated with the C5aR antagonists peptide 3 or 2 for 15 min before the addition of 250 nM C5a. Degranulation was assessed as the release of {beta}-hexosaminidase, as described under "Experimental Procedures." Results are the means of at least three separate experiments performed in triplicate ±S.E.

 
The Effects of Substitution at Residue 5 in Cyclic Antagonist 3—To determine the effects of the size and hydrophobicity of the side chain on the agonist activity of this peptide at WT-C5aR and I116A-C5aR, the critical antagonism determining Trp residue at position 5 of 3 was substituted by a series of amino acids possessing hydrophobic side chain rings. The receptor affinities and antagonist potencies of this series of peptides have been tested on human PMNs (21). Three peptides, 3-benzothiazolealanine (3-Bta), 3-Phe, and 3-1-naphthylalanine (3-1-Nal) bound C5aR with a similar affinity to 3 and were effective antagonists, although 3-Bta had a significantly higher IC50 value than 3. When tested on transfected RBL cells, no agonist activity was detected with any of the peptides at concentrations below 100 µM (data not shown), but at 100 µM one peptide, 3-Bta, caused a significantly higher secretory response from I116A-C5aR relative to the response to 3 (Fig. 4). 3-1-Nal did not stimulate significant levels of secretion from I116A-C5aR relative to 3, but when compared with the effects on WT-C5aR, 3-1-Nal had a significantly greater effect on I116A-C5aR (t test, p < 5%). Neither WT nor V286A-C5aR showed any significant secretory response to any of these peptides.



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  		FIG. 4.
The stimulation of degranulation in transfected RBL cells by cyclic hexapeptides containing substitutions at position 5. RBL cells transfected with wild type (open bars)-, I116A (filled bars)-, or V286A-C5aR (hatched bars) were incubated with the 100 µM peptide 3-XXX5, where XXX = Trp, homophenylalanine (hPhe), Bta, Phe, His, 2-Nal, or 1-Nal. Degranulation was assessed as the release of {beta}-hexosaminidase, as described under "Experimental Procedures" and is shown as a percentage of maximal degranulation in response to 1 µM C5a. Results are the means of three separate experiments performed in triplicate ± S.E. Significantly different to the response to 3. **, p < 0. 5%.

 
Receptor-Ligand Modeling—A previous model for C5aR binding to peptide 2 used a presumed interaction between Arg6 carboxylate of 2 with Arg206 of C5aR to orient the ligand with the receptor (1). Dockings were carried out on a model that allowed for little or no movement of the receptor residues so a different rotamer of a particular residue would produce different docking results. We have now repeated this work (Fig. 5A) using a homology model of the C5aR created without the Cys188–Cys109 disulfide bond so that the loops are open and not available for interaction during the ligand docking. We checked the model of Bourne and co-workers (1) using GOLD and automated dockings of 2 (the positions of the carbonyl oxygens from the NMR turn structure were used to restrain ligand docking). Using this alternative approach to the earlier model, we found that the D-Arg6 side chain did not project deep within the helices (not shown) but, instead, consistently lay close to residues in the extracellular loop between helix 4 and helix 5, particularly Glu199. We think that D-Arg6–Glu199 ligand/receptor salt bridge formation is more likely than insertion of the D-Arg6 side chain into a hydrophobic pocket formed by aromatic residues. If such an interaction between D-Arg6 and Asp282 occurs, as suggested here and elsewhere (14), it would preclude the other interactions with D-Arg6 or suggest a dynamic binding mechanism in which initial transient interactions (e.g. between D-Arg6 and Glu199) are followed by more durable interactions (e.g. between D-Arg6 and Asp282).



