Photoaffinity labeling of human sex hormone-binding globulin using 17alpha-alkylamine derivatives of 3beta-androstanediol substituted with azidonitrophenylamido, azidonitrophenylamino, or trifluoroazidonitrophenylamino chromophores. Localization of Trp-84 in the vicinity of the steroid-binding site.
Purified human SHBG was photoaffinity labeled with 17alpha-aminomethyl (M), 17alpha-aminoethyl (E), and 17alpha-aminopropyl (P) derivatives of [3alpha-(3)H]-5alpha-androstane-3beta,17beta-diol coupled to 5-azido-2-nitrobenzoylamido (ANB), 4-azido-2-nitrophenylamino (ANP), and 5-azido-2-nitro-3,4,6-trifluorophenylamino (ANTFP) chromophores. Successful labeling was achieved in all cases except for the two photoreagents with the shortest side chains, namely, ANP-M and ANTFP-M derivatives. Edman sequencing and mass spectrometry of immunopurified photolabeled tryptic fragments revealed that radioactivity was present either on the sequence of residues 73-94, uniquely at the level of Trp-84 (stable covalent labeling), or on one of the two overlapping sequences of residues 126-134 and 126-135, at the level of Pro-130 (labile labeling) and Lys-134 (either stable or partially labile labeling), respectively. The same Trp-84 was photolabeled with the three ANB derivatives of increasing lengths, and by the ANP-P photoreagent. This residue was the exclusive target for the shortest [(3)H]ANB-M photoreagent but was a minor site for the longest [(3)H]ANB-P photoreagent, essentially recovered at the level of Pro-130. The [(3)H]ANB-E photoreagent of intermediate size also labeled exclusively Trp-84, except in some experiments in which photolabeling was recovered predominantly at the level of Pro-130. The [(3)H]ANP-P photoreagent with an overall length similar to that of the ANB-P photoreagent labeled simultaneously Trp-84 (minor site) and Lys-134. The other [(3)H]ANP-E, [(3)H]ANTFP-E, and [(3)H]ANTFP-P derivatives labeled in all cases Lys-134. These findings indicate that the conserved Trp-84 and the two Pro-130 and Lys-134 residues are all located in the vicinity of the D ring of steroid ligands and remain freely accessible from the C17alpha position, thus providing biochemical data delineating the corresponding region of the steroid-binding site.
Chambon
C
C
Institut National de la Santé et de la Recherche Médicale, Unité INSERM U 329, Pathologie Hormonale Moléculaire, Hôpital Debrousse, 69322 Lyon, France.
Bennat
D
D
Delolme
F
F
Dessalces
G
G
Blachère
T
T
Rolland de Ravel
M
M
Mappus
E
E
Grenot
C
C
Cuilleron
C Y
CY
D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D000595:Amino Acid Sequence; D015113:Androstane-3,17-diol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014316:Tritium; D014357:Trypsin
D000345:Affinity Labels; D010446:Peptide Fragments; D012738:Sex Hormone-Binding Globulin; D014316:Tritium; D015113:Androstane-3,17-diol; D014357:Trypsin