Experimental and structural evolution of hormone receptors

描述(申请人提供)：这项研究的目标是阐明类固醇激素受体这一生物医学重要基因家族中新功能进化的进化动力学和结构基础。我们将结合实验进化、结晶学、祖先基因复活和基因功能的操纵性分析来分析受体如何与新的激素配体进化紧密的分子伙伴关系。具体目标是： 1.使用工程酵母中的实验进化系统来选择人类雌激素受体，就像某些乳腺肿瘤中的受体一样，这种受体会进化为受主要乳腺癌药物泰莫西芬刺激。利用一系列功能分析和进化技术重建受体序列的进化，我们将确定这种新的ER-配体相互作用进化的机制基础和受体-配体共同进化的动力学。 2.我们将复活祖先的类固醇受体(使用系统发育分析和基因合成)，然后在相同的实验进化系统中进化这些受体。将选择祖先受体来概括在数亿年的真实历史进化过程中发生的配基特异性的功能变化。我们将详细研究这一过程的机制基础和进化动力学。 3.我们将表达、结晶和确定祖先受体的三维结构，以及几种功能不同的现存类固醇受体的三维结构。这些结构--连同目标1和目标2的新功能进化的数据--将提供一个丰富的数据库，从中重建受体蛋白质结构的进化。这有望揭示新受体功能进化的结构机制，无论是在我们的实验进化系统中，还是在真实的历史进化中。 这种操纵性、假设检验技术的综合将为基因功能进化的动力学和机制提供前所未有的详细知识。这些数据应该有助于解决进化生物学中长期存在的基本问题，并提高我们对决定类固醇受体功能的结构-功能关系的理解。

DESCRIPTION (provided by applicant): The goal of this research is to elucidate the evolutionary dynamics and structural basis for the evolution of novel functions in a biomedically important gene family, the steroid hormone receptors. We will combine experimental evolution, crystallography, ancestral gene resurrection, and manipulative assays of gene function to analyze how receptors evolve tight molecular partnerships with novel hormonal ligands. The specific aims are: 1. Use an experimental evolution system in engineered yeast to select for human estrogen receptors which, like those in some mammary tumors, evolve to be stimulated by taxmoxifen, a major breast cancer drug. Using a battery of functional assays and evolutionary techniques for reconstructing the evolution of receptor sequences, we will determine the mechanistic basis for the evolution of this new ER-ligand interaction and the dynamics of receptor-ligand coevolution. 2. We will resurrect ancestral steroid receptors (using phylogenetic analysis and gene synthesis) and then evolve these receptors in this same experimental evolution system. Ancestral receptors will be selected to recapitulate the functional shifts in ligand specificity that occurred during real historical evolution over hundreds of millions of years. The mechanistic basis and evolutionary dynamics of this process will be studied in detail. 3. We will express, crystallize, and determine the three-dimensional structures of ancestral receptors, as well as those of several functionally divergent extant steroid receptors. These structures - together with the data on the evolution of new functions from Aims 1 and 2 - will provide a rich database from which to reconstruct the evolution of receptor protein structures. This is expected to reveal the structural mechanisms by which new receptor functions evolve, both in our experimental evolution system and in real historical evolution. This synthesis of manipulative, hypothesis-testing techniques will provide unprecedented detailed knowledge of the dynamics and mechanisms by which gene function evolves. These data should help resolve long-standing fundamental issues in evolutionary biology, and also improve our understanding of the structure-function relationships that determine steroid receptor function.