Structural mechanics of MsbA family ABC transporters

描述(申请人提供)：三磷酸腺苷结合盒(ABC)转运体代表了细胞最常用的分子结构，以三磷酸腺苷(ATP)为能源，驱动溶质跨膜的主动运输。ABC转运蛋白存在于所有活着的生物体中，是全序列微生物基因组中最大的基因家族之一。至少有一种细菌ABC转运蛋白被证明对大肠杆菌的生存是必不可少的，而其他的已经被证明是细菌致病因子。人类ABC转运蛋白的异常功能在多种疾病中起着核心作用，包括囊性纤维化、肾上腺脑白质营养不良、晚期肿瘤和白血病的多药耐药。我实验室之前的研究已经帮助确定了ABC转运蛋白中结构定型的ABC运动域利用ATP的结合和水解来执行机械工作的机制。利用酶学和结晶学相结合的研究，我们发现ATP与保守的序列基序结合推动ABC运动域形成“ATP-夹心二聚体”。这种由ATP诱导的二聚化被认为代表了泵的功率行程，因为在二聚体形成过程中ABC的物理运动被假设为在介导溶质运输的跨膜结构域中驱动必要的构象重排。我们建议扩展我们早期对ABC转运体马达结构域的机械力化学反应周期的研究，以表征驱动一类特定类型的ABC外排泵中的溶质运输的跨膜结构域中这些变构构象变化的结构细节。我们提出的研究涉及遗传学、酶学和结晶学的结合，以解决这一基本的生物物理问题。这项工作的成功完成将提供更深层次的结构和机制理解，这将促进对ABC转运蛋白超家族致病蛋白的分子药理学的研究。

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.