Random mutagenesis of the complement factor 5a (C5a) receptor N terminus provides a structural constraint for C5a docking.
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.
Hagemann
Ian S
IS
Departments of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Narzinski
Kirk D
KD
Floyd
Desiree H
DH
Baranski
Thomas J
TJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D015936:Complement C5a / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry* / Q000502:physiology*; D012984:Software; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; C488033:C5AR1 protein, human; D004220:Disulfides; D008024:Ligands; D008164:Luminescent Proteins; D008565:Membrane Proteins; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; C054562:yellow fluorescent protein, Bacteria; D015936:Complement C5a; D001616:beta-Galactosidase; D003545:Cysteine