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  		FIG. 5.
Modeling the interaction between peptide antagonists and the C5a receptor. A, cyclic antagonist (N-methyl-Phe)-Lys-Pro-D-Cha-Trp-Arg-CO2H(2) docked into the C5a receptor model. The NMR structure of the free ligand was used as a template to constrain the conformation (based on H-bond acceptors) of the docked ligand. A constraint between Arg206 and the C-terminal carboxylate of Arg6 was used to reproduce a previous model (1) in addition to the Trp5–Ile116 constraint. B, cyclic antagonist Ac-Phe-cyclo-[Orn-Pro-D-Cha-Trp-Arg] (Ac-3) docked into the C5a receptor model. Two constraints were used; one fixes a hydrogen bond between the Arg6 of the ligand and Asp282 (helix 7) of the receptor, and the second constrains the Trp5 side chain (C{alpha}2) of the ligand to within 5 Å of the C{alpha} of Ile116 on helix 3 of the receptor. C, cyclic antagonist Ac-3 docked into the C5a receptor model. Two constraints were used; one fixes a hydrogen bond between the Arg6 of the ligand and Glu199 (Helix 5) of the receptor, and the second constrains the Trp5 side chain (C{alpha}2) of the ligand to within5ÅoftheC{alpha} of Ile116 on helix 3 of the receptor.

 
Fig. 5B shows a homology model of the C5aR, created with a Cys188–Cys109 disulfide bond and extracellular loops closed into which compound Ac-3 was fitted using automated docking. Two receptor-ligand constraints were used; C{alpha} Ile116 was restrained to within 5 Å from the center of the indole ring of Trp5 (C{alpha}2), and Asp282 (modeled into the extracellular loop 3 that immediately precedes helix 7) was constrained to form an H-bond with the side chain of Arg6. A consistent result was obtained from 10 iterations. The Ac-Phe side chain inserted itself between helices 6 and 7 of the receptor in the vicinity of Ile263 (helix 6) and Leu207 and Phe211 (helix 7). Arg206 in this case is modeled as having an interaction with the proximal Glu199. The D-Cha residue at position 4 points toward the outlying helix 4 but closer to helix 3 and is close to Pro113, Ser114, Arg197 and the salt bridge between Arg206 and Glu199. Trp5 is adjacent to Val286, Ile116, and Pro113. Because of the constraint imposed on Asp282, Arg6 is close to this as well as Gln259 and Tyr258, with Asp181 also presenting a possible interaction given that this is a closed loop model. Based on this model the Arg206–Glu199 interaction appears to be likely.

However, a feature of this closed loop model is that receptor residues in the loops may form important salt bridges/contacts with one another, although it is not possible based on homology modeling alone to pinpoint which of these might be occurring (e.g. Arg206–Asp191/Glu180, Arg175–Asp191/Glu180, Asp282–Lys185/Arg178). If any of those interactions occur, then the ligand Arg6 residue could speculatively be oriented in two conceivable ways. If Arg6 of Ac-3 is oriented toward Asp282, it may interrupt a potential linkage between Asp282 and Arg178, which are both in the loop region above the ligand binding pocket. Alternatively, if Arg6 is oriented toward Arg206, it may disrupt the possible salt bridge between Arg206 and Asp191 or Glu180.

Another docking experiment was conducted with the same constraints as those presented in Fig. 5B using a homology model lacking the disulfide bridge and, thus, without the closed loops. Compound Ac-3 was docked into the receptor using automated docking, with two receptor-ligand constraints (C{alpha} Ile116 was restrained to <5 Å from the center of the indole ring of Trp5 (C2); Asp282 was constrained to form a H-bond with the side chain of Arg6 (modeled into the extracellular loop 3 that immediately precedes helix 7)). A very consistent result was obtained for five docking iterations. The Arg6 side chain was within 5 Å of Val286, Cys285, Asp282 (constrained), Thr261, Gln259, Gly262, and Tyr258. The Trp5 side chain lies within 6 Å of Leu117, Ile116, Pro113, Val286, Ala289, Cys285, and Tyr258. The remaining interactions were within6Åofthe D-Cha side chain (Arg197, Glu199 (constrained), Arg206, and Pro113). The Ac-Phe side chain is in the same position discussed above, between helices 6 and 7 of the receptor, in the vicinity of Ile263 (helix 6) and Leu207 and Phe211 (helix 7).

