Crystallographic studies of equilibrative nucleoside transporters

描述（由申请人提供）：平衡核苷转运蛋白（ENTs）是人类主要的膜转运蛋白，介导腺苷的摄取和外排，腺苷是一种重要的细胞间信号分子，介导从神经和心脏保护到疼痛感知、癫痫易感性和睡眠周期的广泛作用。尽管它们在人体生理学和病理生理学中具有巨大的意义，但人们对耳鼻喉科细胞识别和转运其疏水性脂质双层底物的机制知之甚少。在本提案中，我们专注于ENT同系物的结晶，这是确定这些分子晶体结构的关键一步。我们已经确定了在洗涤剂中具有极好的结构稳定性的耳鼻喉同源物，并建立了它们的表达和纯化协议，这些协议通常会产生毫克数量的稳定均质蛋白质。我们将进一步优化蛋白质结构、洗涤剂、配体和脂质补充，以实现转运体结晶。一个重要的问题是，膜转运蛋白本质上是动态蛋白，在溶液中，它们可能分布在多种状态，阻碍结晶。为了解决这个问题，我们将系统地突变hENT1中27个高度保守的氨基酸，这是结晶候选物之一，并在酵母中实施功能筛选，以确定功能缺失的突变体，这些突变体高度表达，在洗涤剂中稳定，能够紧密结合底物但不能运输。我们假设这些突变体将缺乏转运，因为它们无法经历部分或全部基本的构象转变，因此构象受限。我们将评估这种突变体是否会在结晶过程中提供明显的好处。该项目的成功将对耳鼻喉科的研究产生重大影响，并可能对动态膜蛋白（如通道和转运蛋白）的结构生物学的更大领域产生重大影响。公共卫生相关性：腺苷是人类重要的信号分子，调节广泛的效应，如血管舒张、血栓形成、疼痛感知、睡眠周期和体温。细胞膜上的特殊蛋白质，平衡核苷转运体控制可用于信号传导的腺苷水平。它们是许多药物的目标，如广泛用于预防继发性中风的双嘧达莫。在这里，我们建议结晶这些分子，这是确定其原子结构的重要步骤，这本身对于理解转运体机制和增加我们从药理学上控制其功能的能力至关重要。

DESCRIPTION (provided by applicant): Equilibrative nucleoside transporters (ENTs) are the predominant membrane transporters in humans mediating the uptake and efflux of adenosine, which is an important inter-cellular signaling molecule mediating a wide range of effects from neuro- and cardioprotection to pain perception, susceptibility to seizures and sleep cycle. Despite their immense significance in human physiology and pathophysiology, little is known about the mechanism by which ENTs recognize and translocate their substrates across hydrophobic lipid bilayers. In this proposal we focus on the crystallization of ENT homologues, a critical step towards determining the crystal structure of these molecules. We have already identified ENT homologues with superb structural stability in detergent and established expression and purification protocols for them that routinely yield milligram quantities of stable homogeneous proteins. We will further optimize the protein constructs, detergents, ligands and lipid supplementation to achieve transporters crystallization. An important concern is that membrane transporters are dynamic proteins by nature, and that in solution they may distribute over multiple state hindering crystallization. To circumvent this problem, we will systematically mutate 27 highly conserved amino acids within hENT1, which is one of the crystallization candidates, and implement a functional screen in yeast in order to identify the loss-of-function mutants, that are highly expressed, stable in detergent and capable of tight substrate binding but not transport. We hypothesize that such mutants will be deficient in transport because they are unable to undergo some or all of the essential conformation transitions and are, thus conformationally constrained. We will evaluate whether such mutants would provide distinct benefits in the crystallization process. The success of this project will have a major impact on the studies of ENTs and possibly on the larger field of the structural biology of dynamic membrane proteins such as channels and transporters. PUBLIC HEALTH RELEVANCE: Adenosine is an important signaling molecule in humans, regulating a wide range of effects such as vasodilatation, thrombus formation, pain perception, sleep cycle, and body temperature. Specialized proteins in the cellular membranes, equilibrative nucleoside transporters control the levels of adenosine available for signaling. They are the targets of many drugs such as dipyridamole, which is widely used to prevent secondary strokes. Here we propose to crystallize these molecules, an essential step toward determination of their atomic structure, which in itself is essential to understand the transporters mechanism and to increase our capacity to pharmacologically control their function.