Remerowski M L, Kellenbach E, Boelens R, van der Marel G A, van Boom J H, Maler B A, Yamamoto K R, Kaptein R
Department of Chemistry, University of Utrecht, The Netherlands.
Biochemistry. 1991 Dec 17;30(50):11620-4. doi: 10.1021/bi00114a003.
The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by Härd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].
利用二维¹H NMR光谱对大鼠糖皮质激素受体（GR）DNA结合结构域（DBD）与共有糖皮质激素反应元件（GRE）半位点序列的复合物进行了研究。DNA片段是一个10个碱基对的寡核苷酸，5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3'，包含结合更强的GRE半位点六聚体，两端为GC碱基对。93个残基的GR-DBD包含一个86个残基的片段，对应于大鼠GR的440-525位残基。已鉴定出蛋白质与DNA之间的11个NOE交叉峰，并分析了复合物形成时DNA质子化学位移的变化。利用这些蛋白质-DNA接触点，可以得出以下结论：（i）由C460-E469残基形成的“识别螺旋”位于DNA的大沟中；（ii）GR-DBD在GRE半位点上的取向使得形成所谓D环的A477-D481残基可用于完整共有GRE上GR-DBD二聚体中的蛋白质-蛋白质相互作用；（iii）半位点中第二个胸腺嘧啶的5-甲基与缬氨酸462相互作用，证实了间接证据[Truss等人（1990年）美国国家科学院院刊87, 7180-7184；Mader等人（1989年）自然338, 271-274]，即两者在GR-DBD与DNA的结合中都起重要作用。这些发现与Härd等人[(1990年) 科学249, 157-160]提出的模型以及Luisi等人[(1991年) 自然352, 497-505]确定的X射线晶体学复合物结构一致。
