| Added by | informatique.ircm |
|---|---|
| Group name | Informatique |
| Item Type | Journal Article |
| Title | Cysteine 530 of the human estrogen receptor alpha is the main covalent attachment site of 11beta-(aziridinylalkoxyphenyl)estradiols |
| Creator | Aliau et al. |
| Author | S. Aliau |
| Author | H. Mattras |
| Author | E. Richard |
| Author | J. L. Borgna |
| Abstract | The efficiency of 11beta-[p(aziridinylethoxy)phenyl]estradiol 1 and 11beta-[p(aziridinylpentoxy)phenyl]estradiol 2 affinity labeling of the estrogen receptor alpha (ERalpha) was evaluated on the basis of their capacity to inhibit [(3)H]estradiol binding to lamb and human ERalphas. Relative to RU 39 411 (11beta-[p(dimethylaminoethoxy)phenyl]estradiol), the most closely related and chemically inert analogue of 1, the two electrophiles irreversibly inhibited [(3)H]estradiol binding to the lamb ERalpha. The fact that the compound effects were prevented (i) when the ERalpha hormone-binding site was occupied by estradiol and (ii) when the ERalpha-containing extracts were pretreated with methyl methanethiosulfonate (an SH-specific reagent) suggested that the compounds specifically alkylated ERalpha at cysteine residues. Wild-type human ERalpha was alkylated as efficiently as lamb ER, whereas the quadruple cysteine --> alanine mutant, in which all cysteines of the hormone-binding domain (residues 381, 417, 447, and 530) were changed to alanines, showed no significant electrophile labeling. The single C530A mutant was much less sensitive to the action of the electrophiles than the three other single mutants (C381A, C417A, and C447A). Moreover, analysis of the three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) showed that only the C381A/C530A mutant was less susceptible to electrophile labeling than the single C530A mutant. We concluded that in the hormone-binding pocket C530 was the main covalent attachment site of aziridines 1 and 2, whereas C381 could be a secondary site. These results agreed with the crystal structure of the hormone-binding domain of the human ERalpha bound to estrogen or antiestrogen, since C381 and C530 appeared to be (i) located in structural elements involved in delineating the hormone-binding pocket and (ii) in spatial proximity to each other, which was closer in the crystal structure of the ER:antiestrogen complex than in that of the ER:estrogen complex. Since C530 and C381 were also the main and secondary covalent attachment sites of tamoxifen aziridine (a nonsteroidal affinity-labeling agent), we propose a selective mode of superimposition of tamoxifen-class antiestrogens with RU 39 411-class antiestrogens, which could account for the relative positioning of the two types of ligands in the ERalpha hormone-binding pocket. |
| Publication | Biochemistry |
| Volume | 38 |
| Issue | 45 |
| Pages | 14752-62 |
| Date | Nov 9 1999 |
| Language | eng |
| ISSN | 0006-2960 (Print) |
| Tags | Animals, Aziridines/chemistry/ metabolism, Binding Sites, Binding, Competitive, COS Cells, Cysteine/chemistry/ metabolism, Estradiol/ analogs & derivatives/chemistry/metabolism, Estrogen Receptor alpha, Female, Humans, Ligands, Receptors, Estrogen/chemistry/ metabolism, Sheep, Tamoxifen/analogs & derivatives/chemistry/metabolism, Uterus/metabolism |
| Date Added | 2019/12/19 - 16:59:46 |
| Date Modified | 2019/12/19 - 16:59:46 |
| Notes and Attachments | (Note) (Note) |
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