How did the substrate cross the membrane? Structural studies of the human erythrocyte anion exchanger and the Escherichia coli glycerol-3-phosphate transporter

Cellular viability relies on the transport of solutes across the lipid bilayer. This function is carried out by transporters with multiple membrane-spanning domains. For secondary active transporters, downhill transport along an electrochemical gradient provides the energy to transport a second substrate uphill against its concentration gradient. The focus of this study was to understand the mechanism of secondary active transport via structural analysis. Two secondary active transporters were examined, both of which are antiporters that exchange two substrates in opposite directions across the membrane.;The human anion exchanger is a member of the anion exchanger family of transporters. Abundant in the erythrocyte, this antiporter assists in carbon dioxide transport by exchanging HCO3- for Cl- ions. The membrane domain of the human erythrocyte anion exchanger (AE1MD) was purified to homogeneity and characterized in the presence of various detergents. Three-dimensional crystallization trials with AE1MD yielded crystals that diffracted X-rays to 14 A. Delipidation and detergent were important factors contributing to AE1MD crystallization.;The E. coli glycerol-3-phosphate transporter, GlpT, couples the efflux of phosphate to the uptake of glycerol-3-phosphate. This transporter is a member of the major facilitator superfamily, MFS. GlpT was overexpressed to homogeneity and characterized in various detergents. GlpT was monomeric in detergent solution and retained transport activity when reconstituted into lipid vesicles. GlpT was subjected to three-dimensional crystallization using an efficient homemade screen. Optimization of cell culture, purification, expression construct, and detergent yielded GlpT crystals diffracting X-rays to 3.0A. The amount of endogenous lipid co-purifying with GlpT provided evidence for the importance of delipidation in membrane protein crystallization. Selenomethionine substituted GlpT also yielded crystals that diffracted X-rays to 3.5A yet no phasing information was obtained. Initial attempts with heavy metals did not yield derivatized GlpT crystals for phasing. While no structural information was obtained, the GlpT transporter was probed with proteases in order to identify protease-sensitive conformations in the presence and absence of substrates. A loop connecting transmembrane segments 6 and 7, conserved amongst MFS transporters, was flexible during substrate binding. This loop may participate in the translocation of substrates, leading us to propose a model for antiport by GlpT.