To be, or not to be two sites: that is the question about LeuT substrate binding

Abstract

Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.

Figure 1.

Putative mechanisms for substrate transport by LeuT. (A) Classical alternating access mechanism for LeuT. Sodium ions (red spheres) and the amino acid substrate (green rhombus) bind to a central binding pocket to form a complex with the transporter. A conformational change closes external access and opens a path to the inside. After dissociation of substrates, the empty transporter undergoes a conformational change to regenerate the outward facing conformation. (B) The mechanism by Shi et al. (2008) requiring binding of two substrate molecules. This cartoon is strictly based on the model proposed by Shi et al. (2008; Fig. 7 is adapted with permission from Molecular Cell).

Figure 2.

LeuT substrate and inhibitor binding sites. x-ray crystal structures of LeuT in complex with leucine (PDB accession no. 2A65; A), leucine and OG (3GJC; B), and leucine and clomipramine, a TCA (2Q6H; C). The ligands in the structures are represented as spheres and the residues forming the S2 site are shown in blue. Residues Y108 and F253, which are occluding leucine bound to S1 from the extracellular solution, are shown in orange. The broken lines pass through S1 and S2 to indicate the position in the binding sites.