Biological membranes define the boundaries of cells and organelles. The lipid bilayer sets a hydrophobic barrier that insulates the cellular or organelle contents from the surrounding environment. Although some low-molecular-weight chemicals can permeate the membrane, most of the water-loving chemicals, such as sugars, amino acids, and ions, require specific transport proteins to traffic through the hydrophobic lipid membrane. Transport proteins play an essential role in a broad spectrum of cellular activities, such as uptake of nutrients, release of metabolites, and signal transduction. Many diseases are associated with malfunction of membrane transport proteins, which have become direct targets for some widely prescribed drugs, such as antidepressants and painkillers. Therefore, structural and mechanistic investigation of transport proteins will shed light on the understanding of fundamental biology and facilitate potential clinical applications.
Membrane crossing is a process accompanied by energy conversion. Ions and chemicals are distributed asymmetrically across the membrane, establishing and maintaining electrochemical potential. Of all the transport proteins, I am particularly interested in the secondary active transporters, which exploit the electrochemical potential to shuttle a variety of substrates against their concentration gradient. Previous work proposed an alternating-access model as a general mechanism for transport proteins. In this model, to upload and release substrate, a transporter adopts at least two conformations: one open exclusively to the outside (outward-open) and the other to the inside of the membrane (inward-open). Several lines of structural and biophysical evidence supported this model. Nevertheless, two fundamental questions remain: First, what is the energy-coupling mechanism for the active transporters? Second, what triggers the obligatory conformational change of the transporter during the transport cycle?
To address these questions, my laboratory has instigated a systematic, structure-based investigation of the secondary active transporters. Our specific aims include the following:
- Determine the crystal structures of representative secondary active transporters, preferably in different conformational states, namely, outward-open, inward-open, ligand-free, ligand-bound, and so on.
- Perform structure-based functional analyses to identify key residues involved in substrate recognition, energy coupling, and conformational switch.
- Integrate various approaches to understand the dynamic nature of the transport cycle for representative transporters and to dissect their energy-coupling mechanisms.
- Investigate the modulation of membrane transport proteins by lipids.
In the past three years, we determined the atomic structures of two proton:nutrient symporters, the L-fucose transporter FucP and the uracil transporter UraA. The proton gradient generated from the electron transport chain, also known as the proton-motive force, was used to shuttle substrates across membrane by the proton symporters. The fucose transporter FucP and uracil transporter UraA belong to distinct transporter families that share no sequence or structure similarity. Yet structure-based functional analyses of these two unrelated transporters revealed a remarkable common feature: conserved glutamate/aspartate/histidine residues along the transport path are involved in substrate binding and proton translocation. Protonation/deprotonation as well as substrate binding may subsequently trigger the conformational change of the proteins. This discovery provides an important framework for understanding the energy-coupling mechanism of proton symport, which also provides insight into understanding the mechanisms of secondary active transporters in general.
Despite identification of the essential residues involved in substrate recognition and proton translocation, our structures provided only static images of FucP and UraA, representing a starting point to understand the transport process and mechanism. We would like to visualize the complete transport cycle. The conventional approach by x-ray crystallography is insufficient to decipher how proton translocation is coupled to substrate transport and what triggers the conformational change of the transporter protein. Therefore, we are combining various approaches, including computational tools and spectroscopic approaches, to dissect the transport process of the representative transporters and to elucidate the energy-coupling mechanism.
My lab will also explore the structure and functional mechanism of the physiologically significant glucose transporters (GLUTs) in mammals.
Glucose is an essential energy source for cells in species ranging from bacteria to mammals. GLUTs mediate glucose uptake, through either sodium-driven active transport (sodium-dependent glucose transporters, or SGLTs) or facilitated diffusion (GLUTs). It is well documented that abnormality in the regulation or function of GLUTs may lead to deleterious diseases such as type 2 diabetes, Fanconi–Bickel syndrome, and cancers. Therefore, GLUTs, in particular GLUT1 and GLUT4, have been under intense investigation in the past six decades. Unfortunately, no atomic resolution structure of any mammalian GLUTs is available to date. GLUTs belong to the major facilitator superfamily. Although the structures of five major facilitator superfamily members, LacY, GlpT, EmrD, FucP, and PepTSo, have been determined, none shares significant sequence similarity with the GLUT proteins. The lack of structural information severely restricts our understanding of this essential class of transporters. We are interested in structure-based functional characterizations of the mammalian GLUTs, especially the physiologically significant GLUT1–4. We wish to reveal the molecular basis of substrate recognition and to supply structural information for potential therapeutic applications. During this process, we hope to accumulate experience and to develop novel approaches to the overexpression, purification, and structural study of eukaryotic membrane proteins, which represents a serious challenge in structural biology.
Finally, we would like to understand the modulation of structure and function of membrane proteins by lipids. All the structures obtained in my laboratory so far are of proteins purified in the presence of detergents. Purifying and crystallizing proteins destroyed the native environment for membrane proteins, the lipid bilayer. The surrounding lipids are integral components for the structure and function of membrane proteins, and the composition of lipids is thought to play an essential role for the function of some membrane transport proteins. We would like to understand how lipid molecules modulate membrane proteins, which involved a combination of various biochemical and biophysical approaches, and even the development of novel techniques.
This work is partially supported by grants from the National Natural Sciences Foundation of China, Ministry of Science and Technology of China, and Tsinghua University.
As of January 17, 2012