Microseconds simulations reveal a new sodium-binding site and the mechanism of sodium-coupled substrate uptake by LeuT.
Bottom Line: We present here results from extensive (>20 μs) unbiased molecular dynamics simulations generated using the latest computing technology.Significantly, Na(+) binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1″, near S1, as an intermediate state.A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na(+) to Na1″, translocation to Na1, a second Na(+) to vacated Na1″ and then to Na2, and substrate to S1; (ii) rotation of Phe(253) aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule.
Affiliation: From the Department of Computational & Systems Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 email@example.com.Show MeSH
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Mentions: Previous studies pointed to the role of hydration in substrate release (49), to passive water conduction of transporters in general (45), and to the distinctive hydration patterns of LeuT OF- and IF states of LeuT (48). To assess the role of hydration in the opening of the substrate-binding pocket, we thoroughly examined the number of water molecules that entered the substrate-binding pocket as a function of the level of separations of TM1b/TM6a from TM10. Trajectories initiated from the occluded conformations (runs 3–4 and 7–9) were analyzed to this end. Results (Fig. 6, A–C) show that prior to EC vestibule opening (ordinate), the initial event is the influx of water molecules: the number of water molecules in the EC vestibule increases from 15–20 to 30–35, which then triggers the distinctive movements of TM1b (upper trajectory in each panel) or TM6a (lower trajectory) away from TM10 by 3–4 Å. In the presence of Leu (Fig. 6D), the opening of the EC vestibule starts with fewer (∼15–20) water molecules. This opening further promotes the influx of water, up to ∼40 water molecules in Na1-bound LeuT and ∼55 in Na1′- and/or Na1″-bound LeuT.
Affiliation: From the Department of Computational & Systems Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 firstname.lastname@example.org.