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孔蛋白选择性渗透的结构基础。这项拟议的研究将探索选择性通透性是如何构建在这些跨膜通道中的，底物渗透实际上是如何在化学水平上发生的，以及某些残基如何赋予人类异构体选择性通透性。为此，我们将使用X射线结晶学来确定两个同源细菌异构体与其底物的复合体的结构。我们将把结构和序列联系起来，以确定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。蛋白质的表达、纯化和重组将被用于产生类似人类的细菌变体，这些变体将被整合到蛋白质脂质体中，其中选择性渗透性将通过停流测量进行检查。Am(L)将确定GlpF-water的结构，GlpF-water是一种与水分子结合的大肠杆菌水甘油磷脂。比较GlpF-水和GlpF-甘油的结构将阐明甘油分子如何在通过跨膜通道时取代水。目的(2)测定核糖醇及其立体异构体木糖醇与GlpF形成的络合物的结构，以阐明GlpF立体选择性的结构基础。Aim(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在甘油上选择性渗透的残基。目的(4)将研究水和甘油渗透的决定因素如何赋予人类异构体通道选择性通透性。人类决定因素将被替换为AqpZ或GlpF。人类决定因素的选择性渗透性将在细菌模板中表征，并通过与同源人类异构体的比较来评估。在健康和疾病中，人类水通道蛋白和水甘油调节蛋白调节水和少量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，而总体发现将在化学水平上阐明通过结晶膜通道选择性通透的机制。

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.