title: Cgamma H2 of Met174 side chain is the site of covalent attachment of a substance P analog photoactivable in position 5.
abstract: Analogs of substance P (H-RPKPQQFFGLM-NH(2)) incorporating a photoreactive para-benzoyl-l-phenylalanine (p-Bzl)Phe at position 4, 5, 6, 9, or 10 of the sequence have been synthesized and pharmacologically characterized previously as full NK-1 receptor agonists. In this study we show that all analogs, [BAPA(0), (p-Bzl)Phe(x), Met(O(2))(11)]SP also display high yields (40-70%) of NK-1 receptor photolabeling. To identify the site of photoinsertion in the receptor, covalent ligand/receptor complexes were digested with enzymes or chemically cleaved with cyanogen bromide and purified with streptavidin-coated magnetic beads before matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Only the analog photoreactive at position 5 gave irreversible, reproducible, and unequivocal covalent linkage. Sequential digestions of the covalent complex, substance P analog photoreactive at position 5/NK-1 receptor, with trypsin, endo-GluC and carboxypeptidase Y, led to the identification of the tripeptide (173)TMP(175) in the second extracellular loop of the hNK-1 receptor as the site of photoinsertion. Reaction of cyanogen bromide on the pentapeptide TMPSR did not yield the expected cleavage on the carboxylic side of methionine. The high precision of mass spectrometry analysis on the mass measured led us to determine that C(gamma)H(2) of Met(174) was the site of covalent linkage of the photoreactive substance P analog. Such an insertion (photolinked ligand) on its C(gamma)H(2) renders methionine refractory to CNBr cleavage.
authors.affiliation: Unité Mixte de Recherches 7613 CNRS, Structure et Fonction de Molécules Bioactives, Université Pierre & Marie Curie, Case 182, 4 place Jussieu, 75252 Paris cedex 05, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Sachon
authors.forename: Gérard
authors.initials: G
authors.lastname: Bolbach
authors.forename: Gérard
authors.initials: G
authors.lastname: Chassaing
authors.forename: Solange
authors.initials: S
authors.lastname: Lavielle
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D006801:Humans; D008715:Methionine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D064729:Neurokinin-1 Receptor Antagonists; D017433:Protein Structure, Secondary; D018040:Receptors, Neurokinin-1 / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013373:Substance P / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D014357:Trypsin
chemical_list: D000345:Affinity Labels; D064729:Neurokinin-1 Receptor Antagonists; D018040:Receptors, Neurokinin-1; D013373:Substance P; D008715:Methionine; D014357:Trypsin