Fig. 5C involves the alternative homology model of C5aR created without a Cys188–Cys109 disulfide bond, leaving the loops open. In these separate experiments Ac-3 was docked into this open model of C5aR, the Arg6 side chain and Glu199 side chain being tethered with a salt bridge (hydrogen bond constraints) and the Trp5 side chain tethered to Ile116 as above. Receptor residues within 5 Å of Arg6 were Arg197, Glu199 (constrained), Arg201, Val177, Leu173, Pro170, Ser110. Trp5 was within 6 Å of receptor residues Pro113, Ile116, Leu117, Val286, and Tyr258. D-Cha4 was within 6 Å of Val286, Tyr258, Asp282, Cys285, Thr261, and Gly262. Essentially the latter interactions are the reverse of that seen in the first two models (i.e. Arg6 interacts where D-Cha was positioned).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Ile116/Val286—The side chains of C5aR residues Ile116 and Val286 in the 3rd and 7th transmembrane helices, respectively, have been proposed to form part of an activation switch for C5aR (1). We have examined this proposal in detail using linear and cyclic peptide antagonists as probes to determine whether this is a general target for C5aR antagonists. The data presented in this paper contrast strikingly with the effects of I116A and V286A mutations in the human C5aR expressed in yeast, where both of these mutations caused complete loss of C5a receptor activity (1). The mutation of such "putative activation switch residues" had the general effect herein of increasing receptor affinity for C5aR ligands. In contrast, the EC50 for I116A-C5aR activation by C5a was increased 3-fold relative to WT-C5aR. Therefore, although I116A-C5aR has a higher affinity for agonists, the mutation decreases the efficiency of receptor activation by C5a, C5a des-Arg74, and agonist peptide 1.

Mutation at Ile116 or Val286 caused no change in affinity for either the linear antagonist 2 or cyclic antagonist 3, although both C5aR mutants had a higher affinity for the cyclic antagonist over the linear form. 3 was a full and potent antagonist of RBL cell degranulation through both receptors, with no significant difference in IC50 values. In contrast, the pattern of antagonism by 2 was more complex. This peptide was a full antagonist for V286A-C5aR and R206A-C5aR but inhibited WT-C5aR and I116A-C5aR by only 60% in response to 250 nM C5a. 2 is actually a partial agonist at I116A-C5aR, and therefore secretion is a balance between the antagonistic effects of peptide on secretion induced by C5a and the agonistic effects of peptide alone. On WT-C5aR, 2 is not an agonist but failed to completely inhibit C5a activity, presumably due to competition with C5a for the ligand binding site. The cyclic peptide 3 remains a potent antagonist with no detectable agonist activity on either I116A- or V286A-C5aR despite also having Trp at position 5. The antagonist activity is unlikely to be related to the lack of a free carboxylate at the C terminus, since 1-amide is a potent agonist at WT-, I116A-, and V286A-C5aR, suggesting that simple deletion of the interaction with the C-terminal carboxylate is insufficient to generate an antagonist.

Substitution of Trp5 by different amino acids generally reduces receptor affinity and antagonist activity (21) but only the sulfur-containing heterocyclic amino acid Bta produces a cyclic peptide with significant agonist activity at I116A-C5aR relative to 3. This weak gain of agonist activity correlates with the loss of antagonist potential, as 3-Bta5 is a less potent antagonist than 3 at WT-C5aR on PMNs. However, the gain in agonist potential at I116A of 3-Bta5 is unexpected because Trp and Bta differ only at the heteroatom. Increased agonist activity is unlikely to be due simply to improved binding of this bulkier side chain to C5aR, because 3-Bta5 binds PMN C5aR with an identical affinity to 3. In contrast 3-Phe5, with a less bulky aromatic side chain, binds to PMNs with a similar affinity to 3 but remains a potent antagonist. Interestingly, a linear peptide containing Phe at position 5 is an agonist even at WT-C5aR (5), which supports the suggestion that the cyclic peptide contains additional determinants of antagonist activity.

