Structural Basis for Mechanism of Secondary Transporters

描述（由申请人提供）：迄今为止测序的基因组中至少有20%编码参与多种关键功能（特别是能量和信号转导）的多位跨膜蛋白。许多是关于人类疾病的重要（例如。抑郁症、糖尿病、耐药性），并且许多药物靶向于膜转运蛋白（例如，氟西汀和奥美拉唑）。然而，膜蛋白，特别是离子偶联转运蛋白的晶体结构的数量非常有限。 最近，与伦敦帝国理工学院的So岩田小组合作，在约3.5 A处解决了大肠杆菌乳糖通透酶（LacY）的一个面向内的构象。这个被深入研究的主要易化因子超家族（MFS）的成员，包含超过1000个成员，由两个对称的6螺旋束组成，具有含有结合糖的大内腔，仅对细胞质开放。基于结构和大量的生物化学和生物物理学证据，提出了一种机制，其中结合位点可选择地进入膜的两侧。本文提出的具体目的是通过构建和获得外向构象的晶体结构来测试这一假设。由于MFS的其他成员表现出类似的整体结构，如建模研究所示，我们还建议确定某些LacY同系物的结构。从目前的结构，以及各种生物化学和生物物理学技术获得的见解，将用于合理设计和表征突变体在结晶试验和结构测定之前的外向构象（S）。 我们还将在不久的将来获得当前结构的更高分辨率，我们预计将在计划的时间内获得其他结构。这些转运蛋白的多种构象的X射线结构将对生物学和医学产生重要影响。

DESCRIPTION (provided by applicant): At least 20 percent of the genomes sequenced thus far encode polytopic transmembrane proteins involved in a multitude of critical functions, particularly energy and signal transduction. Many are important with regard to human disease (eg. depression, diabetes, drug resistance), and many drugs are targeted to membrane transport proteins (eg. fluoxetine and omeprazole). However, the number of crystal structures of membrane proteins, particularly ion-coupled transporters, is very limited. Recently, an inward-facing conformer of the Escherichia coil lactose permease (LacY) was solved at about 3.5 A in collaboration with So Iwata's group at Imperial College London. This intensively studied member of the Major Facilitator Superfamily (MFS), which contains over 1000 members, is composed of two symmetrical 6-helix bundles with a large internal cavity containing bound sugar, which is open to the cytoplasm only. Based on the structure and a large body of biochemical and biophysical evidence, a mechanism is proposed in which the binding site is alternatively accessible to either side of the membrane. The specific aims proposed here are to test this hypothesis by constructing and obtaining a crystal structure(s) in the outward-facing conformation(s). Since other members of the MFS exhibit a similar overall structure, as indicated by modeling studies, we also propose to determine structures for certain LacY homologues. Insights obtained from the current structure, as well as a variety of biochemical and biophysical techniques, will be used to rationally design and characterize mutants in the outward-facing conformation(s) prior to crystallization trials and structure determination. We will also obtain higher resolution of the current structure in the near future, and we expect the other structures proposed to be obtained within the time period planned. X-ray structures of these transport proteins in multiple conformations will have important impact on biology and medicine.