Structural Studies of a Plant Nitrate Transporter and Receptor

微生物氮素是氨基酸和核酸的重要组成部分，对地球上每一种生命形式都是至关重要的。植物在将无机氮素转化为有机氮素的过程中起着关键作用，并通过吸收硝酸盐吸收90%以上的氮素。为了有效地吸收土壤中的硝酸盐，植物能够跨细胞膜运输硝酸盐，并能够感知环境中硝酸盐浓度的变化。拟南芥硝酸盐转运蛋白CHL1中一个意想不到的硝酸盐受体功能的发现，揭示了植物如何感知土壤硝酸盐变化的线索。作为主要促进剂超家族的一员，CHL1是一种双亲和力转运蛋白，对低浓度和高浓度的硝酸盐表现出双相动力学特性。特定苏氨酸残基的磷酸化会在两种亲和模式之间切换转运蛋白。值得注意的是，与其转运蛋白功能无关，CHL1还可以感知和响应不同的硝酸盐浓度，并以双相方式控制基因表达。CHL1的相同磷酸化位点也调节其传感器活性。CHL1是如何感知硝酸盐浓度的？CHL1作为硝酸盐转运体和感受器的分子机制是什么？磷酸化如何调节CHL1活性？这个项目将通过揭示CHL1在不同功能状态下的原子结构来开始解决这些问题。膜蛋白X射线结晶学将作为实现以下特定目标的主要手段：(1)在高浓度硝酸盐存在下确定CHL1的结构；(2)在低浓度硝酸盐存在下确定CHL1的结构。这些结果将有助于揭示CHL1的整体分子结构，对其转运蛋白和受体功能至关重要的潜在硝酸盐结合位点，以及其双亲和力硝酸盐转运蛋白和传感器活动的结构决定因素和机制。布罗德重要：他的研究项目将在两个主要领域影响科学和教育：(1)推进美国植物生物学的基础研究，具有农业和环境意义；(2)形成一个区域性的膜蛋白结构生物群落。粮食、能源和环境是21世纪世界面临的三大挑战。解决这些挑战需要在最基本的层面上对植物生物学有透彻的了解。该项目将通过将尖端结构生物学方法应用于植物科学来促进植物生物学研究。它代表了推进植物结构生物学的一项倡议，重点是解决植物生物学中新出现的关键问题，并在更大的社区中传播植物科学。该项目还将通过提供重组膜蛋白生产、结晶和结构确定方面的培训，促进膜蛋白结构生物学家区域社区的进一步发展。我们的努力包括在实验室之外对本科生和研究生、技术员和PI进行实践培训，以及在研究生项目会议、部门务虚会、特殊兴趣俱乐部和大学联合课程上的定期演讲。该项目由分子和细胞生物科学部的细胞过程集群和化学部的生命过程化学项目共同支持。

INTELLECUTAL MERITNitrogen is an essential building block of amino acids and nucleic acids and is crucial for every single form of life on earth. Plants play a key role in converting nitrogen from its inorganic form to an organic form and assimilate more than 90% of the nitrogen through nitrate absorption. In order to efficiently take up nitrate in soils, plants are capable of transporting nitrate across cellular membranes, as well as sensing environmental nitrate concentration changes. A clue to how plants sense soil nitrate changes was revealed by the finding of an unexpected nitrate receptor function in the Arabidopsis nitrate transporter, CHL1. As a member of the Major Facilitator Superfamily, CHL1 is a dual-affinity transporter, displaying a biphasic kinetic property in response to low and high concentrations of nitrate. Phosphorylation of a specific threonine residue switches the transporter between the two affinity modes. Remarkably, independent of its transporter function, CHL1 can also sense and respond to variable nitrate concentrations and control gene expression in a biphasic manner. The same phosphorylation site of CHL1 also regulates its sensor activity. How does CHL1 sense nitrate concentration? What is the molecular mechanism underlying the functions of CHL1 as both a nitrate transporter and sensor? How does phosphorylation regulate CHL1 activities? This project will start to address these questions by revealing the atomic structures of CHL1 in different functional states. Membrane protein X-ray crystallography will be used as the primary approach to achieve the following specific aims: (1) structure determination of CHL1 in the presence of high nitrate concentration; and (2) structure determination of CHL1 in the presence of low nitrate concentration. The results will help unravel the overall molecular architecture of CHL1, the potential nitrate-binding sites that are important for its transporter and receptor functions, and the structural determinants and mechanisms underlying its dual-affinity nitrate transporter and sensor activities.BROADER IMPACTSThis research project will impact science and education in two major areas: (1) Advancing basic research in plant biology in the US with agricultural and environmental implications and (2) Nucleating a regional membrane protein structural biology community. Food, energy, and environment are the three major challenges the world is facing in the 21st century. Solutions to these challenges require a thorough understanding of plant biology at the very fundamental level. This project will promote plant biology research by applying cutting-edge structural biology approaches to plant sciences. It represents an initiative in advancing plant structural biology, with an emphasis on addressing emerging key questions in plant biology and propagating plant sciences in the larger community. The project will also foster further development of regional community of membrane protein structural biologists by offering training in recombinant membrane protein production, crystallization, and structure determination. Our efforts range from hands-on training of undergraduate and graduate students, technicians, and PIs outside the lab, to regular presentations at graduate program meetings, departmental retreats, special interests clubs, and joint university courses.This project is jointly supported by the Cellular Processes Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.