EAGER: Mechanism of Energy Coupling with a Membrane Symport Protein

University Of California-Los Angeles, Los Angeles CA

Investigators

Abstract

The detailed mechanism of the transport of sugars, amino acids and other nutrients across cell membranes is an unsolved biological problem. This project focuses upon the lactose permease transporter (LacY) of the bacterium Escherichia coli. LacY transports a specific sugar molecule and a proton across the cell membrane and is typical of membrane proteins that catalyze the transport of nutrient molecules such as sugars against a concentration gradient. The goal of this project is to understand precisely how this coupled mechanism works. Current biochemical/biophysical studies provide supporting evidence for a mechanism in which sugar- and proton-binding sites of LacY gain alternating access to either side of the membrane as the result of global structural changes in the protein. Although alternating access is now generally accepted as the mechanism for membrane transport, the chemistry of coupling between sugar and proton transport remains unresolved. The current research project will utilize a number of Camelid nanobodies, which are a special form of antibody, to stabilize LacY in different intermediate states that will provide an in-depth understanding of the mechanism for the first time for this class of transport proteins. Understanding the molecular details of this mechanism will address an important issue for the function of living systems and provide a model for systematically dealing with structural determinations of membrane proteins that are difficult to study. The project will provide training and education to students at the graduate and undergraduate level. The aim of this research is to develop an atomic-level understanding of the mechanism of lactose/proton 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, as well as the demonstration that lactose/proton symport is driven thermodynamically by chemiosmosis, the mechanism of this chemiosmotic process is not completely understood. Thus, it has been demonstrated that sugar binding to highly dynamic and protonated LacY triggers a global conformational change in which sugar- and proton-binding sites gain alternating access to either side of the membrane, but it is apparent that sugar binding and dissociation drive this conformational change through an induced-fit mechanism, while the proton electrochemical gradient accelerates the rate of deprotonation. Therefore, LacY behaves much 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 alternating access mechanism will be studied by applying pre-steady state kinetics, as well as multiple biochemical and spectroscopic approaches pioneered in the PI's laboratory and by using kinetic data obtained in real time for several steps in the transport cycle. This research focuses on the use of Camelid nanobodies to stabilize LacY in different intermediate states to be studied by X-ray diffraction. These studies will provide an in-depth understanding of the symport mechanism.