Structural Basis of Selective Permeability in Aquaporin

我们研究的长期目标是了解水和多元醇通过人水通道蛋白和水甘油通道蛋白选择性渗透的结构基础。 这项拟议的研究将探索如何在这些跨膜通道中建立选择性渗透性、底物渗透实际上如何在化学水平上发生以及某些残基如何赋予人类异构体选择性渗透性。为此，我们将使用 X 射线晶体学来确定两种同源细菌亚型及其底物复合物的结构。 我们将关联结构和序列来识别决定选择性渗透性的残基。然后将使用诱变将人类残留物引入细菌模板中。 蛋白质表达、纯化和重建将用于产生类人细菌变体，这些变体将被纳入脂蛋白体中，通过停流测量来检查选择性渗透性。目标 (l) 将确定 GlpF-water（一种与水分子结合的大肠杆菌水甘油孔蛋白）的结构。 比较 GlpF-水和 GlpF-甘油的结构将阐明甘油分子在通过跨膜通道时如何置换水。 目标（2）将确定 GlpF 与核糖醇及其立体异构体木糖醇复合物的结构，以阐明 GlpF 立体选择性的结构基础。目标 (3) 将结晶 AqpZ（一种大肠杆菌水通道蛋白）并确定其结构。 比较 AgpZ 和 GlpF 的结构将揭示决定水相对于甘油的选择性渗透的残基。 目标 (4) 将研究水和甘油渗透的决定因素如何赋予人类亚型通道选择性渗透性。 人类决定簇将被交换为 AqpZ 或 GlpF。 人类决定簇的选择性渗透性将在细菌模板中进行表征，并通过与同源人类亚型进行比较来进行评估。人类水通道蛋白和水甘油通道蛋白在健康和疾病中调节水和小非电解质的渗透平衡。这项拟议研究的完成将为针对人类渗透调节障碍的基于结构的药物设计奠定基础，而一般研究结果将在化学水平上阐明通过结晶膜通道的选择性渗透机制。

The long-term goal of our research is to understand the structural basis for selective permeation of water and polyols through human aquaporins and aquaglyceroporins. This proposed research will explore how selective permeability is built into these transmembrane channels, how substrate permeation actually occurs at a chemical level and how certain residues confer selective permeability upon human isoforms. Toward these ends, we will use X-ray crystallography to determine structures of two homologous bacterial isoforms in complex with their substrates. We will correlate structure and sequence to identify residues that determine selective permeability. Mutagenesis will then be used to introduce human residues into the bacterial templates. Protein expression, purification and reconstitution will be used to generate human-like bacterial variants, which will be incorporated into proteoliposomes, where selective permeability will be examined by stopped-flow measurements. Aim (l) will determine the structure of GlpF-water, an E. coli aquaglyceroporin bound with water molecules. Comparing structures of GlpF-water and GlpF-glycerol will elucidate how a glycerol molecule displaces water while passing through the transmembrane channel. Aim (2) will determine the structures of GlpF in complex with ribitol and its stereoisomer xylitol to elucidate the structural basis for stereo-selectivity in GlpF. Aim (3) will crystallize AqpZ, an E. coli aquaporin, and determine its structure. Comparing structures of AgpZ and GlpF will reveal residues that determine selective permeation of water over glycerol. Aim (4) will investigate how determinants of water and glycerol permeation confer channel selective permeability upon human isoforms. Human determinants will be swapped into AqpZ or GlpF. The selective permeability of human determinants will be characterized in bacterial templates and evaluated by comparing with that of cognate human isoforms. Human aquaporins and aquaglyceroporins regulate osmotic balance of water and small non-electrolytes in health and disease. Completion of this proposed study will set the stage for structure-based drug design targeting human osmoregulation disorders, while the general findings will elucidate the mechanism of selective permeability through a crystallized membrane channel at a chemical level.