Although the precise mechanism of C5aR antagonism by peptides is still unclear, the differential responses to receptor mutations by cyclic versus linear hexapeptides and also the two polypeptide antagonists, C5aRA and V2[Glu68], reported here suggest that antagonist action is not critically related to interference by Trp5 at a putative activation trigger formed by Ile116 and Val286 (1). However, Ile116 does have a role in receptor activation because the activation efficiency of I116A-C5aR is lower than WT-C5aR. Receptor activation may require the residue at position 5 of the hexapeptide ligand to interact with residues buried deeper in the hydrophobic transmembrane domain of C5aR than Ile116. The size of the Trp5 side chain in antagonist 2 may prevent this occurring due to steric hindrance by Ile116, whereas the substitution of Ile116 by the smaller residue Ala allows deeper penetration of the receptor core by Trp5 and, therefore, receptor activation by peptide 2. Thus, linear antagonist becomes the agonist simply because sufficient space has been created for the more bulky Trp side chain at position 5. However, any hypothesis of C5aR antagonism must accommodate the observation that the cyclic peptide derivative of 2, namely 3, remains an antagonist at I116A. It is, thus, interesting that Trp5 is not essential for antagonism in analogues of 3, as a W5F substitution results in comparable antagonist potency for this ligand (21).

NMR studies have shown that the linear and cyclic peptides have similar turn structures (17, 29) and compete similarly with [125I]C5a or [3H]Ac-3 for the receptor, supporting the idea that they bind at the same or a similar site on the receptor. The significant difference between the peptides is, therefore, likely to be rigidity, cyclization having locked 3 into the turn structure, whereas the linear peptide is capable of significant deformation during binding. This ability to deform may allow side chains at position 5 of the linear peptide to interact correctly with the activation site after binding, whereas the rigidity of 3, although permitting high affinity interactions with other receptor binding sites, may prevent position 5 side chains from penetrating into the Ile116 pocket even when Ile116 is mutated. The cyclic constraint certainly reduces the flexibility of the antagonist, and this may also limit the capacity of the antagonist to alter shape in response to receptor mutations. This hypothesis would explain the lack of agonist activity of 3-Phe5 on WT-C5aR and 3 on I116A-C5aR. The substitution of Trp5 by the larger side chain of Bta (and to a lesser extent 1-Nal) may allow the interaction of residue 5 with the receptor activation site protected by Ile116, producing agonist activity at the I116A mutant.

Arg206/Arg175—It has been proposed that the receptor interaction site for the C-terminal carboxylate of C5aR agonists is at Arg206, a residue at the extracellular face of helix 5 (1). Mutation of Arg206 to Ala has only a small effect on receptor activation by C5a (14) and does not abrogate antagonism by either linear or cyclic antagonist (results herein). This mutation does, however, appear to affect interactions with the agonist peptides, and only the amidated form of 1 has weak (but detectable) activity. Taken together with the observation that C5a des-Arg74 binds to, but does not activate R206A-C5aR (this paper and Ref. 14), it is possible that mutation of this receptor residue perturbs the global structure of the receptor rather than disrupting specific ligand interactions. This view is further supported by the finding that a ligand-independent constitutively active C5aR mutant (I124N/L127Q) can be completely deactivated by substitution of Arg206 by His (1).

