Structure and Function of a Phosphorylation Coupled Saccharide Transporter

描述（由申请人提供）：磷酸烯醇丙酮酸依赖的磷酸转移酶系统（PTS）是一个多组分碳水化合物摄取系统，也参与调节细菌的代谢、趋化性和致病性。PTS通过结合配体在膜上的易位及其伴随的共价磷酸化修饰来驱动糖的主动运输，以防止外排。近半个世纪以来，PTS一直是广泛研究的主题，但由于缺乏负责糖穿过内膜运输的完整膜成分EIIC的任何结构，我们对该系统的理解仍然不完整。EIICs还赋予PTS对特定糖的特异性，并协助磷酸从细胞质PTS蛋白EIIB转移到糖。我们打算通过结合ChbC的结构和功能研究来解决这一机制理解上的空白，ChbC是葡萄糖EIIC超家族的成员，对n - n '-二乙酰壳聚糖的摄取具有特异性。这种糖是由几丁质分解产生的，是霍乱弧菌等病原体生命周期中的重要营养物质。为此，我们已经解决了蜡样芽孢杆菌（bcChbC）的ChbC同源物的结构，这使我们提出了转运蛋白如何选择性地结合糖，将其跨膜转运，并协助磷酸化偶联转运的假设。我们将利用这种结构来了解EIIC的功能机制，有三个目的：(1)通过结合和摄取实验确定bcChbC对底物选择性的结构基础；(2)通过解析bcChbC及其伙伴EIIB、bcChbB的复合物的结构来揭示磷酸化机制；(3)通过解析bcChbC的外向开放态结构来重建转运循环下的构象变化。

DESCRIPTION (provided by applicant): The phosphoenolpyruvate-dependent phosphotranferase system (PTS) is a multicomponent carbohydrate uptake system that is also involved in the regulation of metabolism, chemotaxis, and pathogenicity in bacteria. The PTS drives active transport of sugar by coupling the translocation of the ligand across the membrane with its concomitant covalent modification by phosphorylation to prevent efflux. The PTS has been the subject of extensive study for nearly half a century, but our understanding of the system has remained incomplete due to the lack of any structures for the integral membrane component EIIC responsible for the transport of the sugar across the inner membrane. The EIICs also confer specificity for a particular sugar to the PTS, and assist in the transfer of the phosphate from the cytoplasmic PTS protein EIIB to the sugar. We intend to address this gap in the mechanistic understanding of the PTS by combining structural and functional studies of ChbC, a member of the glucose EIIC superfamily that is specific for the uptake of N.N'- diacetylchitobiose. This sugar is produced by the breakdown of chitin, and as an important nutrient in the life cycle of pathogens such as Vibrio cholerae. To this end, we have solved the structure of a ChbC ortholog from Bacillus cereus (bcChbC), which has led us to propose hypotheses for how the transporter selectively binds sugar, translocates it across the membrane, and assists in coupling phosphorylation to transport. We will use this structure to understand the mechanism of EIIC function with three aims: (1) to determine the structural basis of bcChbC's substrate selectivity with binding and uptake assays, (2) to uncover the mechanism of phosphorylation by solving the structure of an bcChbC in complex with its partner EIIB, bcChbB, and (3) to reconstruct the conformational changes underlying the transport cycle by solving the structure of the outward- facing open state of bcChbC.