Transport proteins are the gatekeepers of cells and cell organelles, and are involved in cellular nutrition, ionic homeostasis, disposal of waste products and uptake of certain drugs. Dysfunction of these membrane proteins leads to a number of serious human diseases. Yet the structural information required to help us understand cellular transport and genetic disorders, and design effective drugs is lacking. High-resolution structures of pro- and eukaryotic transport proteins, and indeed of membrane proteins in general, are sparse compared to structures of water-soluble proteins. The reasons for this are mainly attributed to the amphiphilic nature of membrane proteins and the difficulty to overexpress them heterologously.
This Swiss National Science Foundation (SNSF) project proposal aims to elucidate the structure, the conformational changes upon substrate binding and the supramolecular organization of selected prokaryotic and mammalian transport proteins by X-ray crystallography, cryo-transmission electron microscopy and atomic force microscopy. Certain projects are continuations of our previous SNSF proposal (No. 31003A_125150), while others are new. Promising preliminary results are presented and form the basis of this new SNSF proposal.
Our target transport proteins are prokaryotic and human members of the amino acid/polyamine/organocation superfamily, including two members of the L-type amino acid transporter (LAT) subfamily, the glucose phosphoenolpyruvate: carbohydrate phosphotransferase system transporter from Escherichia coli, and the potassium-chloride cotransporter KCC4 from mouse. We address important open questions from the membrane transport protein field and aim to answer them by determining the low-, medium- and high-resolution structures of our target proteins. These questions include the structures and conformations of antiporters in their native environment, the lipid bilayer (in contrast to detergent-solubilized proteins), the structure and supramolecular organization of human heteromeric amino acid transporters (HATs), the molecular basis of glucose binding and transport, and the supramolecular structure of mammalian cation-chloride cotransporters.
Knowledge of these target structures will increase our general understanding of transporter organization and molecular transport mechanism. Furthermore, 3D structures at high-resolution, e.g. of human HAT and prokaryotic LAT, which has high amino acid identity to human LATs, will facilitate the development of new inhibitors for pharmaceutical applications by structure-based drug design.