Schwartz J A, Skafar D F
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Biochemistry. 1994 Nov 15;33(45):13267-73. doi: 10.1021/bi00249a013.
We have investigated the relation between ligand structure and binding mechanism between the calf uterine estrogen receptor. A series of structurally altered estradiol analogs was used in which either an amino- or a nitro group had been added to the 2 or 4 position on the phenolic A-ring. The binding affinity of both amino analogs and the 4-nitro analog for the estrogen receptor was reduced relative to that of estradiol, as measured by competitive binding assay; the values were between 0.008% and 8% of estradiol's affinity. The slope of the displacement curve for the 4-nitro analog was also significantly different from that of estradiol (p < 0.05), indicating that the binding mechanism of these two ligands was different. The affinity of the 2-nitroestradiol ligand for the receptor was too low to be measured. The binding mechanism was then further investigated by measuring the Hill coefficient of [3H]estradiol binding in the presence of the analog. The presence of a nitro group on C4 eliminated the positive cooperativity of the [3H]estradiol-estrogen receptor interaction; the Hill coefficient of [3H]estradiol binding in the presence of the analog was 0.99 compared with 1.7 for [3H]estradiol alone. Most interestingly, the presence of an amino group on either C2 or C4 brought about a switch from a positive to a negative cooperative binding interaction; the Hill coefficients of [3H]estradiol binding in the presence of the analogs were between 0.6 and 0.7. These results provide additional support for an induced-fit mechanism of ligand-estrogen receptor interactions.
我们研究了配体结构与小牛子宫雌激素受体之间结合机制的关系。使用了一系列结构改变的雌二醇类似物，其中在酚类A环的2位或4位添加了氨基或硝基。通过竞争性结合试验测定，氨基类似物和4-硝基类似物对雌激素受体的结合亲和力相对于雌二醇均有所降低；其值在雌二醇亲和力的0.008%至8%之间。4-硝基类似物的置换曲线斜率也与雌二醇的显著不同（p < 0.05），表明这两种配体的结合机制不同。2-硝基雌二醇配体对受体的亲和力过低，无法测量。然后通过测量在类似物存在下[³H]雌二醇结合的希尔系数，进一步研究结合机制。C4上存在硝基消除了[³H]雌二醇 - 雌激素受体相互作用的正协同性；在类似物存在下[³H]雌二醇结合的希尔系数为0.99，而单独[³H]雌二醇的为1.7。最有趣的是，C2或C4上存在氨基导致从正协同结合相互作用转变为负协同结合相互作用；在类似物存在下[³H]雌二醇结合的希尔系数在0.6至0.7之间。这些结果为配体 - 雌激素受体相互作用的诱导契合机制提供了额外支持。
