Structure and Function of Concentrative Nucleoside Transporters

描述（由申请人提供）：核苷转运蛋白（nt）是一种完整的膜蛋白，用于将核苷转运到人体内。核苷跨细胞膜运输在生理上是重要的，因为它为细胞内DNA/RNA合成提供核苷的主要来源，并负责腺苷信号的终止。关于nt的另一个重要事实是，许多核苷衍生的抗癌和抗病毒药物通过nt转运到细胞中。尽管它们在生理学和药理学上很重要，但由于缺乏转运体的原子结构，其分子机制在很大程度上是未知的。该领域的重大进展取决于纳米碳纳米管的结构确定和转运机制的阐明。我们的长期目标是通过结构和功能研究了解浓缩核苷转运体（CNTs）选择性核苷转运的基本机制。CNTs属于溶质载体转运体超家族（SLC28），利用离子梯度对抗核苷的化学梯度主动转运核苷。我们选择使用来自霍乱弧菌的碳纳米管同源物（vcCNT）作为我们的结构和功能研究的模型系统。vcCNT与hCNTs具有显著的序列同源性和功能特性，是研究hCNTs的良好模型系统。我们求解了vcCNT与尿苷配合物的晶体结构。该结构揭示了转运体的整体结构和核苷结合位点，提供了vcCNT和CNT选择性核苷转运的机制见解。这些新结果使我们提出了一项结构-功能联合研究，其目的有以下三个：1)确定vcCNT在不同核苷和核苷药物复合物中的晶体结构；2)vcCNT与hCNTs的功能表征和比较；3)确定vcCNT在不同构象中的结构。所提出的研究将极大地促进我们对核苷和核苷类药物通过原核和真核碳纳米管转运的机制的理解，最终将促进新的治疗试剂的开发。

DESCRIPTION (provided by applicant): Nucleoside transporters (NTs) are integral membrane proteins that are designed to transport nucleosides into the human body. Nucleoside transport across cell membranes is physiologically important as it provides the major source of nucleosides for DNA/RNA synthesis in cells and it is responsible for the termination of adenosine signaling. Another important fact about NTs is that many nucleoside-derived anticancer and antiviral drugs are transported into cells by way of NTs. Despite their importance in physiology and pharmacology, the molecular mechanism of NTs is largely unknown due to the lack of atomic structures of the transporters. Great advances in the field depend on the structure determination and illumination of the transport mechanism of NTs. Our long- term goal is to understand the fundamental mechanism of selective nucleoside transport by concentrative nucleoside transporters (CNTs) with structural and functional studies. CNTs belong to a family of the solute carrier transporter superfamily (SLC28) and use an ion gradient to transport nucleosides actively against their chemical gradients. We have chosen to use a CNT homolog from Vibrio cholerae (vcCNT) as a model system for our structural and functional studies. vcCNT is an excellent model system to study hCNTs that shares significant sequence homology and functional properties with hCNTs. We solved the crystal structure of vcCNT in complex with uridine. The structure reveals the overall architecture and the nucleoside-binding site of the transporter, providing mechanistic insights of selective nucleoside transport by vcCNT and CNT in general. These new results have led us to propose a combined structure-function study with the following three aims: 1) Determination of the crystal structures of vcCNT in complex with different nucleosides and nucleoside drugs, 2) Functional characterization and comparison of vcCNT with hCNTs, and 3) Structure determination of vcCNT in alternate conformations. The proposed studies will significantly advance our understanding of the mechanism of nucleoside and nucleoside drug transport by prokaryotic and eukaryotic CNTs, which eventually will facilitate the development of new therapeutic reagents.