Modeling and Dynamics of the Inward-Facing State of a Na+/Cl− Dependent Neurotransmitter Transporter Homologue

The leucine transporter (LeuT) has recently commanded exceptional attention due mainly to two distinctions; it provides the only crystal structures available for a protein homologous to the pharmacologically relevant neurotransmitter: sodium symporters (NSS), and, it exhibits a hallmark 5-TM inverted repeat (“LeuT-fold”), a fold recently discovered to also exist in several secondary transporter families, underscoring its general role in transporter function. Constructing the transport cycle of “LeuT-fold” transporters requires detailed structural and dynamic descriptions of the outward-facing (OF) and inward-facing (IF) states, as well as the intermediate states. To this end, we have modeled the structurally unknown IF state of LeuT, based on the known crystal structures of the OF state of LeuT and the IF state of vSGLT, a “LeuT-fold” transporter. The detailed methodology developed for the study combines structure-based alignment, threading, targeted MD and equilibrium MD, and can be applied to other proteins. The resulting IF-state models maintain the secondary structural features of LeuT. Water penetration and solvent accessibility calculations show that TM1, TM3, TM6 and TM8 line the substrate binding/unbinding pathway with TM10 and its pseudosymmetric partner, TM5, participating in the extracellular and intracellular halves of the lumen, respectively. We report conformational hotspots where notable changes in interactions occur between the IF and OF states. We observe Na2 exiting the LeuT-substrate- complex in the IF state, mainly due to TM1 bending. Inducing a transition in only one of the two pseudosymmetric domains, while allowing the second to respond dynamically, is found to be sufficient to induce the formation of the IF state. We also propose that TM2 and TM7 may be facilitators of TM1 and TM6 motion. Thus, this study not only presents a novel modeling methodology applied to obtain the IF state of LeuT, but also describes structural elements involved in a possibly general transport mechanism in transporters adopting the “LeuT-fold”.Author Summary: Elucidating the mechanism of active transport across the membrane is relevant not only to the understanding of physiological processes but also to the rational design of drugs that modulate these processes. In the cell membrane, specialized proteins known as secondary transporters utilize the energy stored in the electrochemical gradient of ionic species across the membrane in order to carry out active transport. The leucine transporter is such a secondary transporter, with the unique distinction of being homologous to clinically relevant neurotransmitter transporters, and also similar in architecture to several other secondary transporters that are unrelated by sequence. This similarity establishes the significance of the typical “LeuT-fold” in secondary transporter function. In this study, we set forth to model and study the dynamics of LeuT in an alternative conformational state of the transport cycle, for which no crystal structure is known. A novel methodology is developed, yielding models of the inward-facing state of LeuT. We discuss several key features of this state, including structural elements and interactions that participate in the transition to this state. The study thus enhances the understanding of the transport mechanism of several families of “LeuT-fold” transporters, most including known and putative drug targets.