Structure and mechanism of the AbgT-family transporters

 描述(由申请人提供)：我们的长期目标是阐明引起AbgT转运蛋白家族跨细胞膜药物转移的结构和基本机制。拟议工作的主要目标是博尔库姆链球菌YdaH和淋病奈瑟菌mtrF转运蛋白。已经确定了大约13,000名该家族的推定运输者。然而，对于这个膜蛋白家族，目前还没有结构信息，甚至功能数据也很少。有人假设，AbgT家族转运蛋白通过引入分解代谢产物对氨基苯甲酰谷氨酸来产生这种必需的维生素，从而参与细菌叶酸的合成途径。为了了解AbgT转运蛋白家族的结构和功能，我们最近克隆、表达、纯化和结晶了博尔库姆支原体YdaH和淋球菌MtrF全长膜蛋白。我们还测定了这两个转运体的X射线结构，其分辨率分别为2.9和3.9。这些结构表明，这两个转运蛋白以二聚体的形式组装在一起，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，有一个充满溶剂的盆，从细胞质延伸到膜双层的一半。YdaH和MtrF的单体含有9个跨膜螺旋和2个发夹。这些结构直接暗示了底物运输的一条可能的途径。结合晶体结构、遗传分析和底物积累分析表明，YdaH和MtrF都具有出口作用，能够从细菌细胞中去除叶酸代谢物对氨基苯甲酸。基于药物敏感性和放射性转运分析的进一步实验数据表明，YdaH和MtrF都参与了抗生素外排泵，重要地介导了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家族的转运蛋白作为输出物，从而赋予细菌对磺胺类药物的耐药性。具体目的是确定AbgT转运蛋白家族的结构和转运机制。我们将使用结构、生化和计算方法来确定YdaH和MtrF转运蛋白的分子机制。淋病奈瑟菌是人类的必备病原体，也是性传播疾病淋病的感染源。虽然淋病是被描述的最古老的疾病之一，但它仍然是一个重大的全球问题，每年全球报告的病例超过1亿例，抗生素耐药性以惊人的速度增加。20世纪30年代末和40年代初，磺胺类药物被用于治疗淋病，但对这类药物产生抗药性的菌株迅速出现，导致磺胺类药物从推荐治疗列表中删除。因此，阐明AbgT家族转运蛋白的结构和机制将为合理设计抗细菌感染的新型抗生素提供重要的新靶点。

 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.