Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

J Biol Chem. 2005 May 6;280(18):17831-40. doi: 10.1074/jbc.M410797200. Epub 2005 Jan 20.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites / drug effects
  • Binding Sites / physiology
  • Cell Line
  • Humans
  • Ligands
  • Membrane Proteins / agonists*
  • Membrane Proteins / antagonists & inhibitors*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mutation*
  • Peptide Fragments / metabolism
  • Peptide Fragments / pharmacology
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement / agonists*
  • Receptors, Complement / antagonists & inhibitors*
  • Receptors, Complement / genetics
  • Receptors, Complement / metabolism

Substances

  • C5AR1 protein, human
  • Ligands
  • Membrane Proteins
  • Peptide Fragments
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement