title: Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site.
abstract: 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 (Ile(116), Arg(175,) Arg(206), Glu(199), Asp(282), and Val(286)). 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 Ile(116) 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 Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) 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. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) 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.
authors.affiliation: Academic Neurology Unit, University of Sheffield Medical School, Sheffield S10 2RX, United Kingdom.
authors.forename: Adrian
authors.initials: A
authors.lastname: Higginbottom
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Cain
authors.forename: Trent M
authors.initials: TM
authors.lastname: Woodruff
authors.forename: Lavinia M
authors.initials: LM
authors.lastname: Proctor
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Madala
authors.forename: Joel D A
authors.initials: JD
authors.lastname: Tyndall
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Taylor
authors.forename: David P
authors.initials: DP
authors.lastname: Fairlie
authors.forename: Peter N
authors.initials: PN
authors.lastname: Monk
mesh_terms: D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: C488033:C5AR1 protein, human; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement