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Transporter mechanism in sight

PSI-SGKB [doi:10.1038/fa_psisgkb.2009.4]
Featured Article - February 2009
Short description: Structural studies of the leucine transporter reveal details of the early steps in substrate transport across the membrane.Science 322, 1655-1661 (2008)

Secondary membrane transporters catalyse the movement of small molecules and ions across cellular membranes by linking substrate passage to transmembrane ion gradients. Although more than 200 different families of secondary transporters have been identified, with diverse protein sequences and a broad range of substrate and ion specificity, they probably work on common mechanistic principles.

These transporters lack a continuous pore for substrates to travel through, and so are thought to change conformation to allow substrates to access the transporter from each side of the membrane. The energy required is obtained from the potential energy stored in ion gradients across the membrane generated by primary, ATP-driven, transporters.

Several recently published structures of secondary transporters have shown that despite low protein sequence identity the core structure is very similar, and they may well share a similar mechanism. The first structure was of the leucine transporter LeuT, a prokaryotic member of the neurotransmitter sodium symporter (NSS) family.

Disruption of human NSS transporters is implicated in human disease, including depression, obsessive-compulsive disorder, epilepsy and autism. Their transport activity is inhibited by several classes of drugs, including tricyclic antidepressants, anticonvulsants and cocaine, but despite their importance, the molecular mechanism of substrate translocation and competitive inhibition has not been clear. Although earlier LeuT structures showed antidepressants binding in an extracellular pocket, or vestibule, they offered an explanation for noncompetitive inhibition only.

Singh et al. now present detailed X-ray crystallographic and functional studies on LeuT that identify a potential mechanism for substrate translocation. First, the authors identified tryptophan as a competitive inhibitor after screening a range of amino acids to find one that could displace leucine from the substrate site but could not be transported. They confirmed this observation using steady-state kinetic experiments.

Second, they examined the molecular basis of ligand specificity by co-crystallizing LeuT with six different amino acids. Five of these act as substrates, and when bound to the transporter, LeuT has the same outward-facing occluded conformation seen in the previously published structures, despite the variation in size of the substrates. As in the earlier structures, access from the extracellular side is blocked by just a few residues, and access from the intracellular side is blocked by almost 25 å of tightly packed protein.

But the tryptophan-bound complex has a different conformation. It is open to the outside of the cell, and it has a widened, solvent-accessible extracellular pocket where the substrate would normally bind. There are several differences between the open and the occluded state, including the rotation of three transmembrane helices 1b, 2a and 6a in the open state. In tryptophan's presence, the extracellular substrate pocket is unable to close, and therefore cannot form the occluded state necessary for transport.

Surprisingly, a second tryptophan-binding site is also observed. Two residues of the extracellular gate of LeuT —arginine 30 and aspartic acid 404 — form a low-affinity binding site for tryptophan. The authors suggest that this site is a temporary binding site for incoming amino acids before they move to the primary substrate site.

Singh et al. present a potential mechanism for LeuT in which incoming amino acids transiently bind to gating residues that are only accessible in the open-to-outside conformation. The substrate amino acid then moves to the primary binding site, and the gate residues then interact and close. This closure, or occlusion, seems to be required for the larger conformational change needed to produce the inward-facing conformation.

The structural similarities between unrelated secondary transporters make it possible that Singh et al. have uncovered principles of mechanism common to this group.

Maria Hodges


  1. S. K. Singh, C. L. Piscitelli, A. Yamashita & E. Gouaux A competitive inhibitor traps LeuT in an open-to-out conformation.
    Science 322, 1655-1661 (2008).

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