Another potential receptor site for interaction with the C-terminal carboxylate is Arg175, located either on the extracellular face of helix 4 or in the adjacent loop. The analogous residue (Arg161) in the closely related C3a receptor has been proposed to interact with the C-terminal carboxylate of C3a (28). We have previously shown that although C5aRs mutated at Arg175 are only weakly activated by C5a, they can be strongly activated by a mutant form of C5a des-Arg74 (V3), isolated from a randomly mutated C5a des-Arg74 library (22), suggesting that a specific and important interaction between C5aR and C5a is lost when Arg175 is mutated to either Ala or Asp. In this paper we have shown that 1-amide is equipotent with 1 as an activator of Arg175 mutants, suggesting that Arg175 may make an interaction with the C-terminal carboxylate of 1. It is also interesting to note that 3, with no free carboxylate, is a weak activator of R175D. This suggests first that the conversion of an antagonist to an agonist is not a unique property of Ile116 and, second, that the C-terminal carboxylate of the ligand interacts in some way with Arg175. Furthermore 1-L-Arg, with the C-terminal carboxylate oriented differently to 1, can activate R175A to a greater extent than C5a. Previously, Arg206 has been suggested to act as a "gatekeeper" that must be displaced by the charged groups on the ligand C-terminal Arg residue to allow the ligand carboxylate to interact elsewhere on the receptor (13). A possible explanation of our data is that the peptide carboxylate makes interactions with both Arg206 and Arg175 at different points in the receptor binding and activation process. Whereas our homology modeling studies are more strongly supportive of a role for Arg175 in stabilization of the inactive receptor state, perhaps by weak polar interaction with residues such as Glu199, we note that only a small (ligand-induced?) decrease in distance between helix 4 and helix 5 in our model could conceivably enable interactions with both Arg175 and Arg206. These proposed mechanisms cannot be distinguished from available data at present.

Asp282/Glu199—We have previously shown that Asp282 at the extracellular face of helix 7 interacts with the side chain of Arg74 of C5a and with the C-terminal Arg in peptide analogs (14, 22). The mutation of D282A decreases the affinity for cyclic antagonist 3 10-fold but has no effect on affinity for the linear antagonist. The mutation E199K has a similarly small effect on antagonism, in contrast to the complete lack of responsiveness of this mutant to agonists lacking a C-terminal Arg, such as C5a des-Arg74 and C5a[Ala74], suggesting that in addition to a previously demonstrated interaction between Lys68 and Glu199 (19, 23), the side chain of the C-terminal Arg74 residue interacts with Glu199. However, the loss of this interaction after mutation of Glu199 has no effect on the responsiveness to C5a, possibly suggesting only a transient interaction between Arg74 and Glu199, with a more important interaction occurring between Arg74 (Arg6 of peptide ligands) and Asp282. This is clearly shown by the mutation D282R, which has a very low responsiveness to C5a but a relatively normal response to C5a des-Arg74 and similar ligands (14, 22).

A New Model for the Interaction of Peptide Ligands with C5aR—Previously, it was proposed by Bourne and co-workers (1) that Ile116 and Val286 of C5aR are key residues in determining whether C5a and its C-terminal analogs are agonists or antagonists. That work involved a model based on manual docking of an early NMR structure of the linear antagonist 2 into the receptor followed by energy minimization (SYBYL®). They initially docked the ligand (NMR-derived structure showing a turn) incorporating the Trp5–Ile116 interaction as well as a presumed ligand/receptor interaction between the carboxylate at the C terminus of D-Arg6 with Arg206. This orientation by default projects the D-Arg side chain deep within the space between helices 3, 5, and 6, adjacent to aromatic residues Tyr121/Phe211/Phe251. Based on the premise that position 5 (Trp) of the small molecule antagonist 2 becomes lodged in a pocket defined by Ile116, Val286, and Ala289, it was conjectured that mutation of the latter residues might alter antagonist/agonist activity. Indeed the mutation I116A, which increases the receptor space available for ligand occupation, does convert 2 from antagonist to agonist. A change in the ligand at position 5 from L-Trp to L-Cha also converted antagonist 2 to the agonist 1. Together, the results were consistent with one site on the receptor being crucial for differentiating between agonist activity and antagonism.

In the present paper we sought to test whether Ile116 and Val286 participate in a general switching mechanism for receptor activation or whether the previous observations (1) were ligand-specific by examining the effects of receptor mutations for both 2 and a different antagonist, Ac-3. Because our data suggests that antagonism by compound Ac-3 is not affected by mutation of Ile116, it is likely that the antagonist mechanism proposed previously may indeed be specific for compound 2 (1). However, our data do suggest that compounds with bulkier residues at position 5 are affected by mutation of Ile116, and so a receptor-ligand interaction in the vicinity of Ile116 is a likely event.

