Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation

Abstract

The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na ؉ ; later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule Ϸ11 Å above the bound leucine and 2 Na ؉ . We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na ؉ -coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-␤-D-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 Å the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na ؉ -coupled symport.crystal ͉ molecular dynamics simulation ͉ neurotransmitter:sodium symport ͉ second substrate (S2)-binding site ͉ scintillation proximity binding assay

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

“…Taken together, our findings provide more evidence against the supposition that there is a second or S2 high-affinity substrate binding site in LeuT [18][19][20][21][22]. The results of the Javitch group related to the S2 site, the properties of the F253A mutant and the role of the putative S2 site in the mechanism of LeuT and NSSs are, at present, without satisfactory explanation.…”

“…Crystal structures of LeuT in detergent micelles [6,14,15] and in lipid bicelles [16], together with ligand binding studies [17], reveal a single high-affinity substrate binding site, termed the S1 site. By contrast, molecular dynamics, substrate binding and singlemolecule experiments have been interpreted in terms of a model in which there is a second, or S2, high-affinity substrate binding site [18][19][20][21][22]. In an effort to disrupt substrate binding to the S1 site, Javitch and colleagues [18,21] studied the F253A mutant, a residue that lines a portion of the S1 binding site [6,14].…”

Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway

“…The increased reactivity of F556C and A330C/F556C to the bifunctional MTS reagent in the presence of 5-HT is consistent with the proposed conformational shifts in TMHs I, II, VI, and VII associated with an inward facing conformation (27). Interestingly, a recent report identifying a potential second substrate binding site on the dopamine transporter suggests a similar conformational shift for TMHs I and V (32).…”

Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP

“…The functional role of EH2 in BetP is unknown. In LeuT, a phenylalanine in the corresponding EH2 segment (EL4) was proposed to contribute to the formation of a periplasmic second substrate-binding (S2) site (44). The S2 site is assumed as being transiently occupied during translocation as the substrate moves towards the primary substrate-binding (S1) site (45).…”