Ekena K, Weis K E, Katzenellenbogen J A, Katzenellenbogen B S
Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 1997 Feb 21;272(8):5069-75. doi: 10.1074/jbc.272.8.5069.
We have previously examined, by alanine scanning mutagenesis, amino acids 515-535 of the estrogen receptor (ER) ligand binding domain to determine which of these residues are important in estradiol binding. Mutation at four sites that potentially lie along one face of an alpha-helix, Gly521, His524, Leu525, and Met528, all significantly impaired estradiol binding by the ER (Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 20053-20059). In this report, we compare the pattern of residues that are important in the recognition of several structurally diverse estrogen agonists and antagonists (the synthetic nonsteroidal agonist hexestrol, an agonist derived from the mold metabolite zearalenone, P1496, and the partial agonist-antagonist trans-hydroxytamoxifen) with those that are predicted to contact estradiol in the receptor-ligand complex. Although there are some similarities in the pattern of residue recognition among all four ligands, each ligand showed distinct differences as well. Interestingly, alanine substitution at only one residue, the leucine at position 525, was found to inhibit binding of all the ligands tested. Another residue, His524, was found to be important in the recognition of three different agonists but not trans-hydroxytamoxifen (the only ligand lacking a second hydroxyl group). The recognition of estradiol and another agonist, P1496, was impaired by the G521A mutation, whereas ligand-induced activity by the two compounds that lack B- and C-rings, hexestrol and trans-hydroxytamoxifen, was unaffected. Our findings demonstrate that these ligands fit into the ER ligand binding pocket differently and that each contacts a distinct set of amino acids. The smaller ligands (estradiol and hexestrol) have a narrower footprint of interacting residues than the larger ligands (P1496 and trans-hydroxytamoxifen). This pattern of interaction is most consistent with the amino acids within this region being in contact with the portion of these ligands that corresponds to the D-ring end of estradiol. The interplay between the shape of an ER ligand and the residues that support its binding to ER may potentially underlie the selective actions of different ER ligands in various cell and promoter contexts.
我们之前通过丙氨酸扫描诱变研究了雌激素受体（ER）配体结合结构域的515 - 535位氨基酸，以确定这些残基中哪些对雌二醇结合很重要。位于一条α-螺旋一个面上的四个位点（Gly521、His524、Leu525和Met528）发生突变，均显著损害了ER对雌二醇的结合（埃凯纳，K.，韦斯，K. E.，卡曾内伦博根，J. A.，以及卡曾内伦博根，B. S.（1996年）《生物化学杂志》271卷，20053 - 20059页）。在本报告中，我们比较了在识别几种结构多样的雌激素激动剂和拮抗剂（合成非甾体激动剂己烯雌酚、一种源自霉菌代谢产物玉米赤霉烯酮的激动剂P1496以及部分激动剂 - 拮抗剂反式羟基他莫昔芬）中重要的残基模式与在受体 - 配体复合物中预计与雌二醇接触的残基模式。尽管所有四种配体在残基识别模式上有一些相似之处，但每种配体也显示出明显差异。有趣的是，仅在一个残基（525位的亮氨酸）处进行丙氨酸取代就被发现会抑制所有测试配体的结合。另一个残基His524被发现对识别三种不同激动剂很重要，但对反式羟基他莫昔芬（唯一缺少第二个羟基的配体）不重要。G521A突变损害了对雌二醇和另一种激动剂P1496的识别，而缺少B环和C环的两种化合物（己烯雌酚和反式羟基他莫昔芬）的配体诱导活性未受影响。我们的研究结果表明，这些配体以不同方式契合ER配体结合口袋，并且每种配体接触一组不同的氨基酸。较小的配体（雌二醇和己烯雌酚）与较大的配体（P1496和反式羟基他莫昔芬）相比，相互作用残基的覆盖范围更窄。这种相互作用模式与该区域内的氨基酸与这些配体中对应于雌二醇D环末端的部分接触最为一致。ER配体的形状与支持其与ER结合的残基之间的相互作用可能潜在地构成了不同ER配体在各种细胞和启动子环境中的选择性作用的基础。
