Active transport driven by electrochemical ion gradients (i.e., secondary active transport) is a fundamental biological process found in all life forms that plays an essential role in many aspects of cell function, such as nutrient uptake, signal transduction and resistance to noxious components in the environment. The aim of this project is to obtain an x-ray structure of the melibiose permease of Escherichia coli (MelB). Recently, a breakthrough was achieved in this general area. X-ray structures of two secondary transporters, LacY and the GlpT, were determined. However, there is no x-ray crystal structure available for any Na+-coupled secondary transport protein. MelB consists of 469 amino acid residues and is a biochemically well-studied member of the glycoside-pentoside-hexuronide:cation symport family. MelB catalyzes the accumulation of galactopyranosides by utilizing the free energy from the energetically downhill inward movement of Na+, Li+ or H+ to drive stoichiometric uphill transport of galactosidic sugars, and the coupling ion is dependent on the nature of the sugar transported. Generally, alpha-galactosides (melibiose, raffinose and p-nitrophenyl-alpha-galactoside) are symported with either H+ or Na+, while beta-galactosides (lactose, methyl-1-D-galactopyranoside or p-nitrophenyl-beta-D-galactoside) are symported with Na+ but not with H+, which makes MelB a highly unique transporter. Like LacY, MelB appears to contain 12-transmembrane domains with the N- and C-termini facing the cytoplasm. 2D crystals have been obtained, and a projection map at low resolution displays a molecule consisting of two domains lining a central cleft, similar to the overall structure of LacY; however, neither the location of the sugar nor the cation binding sites are resolved, making the mechanism of Na+-driven transport unresolved. Lately, manipulation of the concentration of phospholipids during co-crystallization has led to reproducible crystals of LacY that diffract to higher resolution, as well as a number of important mutants defective in H+ translocation. By using the approaches described, initial crystals of MelB have been obtained. The structure anticipated will provide important insights. Cocrystallization will also be attempted with IIA-Glc, a soluble regulatory component of the phosphoenolpyruvate:sugar phosphotransferase system that binds to MelB and LacY and co-crystallizes with glycerol kinase.
Membrane proteins, particularly those that catalyze ion-coupled transport, are notoriously difficult to crystallize presumably because of their hydrophobic nature and conformational flexibility. This is reflected by the fact that there are some 40,000 structures of soluble proteins in the Protein Data Bank (PDB), but only about 45 independent membrane protein structures. Understanding of the mechanism of the cation coupled transport is a major challenge in the field of bioenergetics. This project extends a recent breakthrough, and these activities are expected to lead to an x-ray structure of a unique transporter that utilizes either H+ or Na+. The expected results will significantly improve overall knowledge of active transport and bioenergetics. In addition, manipulation of phospholipid concentrations to obtain and improve the quality of membrane protein crystals is a novel approach and further characterization of the phospholipid effect may provide a basic guideline for a general method of membrane protein crystallization, which remains a major barrier in structural biology.
Broader Impacts: Structure/function studies on LacY, an obligate H+/sugar symporter, have served as a model for studies on active transport and bioenergetics. These studies have been widely selected for inclusion in various textbooks, reference books and teaching materials in many languages for both undergraduate and graduate teaching worldwide. The project will involve outreach to high school and college audiences in order to convey scientific knowledge to young people and stimulate their interest in basic science. The progress expected will directly provide databases for public access by submission of original data to the PDB. Invited lectures, oral presentations at symposia, multi-disciplinary conferences held around the world will serve as multiple channels to convey this novel knowledge to society in a timely manner.
