Ion-controlled conformational dynamics in the outward-open transition from an occluded state of LeuT

By Chunfeng Zhao, Sebastian Stolzenberg, Luis Gracia, Harel Weinstein, Sergei Noskov, and Lei Shi.

Published in Biophysical Journal, 103 (5): 878-888 (2012). PMID: 23009837. PMCID: PMC3433624. Link to Pubmed page.

Project: The Transport Cycle in Neurotransmitter Uptake Systems

Figure 1. Structural elements correlated with the conformational changes along the first principal component vector (PC1). (A) The conformational transition observed in the MD1 simulation is represented as a superposition of 15 frames, selected equidistantly along the γ1(t) of MD1, from the occluded (dark green) to the outward-open conformation (gray). The eigenvectors of PC1 of MD1 and MD2 are also visualized in Movie S1 and Movie S2, respectively. (B and C) Zoomed-in views of the region near the Na1 site in the most occluded (B) and most open (C) frames in panel A. Water molecules are represented as red beads. In panel C, nonbonded interactions between Asn-27 and Na+ are marked by dashed lines. (D) Distribution of the HAs and HDs that are correlated with the γ1(t) of MD1. The top 40 HA or HD segment pairs that correlated directly (positive values, green) and inversely (negative values, orange) are mapped onto an outward-open LeuT model viewed from the extracellular side. The bars connect the centers of mass of the segment pairs; the radii of the spheres are drawn in proportion to the frequencies of the involved segments. The most frequently involved segments are EL3b and TM6a (green) and TM11 (orange). (E and F) Distributions of 40 HAs and HDs that are most correlated with the Na1 and Na2 binding enthalpies, respectively. The viewing angle, representation, and color schemes are the same as panel A. No HA or HD that is correlated with the Na2 enthalpy involves TM10a (dotted circle in F). This is a distinct difference from the correlation patterns observed for the Na1 enthalpy and γ1(t). Correlation coefficients are given in Table S2.


Neurotransmitter:sodium symporter (NSS) proteins are secondary Na+-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs—Leucine Transporter (LeuT)—and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na+ binding site (Na1 site). In the course of MD simulations starting from the occluded state with bound Na+, but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu-290. Consistently, the Na+ affinity for the Na1 site is inversely correlated with the extent of outward-open character and is lower than in the occluded state with bound substrate; however, the Na1 site retains its selectivity for Na+ over K+ in such conformational transitions. To the best of our knowledge, our findings shed new light on the Na+-driven transport cycle and on the symmetry in structural rearrangements for outward- and inward-open transitions.


Figure 1. Analysis of the conformational transition in the Na+-only trajectory

Figure 3. Na+ binding and selectivity in the Na1 site.