An integrated approach to understanding the molecular mechanisms of cation-coupled transport

Project Summary/Abstract: Cation-coupled secondary active transporters play critical roles in many aspects of cell physiology, and pharmacokinetics, and are also involved in many serious diseases. Based upon decades of effort in structural biology and biochemistry, we now know the architecture, folding, substrate binding, and global conformational change of many transporters. However, for many transporters, we still have a deeply incomplete understanding of transport mechanisms. Critically, many important mammalian transporters remain structurally and functionally uncharacterized. My laboratory has been working on fundamental questions of cation-coupled sugar symport using a prototype bacterial transporter, the Na+-coupled melibiose symporter MelB. In the past 5 years, we have achieved important milestones, including the determination of MelB structures in two distinct kinetic states with bound sugar or Na+ and the construction of novel mechanisms of symport utilizing structural, functional, biochemical, and biophysical methods. By further applying and extending these successful methods in the next 5 years, we will expand our research to include the study of the mammalian Na+-coupled epithelial amino acid co-transporter SLC6A14, involved in several chronic diseases, including cancer. Significantly, we have made critical breakthroughs and have succeeded in the expression, purification, and functional reconstitution of SLC6A14. The use of purified SLC6A14 is a critical complement for validating the published data from cell-based assays where other amino acid transporters may be present. For cryoEM-single particle analysis, we will develop suitable fiducial tools to increase structure determination throughput; these tools will be of great utility to the more general membrane protein structural biology community. Our SLC6A14 structures, combined with results from other approaches we regularly utilize, will provide insight into its broad substrate specificity and inhibitory mechanisms by its inhibitor, an important input for potential inhibitor development. For MelB, we will further examine our proposed cooperative binding-based core symport mechanism by determining missing key structure(s) and analyzing conformational dynamics using hydrogen/deuterium exchange-mass spectrometry and molecular dynamics simulations. We will stabilize MelB at specific state(s) using nanobodies or binding proteins obtained from different scaffolds and will determine their structures by cryoEM. Biochemically, these stabilized states will uniquely enable a better understanding of relationships between substrate binding and conformational dynamics. The expected new structures and functional studies of both Na+-coupled nutrient symporters SLC6A14 and MelB will provide critical novel knowledge about the molecular basis of ion-coupled transport and coupling mechanisms. This research can significantly impact the development of new therapeutic strategies for a broad range of chronic diseases including cancer. 1