Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using .DELTA.6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents

Abstract

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.

Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain

“…The structure of the human SHBG steroid-binding pocket is in agreement with mutagenesis data (Bocchinfuso et al, 1992;Sui et al, 1992;Bocchinfuso and Hammond, 1994) and photolabelling experiments (Grenot et al, 1992;Kassab et al, 1998). In particular, it explains why the substitution of Ser42 with leucine causes a complete loss of steroid binding with no apparent effect on the dimerization potential of intact human SHBG expressed in CHO cells (Hammond and Bocchinfuso, 1996).…”

“…In particular, it explains why the substitution of Ser42 with leucine causes a complete loss of steroid binding with no apparent effect on the dimerization potential of intact human SHBG expressed in CHO cells (Hammond and Bocchinfuso, 1996). In the structure, atom C6 of steroid ring B and the sulfur atom of Met139 are only 4.5 Å apart, and photoaffinity labelling with Δ 6 -testosterone or Δ 6 -oestradiol specifically marks Met139 (Grenot et al, 1992). The relative importance of residues in contact with the steroid is also emphasized by their across-species conservation (Hammond, 1993).…”

Sex Hormone-Binding Globulin: Gene Organization and Structure/Function Analyses

“…Affinity-labeling studies have shown that Met139 in human SHBG is probably important for interaction with the steroid B ring [27], We and others have confirmed this using site-directed mutagenesis [32. 33], and the steroidbinding properties of human SHBG/rat ABP chimeras led us to conclude that residues important for high-affinity steroid binding arc probably situated within the amino terminal 205 amino acids of human SHBG [23,32], Con sequently, we decided to introduce amino acid substitu tions in a region encompassing Met139, as well as other locations in the SHBG molecule, in an attempt to identify residues that influence its ability to bind steroid and/or dimerize.…”

“…The requirements for steroid binding to human SHBG and rat ABP have been studied in great detail [24], but attempts to identify their steroid-binding sites have raised questions as to whether amino acids that interact with ste roid ligands are confined to a discrete region [25][26][27] or are dispersed throughout the molecule [4,28,29], Dimeri zation of SHBG is thought to be necessary for the forma tion of a single steroid-binding site, and ligand binding appears to stabilize the homodimer [30], The fact that the steroid-binding activity of SHBG is preserved more effec tively during storage in the presence of both androgen and calcium [31] also suggests that an interaction with diva lent cation, as well as occupancy of the steroid-binding site, may help maintain its structural integrity.…”

Synthesis of New Photoactivatable Phenylalanine Analogues and Their Incorporation into a Model Peptide − Phenylseleno Derivatives as Precursors of α,β-Unsaturated Ketones in Peptide Synthesis

“…Acetophenone derivatives of guanosine-5Ј-triphosphate peptides for photoaffinity labeling of GTP-binding proteins and human serum albumin have been reported, and steroids bearing an enone function have been developed for photoaffinity labeling of sex hormone binding globulin. [12,13] In this study, we report the syntheses of three N-protected phenylalanine derivatives: (S)-p-(propanoyl)phenylalanine (10), (S)-Boc-p-[2Ј-(phenylselenenyl)propanoyl]phenylalanine (11), and (S)-Boc-p-[3Ј-(phenylselenenyl)propanoyl]-phenylalanine (13) (Scheme 1). The protected amino acids 11 and 13 were introduced into the sequence of the model pentapeptide thymopentin TP5 (N-acetylated, C-terminal carboxamide), which, after HF cleavage, yielded peptides bearing either a p-propanoyl or a p-propenoyl group on the aromatic residue.…”