Structural Basis for Mechanism of Secondary Transporters

二级转运蛋白机制的结构基础

• 批准号: 7332228
• 负责人: Howard Ronald KABACK
• 金额: $ 44.56万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7332228
• 关键词: Affinity; Affinity Labels; Aspartic Acid; Attention; Binding; Binding Sites; Biochemical; Biology; C-terminal; Carrier Proteins; Charge; Closure; Collaborations; Condition; Crystallization; Cysteine; Cytoplasm; Data; Dehydration; Diabetes Mellitus; Doctor of Medicine; Drug Delivery Systems; Drug resistance; Escherichia; Event; Exhibits; Fab Immunoglobulins; Family member; Fluoxetine; Future; Galactosides; Glycine; Goals; Heart; Helix (Snails); Histidine; Homologous Gene; Homology Modeling; Integral Membrane Protein; Ion Cotransport; Ions; Lactose; Ligand Binding; London; Lysine; Medicine; Membrane; Membrane Proteins; Membrane Transport Proteins; Mental Depression; Molecular Conformation; Monoclonal Antibodies; Mutation; Numbers; Omeprazole; Play; Property; Range; Research Personnel; Resolution; Roentgen Rays; Role; Sequence Homology; Side; Signal Transduction; Structure; Study models; Techniques; Testing; Time; Tryptophan; affinity labeling; base; college; conformer; design; divalent metal; ear helix; genome sequencing; human disease; improved; insight; lactose permease; member; mutant; oxidation; periplasm; programs; sugar

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.

描述（由申请人提供）：到目前为止，至少有20％的基因组编码了参与多种关键功能的多元跨膜蛋白，尤其是能量和信号转导。对于人类疾病（例如抑郁症，糖尿病，耐药性），许多药物针对膜转运蛋白（例如氟西汀和奥美拉唑）很重要。但是，膜蛋白的晶体结构的数量，尤其是离子偶联转运蛋白的数量非常有限。 最近，与So iwata的伦敦帝国学院的IWATA小组合作解决了大约3.5 a的Escherichia coil乳糖粘酶（Lacy）的向内构象体。该大量研究的主要促进剂超家族（MFS）的成员包含1000多个成员，由两个对称的6螺旋束组成，其中含有大型内腔，其中包含结合糖，该糖仅针对细胞质开放。根据结构和大量的生化和生物物理证据，提出了一种机制，其中结合位点可用于膜的两侧。这里提出的具体目的是通过在面向外构象中构造和获得晶体结构来检验这一假设。正如建模研究所示，由于MF的其他成员表现出相似的总体结构，因此我们还建议确定某些蕾丝同源物的结构。从当前的结构中获得的见解以及多种生化和生物物理技术将在结晶试验和结构确定之前合理设计和表征突变体。 我们还将在不久的将来获得当前结构的更高分辨率，我们希望在计划的时间段内获得其他结构。这些运输蛋白多种构象的X射线结构将对生物学和医学产生重要影响。