Structural mechanics of MsbA family ABC transporters

MsbA家族ABC转运蛋白的结构力学

• 批准号: 6969318
• 负责人: JOHN Francis HUNT
• 金额: $ 29.73万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6969318
• 关键词: Bacillus subtilis; Escherichia coli; SDS polyacrylamide gel electrophoresis; active sites; adenosine triphosphate; adenosinetriphosphatase; allosteric site; bacterial proteins; conformation; crystallization; enzyme activity; gel filtration chromatography; gene mutation; lipid transport; membrane transport proteins; method development; microorganism culture; phospholipids; protein protein interaction; protein purification; protein reconstitution; protein structure function; Bacillus subtilis; Escherichia coli; SDS polyacrylamide gel electrophoresis; active sites; adenosine triphosphate; adenosinetriphosphatase; allosteric site; bacterial proteins; conformation; crystallization; enzyme activity; gel filtration chromatography; gene mutation; lipid transport; membrane transport proteins; method development; microorganism culture; phospholipids; protein protein interaction; protein purification; protein reconstitution; protein structure function

DESCRIPTION (provided by applicant): ATP-binding cassette (ABC) Transporters represent the molecular architecture most commonly employed by cells to drive the active transport of solutes across membranes using adenosine triphosphate (ATP) as an energy source. ABC Transporters occur in all living organisms and comprise one of the largest gene family in fully sequenced microbial genomes. At least one bacterial ABC Transporter has been shown to be essential for the viability of E. coli, while others have been shown to function as bacterial pathogenicity factors. The aberrant function of human ABC Transporters plays a central role in a variety of diseases including cystic fibrosis, adrenoleukodystrophy, and multidrug resistance in advanced tumors and leukemias. Previous research from my lab has helped to define the mechanism by which the structurally-stereotyped ABC motor domains in ABC Transporters employ the binding and hydrolysis of ATP to perform mechanical work. Using combined enzymological and crystallographic studies, we showed that ATP binding to conserved sequence motifs drives the formation an "ATP-sandwich dimer" by the ABC motor domains. This ATP-induced dimerization is believed to represent the power-stroke of the pumps because the physical movements of the ABC's during dimer formation is hypothesized to drive the essential conformational rearrangements in the transmembrane domains that mediate solute transport. We propose to extend our earlier studies of the mechanochemical reaction cycle of ABC Transporter motor domains to characterize the structural details of these allosteric conformational changes in the transmembrane domains that drive solute transport in one specific class of ABC efflux pumps. Our proposed studies involve a comgination of genetic, enzymological, and crystallographic approached to this fundamental biophysical problem. Successful completion of this work would provide deeper structural and mechanistic understanding that would facilitate studies on the molecular pharmacology of the disease-causing proteins in the ABC Transporter superfamily.

描述（由申请人提供）：ATP结合盒（ABC）转运蛋白代表细胞最常用的分子结构，以使用Triphosphate（ATP）作为能源来驱动溶质跨膜的主动转运。 ABC转运蛋白发生在所有生物体中，并构成完全测序的微生物基因组中最大的基因家族之一。至少一种细菌ABC转运蛋白已被证明对于大肠杆菌的生存能力至关重要，而其他细菌则是细菌致病因素。人类ABC转运蛋白的异常功能在各种疾病中起着核心作用，包括囊性纤维化，肾上腺素肌营养不良和晚期肿瘤和白血病中的多药耐药性。我实验室的先前研究有助于确定ABC转运蛋白中结构型的ABC运动结构域采用ATP的结合和水解进行机械工作的机制。使用酶学和晶体学研究结合，我们表明ATP与保守序列基序的结合驱动了ABC运动域的“ ATP-Sandwich二聚体”的形成。据信这种ATP诱导的二聚化代表了泵的强力冲程，因为假设ABC在二聚体形成过程中的物理运动可以驱动介导溶质传输的跨膜域中的必要构象重排。我们建议扩展对ABC转运蛋白运动结构域的机械化学反应周期的早期研究，以表征跨膜结构域中这些变构构象变化的结构细节，这些变化构象变化在一类特定类别的ABC外排泵中驱动溶质转运。我们提出的研究涉及对这种基本生物物理问题的遗传，酶学和晶体学的复杂化。这项工作的成功完成将提供更深入的结构和机械理解，从而有助于研究ABC转运蛋白超级家族中引起疾病​​蛋白的分子药理学。