EAGER: Molecular Mechanism of Permeases

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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.