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 a s 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-c oupled electrospray ionization mass spectrometry (LC/ESI-MS)). Rapid c leavage 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 tryp sin, chymotrypsin, thermolysin, V8, and Asp-N proteinases are observed to occur in the region 553-571, followed by further cleavage with the rmolysin (548) and trypsin (548, 531, and 529). Thus, N-304 and K-529 define the protease-resistant N- and C-termini of a core structure for this domain that appears to contain the elements sufficient for ligan d binding. The remaining segment of this domain (530-553), which is kn own to embody elements essential for ligand-modulated transcription ac tivation (AF-2), is likely a surface-exposed region that, through thes e studies, is shown to be accessible to proteases. Only a single regio n within the 26 kDa ligand-binding core (N-304-K-529) has been identif ied 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 K-467, L(466), a nd both T-465 and S-468, respectively. The flexibility implied by the cuts in this internal 465-468 region suggest that the hER-LBD may actu ally consist of two subdomains. These proteolysis studies provide a su bstantially refined view of the conformational nature of the human est rogen receptor ligand binding domain.