Data presented in this paper do not support the earlier model of the interaction between receptor and ligand proposed in (1), and recent work strongly suggests an alternative interaction between Asp282, a residue at the extracellular face of helix 7 of C5aR, and the side chain of Arg6 (14). This has a profound effect on the orientation of peptide ligand with respect to receptor. There is also evidence of an interaction between the C-terminal carboxylate and Arg175, which means that the C-terminal carboxylate cannot be located in the vicinity of Arg206 with any degree of certainty. The position of Arg175 is uncertain in the transmembrane homology model of C5aR reported herein because it is not in a transmembrane helix but rather in the extracellular loop at the top of helix 4. However, by positioning the ligand Arg6 side chain close to Asp282 and ligand Trp5 side chain in the vicinity of Ile116/Val286, new models of C5aR complexed with antagonist peptides 2 and 3 have been derived (Fig. 4). The interaction between Asp282 and Arg6 precludes the guanidinium group of Arg6 from binding deep within the helical bundle. Given the length and flexibility of the arginine side chain, there are more feasible interactions than the proposed {pi}-cation interaction (1) deep within a hydrophobic channel. Furthermore, movement of only selected receptor side chains is highly subjective given the dynamic nature of this system.

The roles of Arg175 and Arg206 might be to stabilize helices 4 and 5. Whereas peptide Ac-3 does not contain a free carboxylate group, peptide 2 does and if positioned in the vicinity of Arg206 as proposed, it may interrupt salt bridges that help stabilize the transmembrane helices. This may be a key mechanistic feature of agonist action. The mutation E199K affects the response to all ligands except C5a, so it is possible that this is because Glu199 interacts with Arg206 (as shown in Fig. 4A) to help stabilize helix 5 of the receptor. Perhaps C5a can itself help stabilize this helix and, thus, is insensitive to this mutation. It is notable that C5aR doubly mutated at Glu199 and Arg206 responds to agonists such as C5a and C5a des-Arg74 (14) and antagonists 2 and 3 in a similar fashion to R206A (data presented here).

Conclusions and Comments—Based on the balance of evidence for effects of mutations in the C5a receptor on the affinity and agonist/antagonist potencies of C5a and small peptide ligands, together with our receptor-ligand modeling studies, we suggest that terminal Arg residues of such ligands do not insert into the hydrophobic pocket in the interior of the helix bundle as previously proposed (1). The receptor and ligand mutagenesis and modeling studies in this paper present some new testable hypotheses about the importance of charged residues in the receptor, particularly Arg206, Glu199, Arg175, and Asp282, and shed further light on the fitting of Arg side chains of small peptide ligands into the C5a receptor.


   FOOTNOTES
 
* This work is supported by Arthritis Research Campaign Project Grant M0648, Wellcome Trust Project Grant 007521 (to P. N. M.), and Australian Research Council and National Health and Medical Research Council Australian Project Grants DP0210598 (to D. P. F.) and 9937208 (to S. M. T./D. P. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Current address: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK. Back

** Current address: School of Pharmacy, University of Otago, P. O. Box 913, Dunedin, New Zealand. Back

{ddagger}{ddagger} These authors contributed equally to this work. Back

§§ To whom correspondence should be addressed: Academic Neurology Unit, University of Sheffield Medical School, Beech Hill Rd., Sheffield S10 2RX, UK. Tel: 44-114 2261312; Fax: 44-114 2760095; E-mail: p.monk{at}shef.ac.uk.

1 The abbreviations used are: C5aR, human complement fragment 5a receptor; WT, wild type; PMN, polymorphonuclear granulocytes; Bta, benzothiazolealanine; Nal, naphthylalanine; Cha, cyclohexylalanine; Ac-3, Ac-Phe-cyclo-[Orn-Pro-D-Cha-Trp-Arg]. Back

2 P. K. Madala, J. D. A. Tyndall, and D. P. Fairlie, manuscript in preparation. Back



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 RESULTS
 DISCUSSION
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