Structural Basis for Mechanism of Secondary Transporters

描述(由申请人提供)：到目前为止，至少有20%的基因组序列编码多位跨膜蛋白，参与多种关键功能，特别是能量和信号转导。许多对于人类疾病来说是重要的(例如，抑郁症、糖尿病、耐药性)，许多药物是针对膜转运蛋白的(例如。氟西汀和奥美拉唑)。然而，膜蛋白的晶体结构，特别是离子耦合转运蛋白的数量是非常有限的。 最近，在伦敦帝国理工学院与岩田先生的研究小组合作下，一种内向的大肠杆菌螺旋乳糖渗透酶(Lacy)构象在3.5A的温度下被解决。这个主要促进因子超家族(MFS)的成员被广泛研究，包含1000多个成员，由两个对称的6-螺旋束组成，内腔大，内含结合糖，只对细胞质开放。基于这种结构和大量的生化和生物物理证据，提出了一种结合部位可交替进入膜两侧的机制。这里提出的具体目的是通过构建和获得外向构象(S)中的晶体结构(S)来检验这一假说。由于MFS的其他成员表现出类似的整体结构，正如建模研究所表明的那样，我们还建议确定某些Lacy同源物的结构。从目前结构中获得的见解，以及各种生化和生物物理技术，将在结晶试验和结构确定之前，用于合理设计和表征向外构象(S)中的突变体。 我们还将在不久的将来获得当前结构的更高分辨率，我们预计拟议的其他结构将在计划的时间段内获得。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的影响。

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.