title: Characterization of the binding site on the formyl peptide receptor using three receptor mutants and analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu.
abstract: The formyl peptide receptor (FPR) is a chemotactic G protein-coupled receptor found on the surface of phagocytes. We have previously shown that the formyl peptide binding site maps to the membrane-spanning region (Miettinen, H. M., Mills, J. S., Gripentrog, J. M., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054). Recent reports have indicated that non-formylated peptides, such as MMWLL can also activate this receptor (Chen, J., Bernstein, H. S., Chen, M., Wang, L., Ishi, M., Turck, C. W., and Coughlin, S. R. (1995) J. Biol. Chem. 270, 23398-23401.) Here we show that the selectivity for the binding of different NH(2)-terminal analogs of MMWLL or MLF can be markedly altered by mutating Asp-106 to asparagine or Arg-201 to alanine. Both D106N and R201A produced a similar change in ligand specificity, including an enhanced ability to bind the HIV-1 peptide DP178. In contrast, the mutation R205A exhibited altered specificity at the COOH terminus of fMLF, with R205A binding fMLF-O-butyl > fMLF-O-methyl > fMLF, whereas wt FPR bound fMLF > fMLF-O-methyl approximately fMLF-O-butyl. These data, taken together with our previous finding that the leucine side chain of fMLF is probably bound to FPR near FPR (93)VRK(95) (Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., and Jesaitis, A. J. (1998) J. Biol. Chem. 273, 10428-10435.), indicate that the most likely positioning of fMLF in the binding pocket of FPR is approximately parallel to the fifth transmembrane helix with the formamide group of fMLF hydrogen-bonded to both Asp-106 and Arg-201, the leucine side chain pointing toward the second transmembrane region, and the COOH-terminal carboxyl group of fMLF ion-paired with Arg-205.
authors.affiliation: Department of Microbiology, Montana State University, Bozeman, Montana 59717, USA. umbmj@montana.edu
authors.forename: J S
authors.initials: JS
authors.lastname: Mills
authors.forename: H M
authors.initials: HM
authors.lastname: Miettinen
authors.forename: D
authors.initials: D
authors.lastname: Cummings
authors.forename: A J
authors.initials: AJ
authors.lastname: Jesaitis
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D016466:CHO Cells; D002633:Chemotaxis / Q000187:drug effects; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D000077560:Enfuvirtide; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008715:Methionine / Q000737:chemistry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010452:Peptide Biosynthesis; D010446:Peptide Fragments / Q000494:pharmacology; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism; D018000:Receptors, Peptide / Q000737:chemistry* / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
chemical_list: D019380:Anti-HIV Agents; D015700:HIV Envelope Protein gp41; D008024:Ligands; D010446:Peptide Fragments; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D000077560:Enfuvirtide; D001224:Aspartic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010649:Phenylalanine; D001216:Asparagine; D014364:Tryptophan; D001120:Arginine; D012964:Sodium; D008715:Methionine; D007930:Leucine; D000409:Alanine