A conserved leucine occupies the empty substrate site of LeuT in the Na(+)-free return state.

Lina Malinauskaite,Azadeh Shahsavar,Birgit Schiøtt,Kasper Severinsen,Henriette Bjerregaard,Poul Nissen,Caglanur Sahin,Julie Grouleff,Steffen Sinning,Thomas Boesen,Pernille Noer,Saida Said

doi:10.1038/ncomms11673

Lina Malinauskaite, Azadeh Shahsavar + Show 10 more

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https://doi.org/10.1038/ncomms11673

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Abstract

Bacterial members of the neurotransmitter:sodium symporter (NSS) family perform Na+-dependent amino-acid uptake and extrude H+ in return. Previous NSS structures represent intermediates of Na+/substrate binding or intracellular release, but not the inward-to-outward return transition. Here we report crystal structures of Aquifex aeolicus LeuT in an outward-oriented, Na+- and substrate-free state likely to be H+-occluded. We find a remarkable rotation of the conserved Leu25 into the empty substrate-binding pocket and rearrangements of the empty Na+ sites. Mutational studies of the equivalent Leu99 in the human serotonin transporter show a critical role of this residue on the transport rate. Molecular dynamics simulations show that extracellular Na+ is blocked unless Leu25 is rotated out of the substrate-binding pocket. We propose that Leu25 facilitates the inward-to-outward transition by compensating a Na+- and substrate-free state and acts as the gatekeeper for Na+ binding that prevents leak in inward-outward return transitions.

Highlights

Bacterial members of the neurotransmitter:sodium symporter (NSS) family perform Na þ -dependent amino-acid uptake and extrude H þ in return
No structural information is available on the Na þ - and substrate-free return steps associated with the inward-to-outward transition of the NSS family
A highly flexible TM1b lines the extracellular cavity as indicated by relatively large B-factors of this region compared with the general structure (Fig. 1e) and as compared with the Na þ -bound states[2,3] (Supplementary Fig. 3)

Summary

Introduction

Bacterial members of the neurotransmitter:sodium symporter (NSS) family perform Na þ -dependent amino-acid uptake and extrude H þ in return. The main body of structural information for the NSS family comes from the Aquifex aeolicus amino-acid transporter LeuT with crystal structures of Na þ -bound, outward-oriented states[2], with bound substrates at a central S1 site[3] and inhibitors bound at a hydrophobic, extracellular cavity[4,5]. Current structural information depicts a consistent mechanism of Na þ -gradient-dependent binding and occlusion of extracellular Na þ and substrate followed by a switch to the inward-oriented state and intracellular opening and release of Na þ and substrate. No structural information is available on the Na þ - and substrate-free return steps associated with the inward-to-outward transition of the NSS family This transition is especially intriguing because it takes place without Na þ and substrate, so how will it proceed from the inward-open state and is it sensitive to the Na þ gradient?

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