Surface mapping of the ligand-filled C-terminal half of the porcine estradiol receptor by restricted proteolysis.
The ligand-filled 32-kDa fragment of the porcine estradiol receptor extending from His267 to the C-terminal Ile595 was purified to homogeneity by adsorption to mAb 13H2. The native protein was exposed at 4 degrees C to a panel of proteases: thermolysin, subtilisin, pronase, elastase, ficin, bromelain, endopeptidase Lys-C, both in the dimer and the monomer state, and chymotrypsin at pH 8.2 only. The digests were analysed by SDS/PAGE/Western blotting for Coomassie staining and immunostaining. Peptides were sequenced from blots. The majority of cleavage sites in upper domain E (8 out of 11) amassed in the Leu296-Leu310 stretch. Cleavage at Leu319 was seen with subtilisin and at Tyr328 with chymotrypsin. Susceptability to enzymic proteolysis was also pronounced in Thr465-Glu470 at the center of domain E. Three peptides, 13 kDa with thermolysin, beginning at Leu337, 6 kDa and, in low yield, 5 kDa with endopeptidase Lys-C beginning at Asp473 resp. Cys417 were only obtained from the monomer substrate. The various digests featured either 27-23-kDa peptides or mixtures of 17-13-kDa and 12-7-kDa peptides separable by SDS/PAGE. All peptides with N-termini between Leu297 and Ser329 reacted with mAb 13H2. The digests showed high peaks of bound estradiol in the dimer position of 32-kDa fragment controls on density gradient centrifugation at pH 7.4. However, the property of proton-driven dissociation was only preserved in the pronase, elastase and chymotrypsin digests with peptides extending beyond the His547-ArgLeuHis550 motif. The preservation of the estradiol-binding niche in the tightly complexed peptides of domain E was also demonstrated by refilling after steroid removal. The sites exposed to proteolytic enzymes and the epitope for 13H2 attachment are in good agreement with surface probability plots.
Thole
H H
HH
Max-Planck-Institut für experimentelle Endokrinologie, Hannover, Germany.
Maschler
I
I
Jungblut
P W
PW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004586:Electrophoresis; D010450:Endopeptidases / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism; D005260:Female; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D011959:Receptors, Estradiol / Q000737:chemistry* / Q000378:metabolism; D017421:Sequence Analysis; D013552:Swine; D014599:Uterus / Q000737:chemistry*
D008024:Ligands; D010446:Peptide Fragments; D011959:Receptors, Estradiol; D004958:Estradiol; D017830:Octoxynol; D010450:Endopeptidases