Analysis of the structural core of the human estrogen receptor ligand binding domain by selective proteolysis/mass spectrometric analysis.
The structure of the ca. 250 amino acid hormone binding domain of the human estrogen receptor (hER-LBD), expressed in E. coli and purified as a complex with estradiol, has been probed by selective proteolysis, with analysis of the protein fragments both by classical methods (SDS-PAGE and Edman N-terminal sequencing) and by mass spectrometry (HPLC-coupled electrospray ionization mass spectrometry (LC/ESI-MS)). Rapid cleavage by several proteases (trypsin, chymotrypsin, thermolysin, and Asp-N endoproteinase) is observed within a localized region (residues 297-303) at the N-terminus. In contrast, proteolytic scission at the C-terminus is less localized and more progressive; initial cuts by trypsin, chymotrypsin, thermolysin, V8, and Asp-N proteinases are observed to occur in the region 553-571, followed by further cleavage with thermolysin (548) and trypsin (548, 531, and 529). Thus, N304 and K529 define the protease-resistant N- and C-termini of a core structure for this domain that appears to contain the elements sufficient for ligand binding. The remaining segment of this domain (530-553), which is known to embody elements essential for ligand-modulated transcription activation (AF-2), is likely a surface-exposed region that, through these studies, is shown to be accessible to proteases. Only a single region within the 26 kDa ligand-binding core (N304-K529) has been identified as being readily accessible to proteases; rapid proteolysis using the proteases trypsin, chymotrypsin, and thermolysin, is localized to residues 465-468, with cleavage occurring at residues K467, L466, and both T465 and S468, respectively. The flexibility implied by the cuts in this internal 465-468 region suggest that the hER-LBD may actually consist of two subdomains. These proteolysis studies provide a substantially refined view of the conformational nature of the human estrogen receptor ligand binding domain.
Seielstad
D A
DA
Department of Chemistry, University of Illinois, Urbana 61801, USA.
Carlson
K E
KE
Kushner
P J
PJ
Greene
G L
GL
Katzenellenbogen
J A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D008024:Ligands; D011960:Receptors, Estrogen; D004958:Estradiol; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; D013820:Thermolysin; C058921:endoproteinase Asp-N