Identification of lysine 134 in the steroid-binding site of the sex steroid-binding protein of human plasma.
The sex steroid-binding protein of human plasma SBP (or sex hormone-binding globulin, SHBG) was specifically inhibited with the alkylating affinity label, 17 beta-[[( 2-14C]bromoacetyl)oxy]-5 alpha-androstan-3-one. The natural ligand, 5 alpha-dihydrotestosterone, was shown to protect against inactivation and labeling. The steroid-binding activity of the protein was abolished when approximately 1 mol of label was incorporated into 1 mol of dimeric SBP. In order to identify and locate the labeled amino acid in the steroid-binding site, the steroidal portion of the bound label was first removed and the protein was digested with Achromobacter protease and subdigested with trypsin. Seven radioactive peptides were isolated, sequenced, and found to contain the common sequence QVSGPLTSXR. Residue X was identified as lysine-134 from the SBP amino acid sequence (Walsh, K. A., Titani, K., Kumar, S., Hayes, R., and Petra, P. H. (1986) Biochemistry 25, 7584-7590). The results indicate that only 1 of the 2 lysine-134 residues in the homodimer was labeled. This suggests that the steroid-binding site is constructed from an association of the two subunits in an AB to BA "sandwich" configuration with lysine-134 residue of one subunit on one surface near the D-ring and the lysine-134 of the other subunit at the opposite end of the steroid, or well away from the steroid-binding site. Although the nature of the data does not allow description of a specific role for lysine-134, its proximity to the 17 beta-OH of the steroid nucleus suggests participation in the binding process through direct or indirect hydrogen bonding.
Namkung
P C
PC
Department of Obstetrics/Gynecology, University of Washington, Seattle 98195.
Kumar
S
S
Walsh
K A
KA
Petra
P H
PH
D000345:Affinity Labels; D000477:Alkylating Agents; D000595:Amino Acid Sequence; D001665:Binding Sites; D013196:Dihydrotestosterone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D000477:Alkylating Agents; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; C023562:dihydrotestosterone 17-bromoacetate; D008239:Lysine