Residues 21–30 within the Extracellular N-terminal Region of the C5a Receptor Represent a Binding Domain for the C5a Anaphylatoxin

Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.One of the most biologically important families of receptors is the G protein-coupled receptors (GPCRs), 2 which eukaryotic cells use to sense signals as diverse as light, odorants, small molecules, and polypeptide hormones (1). The GPCRs, which number ϳ1000 in the human genome (2), have a shared topology made up of seven transmembrane domains (TMs) linked by intracellular and extracellular (EC) loops, along with an extracellular N terminus and intracellular C terminus (Fig. 1). These receptors also have a shared mechanism of action, whereby the activated receptor serves as a guanosine exchange factor on the ␣ subunit of a heterotrimeric G protein, thus transmitting the signal to the interior of the cell. Many drugs are directed against GPCRs, including adrenergic, muscarinic, dopaminergic, serotonergic, GABAergic, and histaminergic receptors. Taken together, drugs acting on GPCRs make up an estimated 50% of the current pharmacopoeia (3).The structural basis of receptor-ligand interaction is of great interest in both basic and applied pharmacology. In the case of GPCRs, a central question is how receptors with a single common topology can be activated by such a wide variety of ligands. A related question is how specificity is built into these receptors, so that each is activated only by the appropriate ligands.Many GPCRs are activated by polypeptide ligands, including chemokines such as SDF-1 (CXCL12), physiologic modulators such as angiotensin II, glycoprotein hormones such as luteinizing hormone, and chemotactic factors such as complement factor 5a (C5a). These ligands are too large to fit entirely in an interhelical cleft of a GPCR, so their bulk must serve some adaptive function other than receptor activation per se. In some cases, the ligand is recruited by its affinity for the receptor extracellular domains, and a second discret...

Section: Use Of C5a C27r/r40c To Trap Interactions With the C5ar Nt-c5asupporting

confidence: 71%

mentioning

confidence: 99%

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

Hagemann

Miller

Klco

et al. 2008

“…Although in a linear polypeptide this would correspond to a distance of roughly 17.5 Å, these residues form a loop in the crystal structure of rhodopsin (3), bringing them considerably closer together. NMR spectroscopic studies of C5aR fragment 1-34 showed nonsequential nuclear Overhauser effect connectivities in the region 20 -30, confirming that some folded conformation is present in this region (26). Furthermore, the receptor N terminus may be conformationally flexible.…”

Section: Discussionmentioning

confidence: 68%

“…Approximately half of the binding energy for C5a appears to be derived from interactions with the receptor N terminus (25). NMR studies showed that a peptide identical to the first 34 amino acids of C5aR retained its ability to bind C5a and that proton resonances in receptor positions 21-30 were most strongly affected by the binding event (26). Although these data implicate the N terminus of the C5aR in ligand binding, it remains unclear which region of C5a is involved in the interaction.…”

Section: G Protein-coupled Receptors (Gpcrs)mentioning

confidence: 82%

Random Mutagenesis of the Complement Factor 5a (C5a) Receptor N Terminus Provides a Structural Constraint for C5a Docking

Hagemann¹,

Narzinski

Floyd

et al. 2006

The N terminus of G protein-coupled receptors has been implicated in binding to peptide hormones. We have used random saturation mutagenesis to identify essential residues in the N terminus of the human complement factor 5a receptor (C5aR). In a library of N-terminal mutant C5aR molecules screened for activation by C5a, residues 24 -30 of the C5aR showed a marked propensity to mutate to cysteine, most likely indicating that sulfhydryl groups at these positions are appropriately situated to form disulfide interactions with the unpaired Cys 27 of human C5a. This presumptive spatial constraint allowed the ligand to be computationally docked to the receptor to form a model of the C5a/C5aR interaction. When the N-terminal mutant C5aR library was rescreened with C5a C27R, a ligand incapable of disulfide interactions, no individual position in the N terminus was essential for receptor signaling. However, the region 19 -29 was relatively highly conserved in the functional mutants, further demonstrating that this region of the C5aR makes a productive physiologic interaction with the C5a ligand.G protein-coupled receptors (GPCRs) 3 transduce signals from a wide variety of biological stimuli, including small molecules, lipids, peptides, proteins, and environmental cues such as odorants and light (1). As the largest family of cell-surface receptors, they have provided fertile ground for pharmacologic modulation. GPCRs share a secondary structure consisting of seven transmembrane helices (TMs 1-7) joined by three intracellular and three extracellular loops, along with N-and C-terminal domains (Fig. 1). The receptors serve as binary switches that, when activated by ligand, exhibit guanine nucleotide exchange factor activity toward heterotrimeric G proteins (2). Because they are large transmembrane proteins, GPCRs are difficult to study by direct structural methods such as x-ray crystallography. A series of crystal structures of bovine rhodopsin has allowed major progress in the understanding of GPCR structure (3-7) but has not made clear the mechanism by which these receptors serve as switches, because rhodopsin has only been crystallized in its resting state. The mechanism of GPCR activation has, however, been productively explored using a variety of biochemical, biophysical, and computational techniques. Overall, ligand binding by GPCRs appears to induce a rigid-body movement of TM6 away from TM3, exposing new receptor surfaces that allow coupling to downstream effectors (8 -11).Our laboratory has studied the human complement factor 5a receptor (C5aR) (12) as a model system because it, like rhodopsin and many other clinically important receptors, belongs to the large family A of GPCRs (13). The C5aR has 19% amino acid identity with rhodopsin and has loop and transmembrane helix lengths similar to those of rhodopsin. Furthermore, the C5aR can be expressed in Saccharomyces cerevisiae, making it amenable to genetic studies.In addition to its role as a model receptor, the C5aR has intrinsic biological interest. The vertebrate...

“…For the C5a receptor, although the ligand-binding site was assigned to residues 21-30 in NT, a deletion of residues 1-22 abolished signaling (59), like the ⌬2-9 deletion in our study. The first residues of NT of CXCR4 could be actually part of site II, possibly interacting with the extracellular loops, as has been proposed for the C5a receptor (60). Alternatively, residues 2-9, although not essential for access of the chemokine to site I, may be required for its proper orientation toward site II, and thus for signaling.…”

Section: Role Of Third Intracellular Loop In Signal Transduction-mentioning

confidence: 99%

Identification of Residues of CXCR4 Critical for Human Immunodeficiency Virus Coreceptor and Chemokine Receptor Activities

Brelot¹,

Heveker²,

Montes³

et al. 2000

231

287

CXCR4 is a G-coupled receptor for the stromal cellderived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH 2 -terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/ Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu 14 and/or Glu 15 and Tyr 21 . Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp 187 ), TMII (Asp 97 ), and TMVII (Glu 288 ). The first residues (2-9) of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.