Structure and mechanism of the AbgT-family transporters

AbgT 家族转运蛋白的结构和机制

• 批准号: 9137611
• 负责人: EDWARD W YU
• 金额: $ 36.39万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-04 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9137611
• 关键词: 4-Aminobenzoic Acid; Anabolism; Antibiotic Resistance; Antibiotics; Antimetabolites; Architecture; Bacteria; Bacterial Infections; Biochemical; Biological Assay; Calorimetry; Case Study; Cell membrane; Cells; Clinical; Communicable Diseases; Comprehension; Cytoplasm; Data; Databases; Development; Disease; Drug Targeting; Drug resistance; Excision; Family; Folic Acid; Glutamates; Goals; Gonorrhea; Growth; Hand; Health; Human; Infection; Infectious Agent; Knowledge; Length; Light; Mediating; Membrane; Membrane Proteins; Molecular; Neisseria gonorrhoeae; Pathway interactions; Pharmaceutical Preparations; Predisposition; Proteins; Radioactive; Research; Resistance; Resistance development; Resolution; Roentgen Rays; Sexually Transmitted Diseases; Shapes; Site-Directed Mutagenesis; Solvents; Structure; Sulfonamides; Therapeutic; Titrations; Vitamins; Work; antibiotic efflux; antimicrobial; antimicrobial drug; bacterial resistance; base; clinical application; combat; design; dimer; drug mechanism; efflux pump; genetic analysis; monomer; mutant; novel; pathogen; pathogenic bacteria; resistant strain; simulation; uptake

 DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to drug transfer across the cell membrane by the AbgT-family of transporters. The primary targets of the proposed work are the Alcanivorax borkumensis YdaH and Neisseria gonorrhoeae MtrF transporters. Approximately 13,000 putative transporters of the family have been identified. However, no structural information has yet been available and even functional data are minimal for this family of membrane proteins. It has been hypothesized that the AbgT-family transporters contribute to the bacterial folate synthesis pathway by importing the catabolite p-aminobenzoyl- glutamate for producing this essential vitamin. To understand the structure and function of the AbgT family of transporters, we have recently cloned, expressed, purified and crystallized the full-length A. borkumensis YdaH and N. gonorrhoeae MtrF membrane proteins. We have also determined the X-ray structures of these two transporters at 2.9 and 3.9 Å resolutions, respectively. The structures reveal that these two transporters assemble as dimers with architectures distinct from all other families of transporters. Both YdaH and MtrF are bowl- shaped dimers with a solvent-filled basin extending from the cytoplasm halfway across the membrane bilayer. The monomers of YdaH and MtrF contain nine transmembrane helices and two hairpins. These structures directly suggest a plausible pathway for substrate transport. A combination of the crystal structure, genetic analysis and substrate accumulation assay indicates that both YdaH and MtrF behave as exporters, capable of removing the folate metabolite p-aminobenzoic acid from bacterial cells. Further experimental data based on drug susceptibility and radioactive transport assay suggest that both YdaH and MtrF participate as antibiotic efflux pumps, importantly mediating bacterial resistance to sulfonamide antimetabolite drugs. Our hypothesis is that many of these AbgT-family transporters act as exporters, thereby conferring bacterial resistance to sulfonamides. The specific aim is to define the structures and transport mechanisms of the AbgT family of transporters. We will use the structural, biochemical and computational approaches to determine the molecular mechanisms of the YdaH and MtrF transporters. Neisseria gonorrhoeae is an obligate human pathogen and the infectious agent for the sexually-transmitted disease gonorrhea. Although gonorrhea is one of the oldest described diseases, it remains a significant global problem with more than 100 million cases reported annually worldwide, with antibiotic resistance increasing at an alarming rate. Sulfonamides were used in the late 1930s and early 1940s to treat gonorrhea, but the rapid emergence of strains resistant to this class of drug resulted in the removal of sulfonamides from lists of recommended therapies. Therefore, elucidating the structures and mechanisms of the AbgT-family transporters will provide important new targets for the rational design of novel antibiotics to combat these bacterial infections.

 描述（由申请人提供）：我们的长期目标是阐明通过转运蛋白AbgT家族引起药物跨细胞膜转移的结构和基本机制。所提出的工作的主要目标是Alcanivorax borneuronensis YdaH和淋病奈瑟菌MtrF转运蛋白。大约有13，000个推定的转运蛋白已被确定。然而，还没有结构信息，甚至功能数据是最小的膜蛋白家族。据推测，AbgT家族转运蛋白通过输入分解代谢产物对氨基苯甲酰谷氨酸盐来产生这种必需维生素，从而有助于细菌叶酸合成途径。为了了解AbgT家族转运蛋白的结构和功能，我们最近克隆、表达、纯化并结晶了全长的A. borkanensis YdaH和N.淋病MtrF膜蛋白。我们还确定了这两个转运蛋白的X射线结构，分别在2.9和3.9 μ m的分辨率。结构显示，这两个转运蛋白组装为二聚体，其结构与所有其他转运蛋白家族不同。YdaH和MtrF都是碗状二聚体，其中充满溶剂的盆从细胞质延伸穿过膜双层的一半。YdaH和MtrF的单体含有九个跨膜螺旋和两个发夹。这些结构直接表明了一个合理的途径为底物运输。晶体结构，遗传分析和底物积累试验的组合表明，YdaH和MtrF都表现为出口商，能够从细菌细胞中去除叶酸代谢产物对氨基苯甲酸。基于药物敏感性和放射性转运测定的进一步实验数据表明，YdaH和MtrF都作为抗生素外排泵参与，重要地介导细菌对磺胺类抗代谢药物的耐药性。我们的假设是，许多这些AbgT家族转运蛋白作为出口商，从而赋予细菌耐药性磺胺。具体的目的是定义的结构和运输机制的AbgT家族的转运蛋白。我们将使用结构，生物化学和计算方法来确定YdaH和MtrF转运蛋白的分子机制。淋病奈瑟氏菌是一种专性人类病原体，是性传播疾病淋病的传染源。虽然淋病是最古老的疾病之一，但它仍然是一个重大的全球性问题，全球每年报告的病例超过1亿例，抗生素耐药性以惊人的速度增加。磺胺类药物在20世纪30年代末和40年代初被用于治疗淋病，但对这类药物耐药的菌株的迅速出现导致磺胺类药物从推荐治疗名单中删除。因此，阐明AbgT家族转运蛋白的结构和机制将为合理设计新型抗生素以对抗这些细菌感染提供重要的新靶点。