STRUCTURE/FUNCTION STUDIES OF GLUCOCORTICOID RECEPTOR

由于缺乏对其主要反式激活结构域(AF1)结构的了解，阻碍了对糖皮质激素受体功能机制的充分了解。据推测，AF1与蛋白质的“转录机制”复合体直接或通过中间蛋白质相互作用。这需要蛋白质和结构之间的表面到表面的相互作用才能产生它们。然而，当作为重组蛋白单独表达时，AR显示很少或没有结构，并且充其量与少数这样的蛋白结合得很弱。我们已经做了两个观察，这为理解AF1是如何获得其假定功能所需的结构和发现其天然结合伙伴提供了令人兴奋的前景。首先，我们发现在三甲胺氧化物(TMAO)的存在下，人GR的AF1具有类似天然的结构，并与其他某些蛋白质的结合大大增强。我们将使用TMAO和其他渗透分子来研究AF1的结构并寻找其结合伙伴。其次，我们已经证明，当含有AF1和DNA结合域的GR的两个结构域片段与其同源DNA结合部位结合时，AR似乎获得了结构并能够结合某些其他蛋白质。在这个项目中，我们将确定控制这一事件的蛋白质和DNA参数。我们将比较AF1的结构和两种技术发现的蛋白质结合伙伴。在我们使用的条件下，显示与AF1结合的核蛋白之一似乎是核受体共激活蛋白家族的成员。将使用的方法包括圆二色谱、本征荧光发射光谱、肽图、蛋白质的热力学严谨研究：DNA结合核磁共振和IF晶体获得、X射线衍射。

Lack of knowledge of the structure of its major transactivation domain (AF1) has hampered full understanding of the mechanism by which the glucocorticoid receptor functions. It is presumed that AF1 interacts directly, or through intermediary proteins, with the "transcription machinery" complex of proteins. This requires surface-to-surface interactions between proteins and the structure to produce them. When expressed separately as a recombinant protein, however, AR displays little or no structure and binds weakly at best with a few such proteins. We have made two observations that offer exciting promise for understanding how AF1 obtains the structure necessary for its presumed function and for discovering its natural binding partners. First, we found that in the presence of an osmolyte, trimethylamine oxide (TMAO), the AF1 of the human GR acquires native-like structure and shows greatly enhanced binding to certain other proteins. We will use TMAO and other osmolytes to study the structure of AF1 and to find its binding partners. Second, we have shown that when a two-domain fragment of the GR containing AF1 and the DNA-binding domain is bound to its cognate DNA binding site, AR seems to acquire structure and be able to bind certain other proteins. In this project, we will determine the protein and DNA parameters that control this event. We will compare the AF1 structure and the protein binding partners uncovered by the two techniques. One of the nuclear proteins shown to bind AF1 under the conditions we employ appears to be a member of the nuclear receptor co- activator family of proteins. Methods used will include circular dichroism, intrinsic fluorescence emission spectroscopy, peptide mapping, thermodynamically rigorous studies of protein:DNA binding NMR and if crystals are obtained, X-ray diffraction.