Abstract
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”.
Highlights
Leucine transporter (LeuT) is a bacterial amino acid transporter, homologous to Naz/Cl{ dependent neurotransmitter transporters of the solute carrier 6 (SLC6) family, which is known as the neurotransmitter:sodium symporter (NSS) family [1,2,3]
It is possible that newly formed interactions with TM7 may play a favorable role in the vertical movement of TM5, which brings the initially distant residue E192 close to the IC lumen. The significance of this motion is discussed. These results suggest that all 10 helices of the LeuT fold, as well as several intermediate loop regions, participate in the conformational changes involved in the OF-to-IF transition observed in targeted molecular dynamics (TMD)-1eq and TMD-2eq
Incorporating dynamics in the method has resulted in revelation of novel functionally-relevant features of ‘‘LeuT-fold’’ transporters
Summary
Leucine transporter (LeuT) is a bacterial amino acid transporter, homologous to Naz/Cl{ dependent neurotransmitter transporters of the solute carrier 6 (SLC6) family, which is known as the neurotransmitter:sodium symporter (NSS) family [1,2,3] This family of transporters includes several clinically relevant drug targets for neurological conditions such as depression, ADHD, anxiety and drug abuse; e.g., transporters for serotonin (SERT), dopamine (DAT), norepinephrine (NET) and c-aminobutyric acid (GAT-1) [4,5,6]. The first structure of LeuT [2] (Fig. 1) presented a high resolution (1.65 A ) picture to complement several earlier functional studies on NSS family members that revealed residues involved in gating, and in binding and translocation of the ions and the substrate/inhibitor [12,13,14,15,16]. Notable computational studies taking advantage of these structures include the prediction of the Cl{ binding site in SERT [22] and GAT-1 [23], the prediction of a model for the inward-facing conformation of LeuT [20], prediction and confirmation of a second substrate binding site in the extracellular lumen [10,24,25], and computational simulations designed to probe substrate/ion binding [26,27,28] and the putative transport mechanism [24,28]
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