EAGER: Molecular Mechanism of Permeases

EAGER：渗透酶的分子机制

• 批准号: 1747705
• 负责人: Ronald Kaback
• 金额: $ 30万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1747705&HistoricalAwards=false
• 关键词: EAGER; Molecular; Mechanism; Permeases

Transport of sugars, amino acids and other nutrients across cell membranes is an unsolved biological problem. This process is mediated by proteins. LacY is one of such proteins and it is involved in the transport of lactose and protons. LacY operates in a manner typical of membrane proteins that catalyze transport against a concentration gradient (i.e. active transport). In this process, in the presence of a proton motive force, downhill transport of the protons is used by LacY to drive uphill concentration of sugar against a gradient. The aim of this project is to understand precisely how this coupled mechanism works. The PI has obtained LacY crystal structures at the atomic level, and biochemical/spectroscopic studies show that sugar- and proton-binding sites gain alternating access to either side of the membrane as the result of reciprocal opening and closing of cavities on either side of LacY. This proposal will investigate structural aspects of LacY at different states of the transport cycle by using crystallographic approaches. This project will also provide training to undergraduate students and research associates by directly involving them in experimental aspects of the research. The aim of this research is to develop an atomic-level understanding of the mechanism of lactose/H+ symport by the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily (MFS), the largest family of membrane transport proteins. Members of the MFS are found in the membranes of all living cells. However, despite an increasing number of X-ray structures of MFS members, including 7 of LacY from the PI's laboratory, as well as the quantitative demonstration that lactose/H+ symport is driven thermodynamically by chemiosmosis, the mechanism of this chemiosmotic process is not completely understood. Thus, it has been demonstrated that galactoside binding to highly dynamic protonated LacY triggers a global conformational change in which sugar- and proton-binding sites gain alternating access to either side of the membrane, while the proton electrochemical gradient accelerates the rate of deprotonation, but has no effect on alternating access. Therefore, LacY behaves like an enzyme except that the transition state(s) involves the protein rather than the substrate. X-ray structures of LacY inward- and almost occluded outward-facing conformations provide the structural basis for studying the alternating access mechanism. The PI plans to study the alternating access mechanism by applying pre-steady state kinetics, as well as multiple biochemical and spectroscopic approaches, and by using kinetic data obtained in real time for several steps in the transport cycle. The PI plans to determine whether the findings regarding the effects of electrochemical gradient are generalized by determining whether exchange and counterflow are affected by this gradient with other MFS symporters. This research also focuses on the use of thirty-one camelid nanobodies to stabilize LacY in different intermediate states to be studied by X-ray diffraction. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.

糖、氨基酸和其他营养物质跨细胞膜的运输是一个尚未解决的生物学问题。这个过程是由蛋白质介导的。 LacY 就是此类蛋白质之一，它参与乳糖和质子的运输。 LacY 以膜蛋白典型的方式运作，催化逆浓度梯度的转运（即主动转运）。 在此过程中，在质子动力存在的情况下，LacY 利用质子的下坡传输来驱动糖浓度逆梯度上坡。该项目的目的是准确理解这种耦合机制的工作原理。 PI 获得了原子水平的 LacY 晶体结构，生化/光谱研究表明，由于 LacY 两侧空腔的相互打开和关闭，糖和质子结合位点交替进入膜的两侧。该提案将利用晶体学方法研究 LacY 在不同运输循环状态下的结构方面。该项目还将为本科生和研究助理提供培训，让他们直接参与研究的实验方面。本研究的目的是从原子水平上理解大肠杆菌乳糖通透酶 (LacY) 的乳糖/H+同向机制，LacY 是最大的膜转运蛋白家族主要促进子超家族 (MFS) 的范例。 MFS 的成员存在于所有活细胞的膜中。然而，尽管越来越多的MFS成员的X射线结构，包括PI实验室的7个LacY，以及定量证明乳糖/H+同向转运是由化学渗透热力学驱动的，但这种化学渗透过程的机制尚不完全清楚。因此，已经证明，半乳糖苷与高度动态质子化的 LacY 结合会引发整体构象变化，其中糖和质子结合位点交替进入膜的任一侧，而质子电化学梯度会加速去质子化的速率，但对交替进入没有影响。因此，LacY 的行为类似于酶，只是过渡态涉及蛋白质而不是底物。 LacY向内和几乎闭塞的向外构象的X射线结构为研究交替访问机制提供了结构基础。 PI 计划通过应用前稳态动力学以及多种生化和光谱方法，并使用传输周期中多个步骤实时获得的动力学数据来研究交替访问机制。 PI 计划通过确定与其他 MFS 同向转运蛋白的交换和逆流是否受到该梯度的影响，来确定有关电化学梯度影响的发现是否可以推广。这项研究还重点关注使用 31 种骆驼纳米体将 LacY 稳定在不同的中间态，并通过 X 射线衍射进行研究。该项目得到生物科学理事会分子和细胞生物科学部分子生物物理学集群的支持。

项目成果

Engineered occluded apo-intermediate of LacY

LacY 的工程化封闭脱辅基中间体

• DOI: 10.1073/pnas.1816267115
• 发表时间: 2018
• 期刊: Proceedings of the National Academy of Sciences
• 作者: Smirnova, Irina;Kasho, Vladimir;Kaback, H. Ronald
• 通讯作者: Kaback, H. Ronald

Oversized galactosides as a probe for conformational dynamics in LacY

• DOI: 10.1073/pnas.1800706115
• 发表时间: 2018-04-17
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Smirnova，Irina;Kasho，Vladimir;Kaback，H. Ronald
• 通讯作者: Kaback，H. Ronald