The Amino Terminus of LeuT Changes Conformation in an Environment Sensitive Manner

Abstract

Neurotransmitter:sodium symporters are highly expressed in the human brain and catalyze the uptake of substrate through the plasma membrane by using the electrochemical gradient of sodium as the energy source. The bacterial homolog LeuT, a small amino acid transporter isolated from the bacteria Aquifex aeolicus, is the founding member of the family and has been crystallized in three conformations. The N-terminus is structurally well defined and strongly interacts with the transporter core in the outward-facing conformations. However, it could not be resolved in the inward-facing conformation, which indicates enhanced mobility. Here we investigate conformations and dynamics of the N-terminus, by combining molecular dynamics simulations with experimental verification using distance measurements and accessibility studies. We found strongly increased dynamics of the N-terminus, but also that helix TM1A is subject to enhanced mobility. TM1A moves towards the transporter core in the membrane environment, reaching a conformation that is closer to the structure of LeuT with wild type sequence, indicating that the mutation introduced to create the inward-facing structure might have altered the position of helix TM1A. The mobile N-terminus avoids entering the open vestibule of the inward-facing state, as accessibility studies do not show any reduction of quenching by iodide of a fluorophore attached to the N-terminus.

Conserved Lysine in transmembrane helix 5 is key for the inner gating of the LAT transporter BasC

“…The equivalent position to Ile7 in BasC is Leu53 in human LAT SLC7A5, which differs by 11.5 Å between the inward-open (Protein Data Bank, PDB ID 6IRS) (Yan et al , 2019) and outward occluded structures (PDB ID 7DSQ) (Yan et al , 2021). Moreover, smFRET studies of LeuT seeking to track the inner gate movement used a similar position in TM1a (Zhao et al , 2010; Tavoulari et al , 2016; Terry et al , 2018; Khan et al , 2020). In BasC, Ile7 in TM1a was combined with two other residues in the protein: Thr120 in TM4, forming the TM1a-TM4 pair, and Cys427, the only natural cysteine in BasC, in TM12, forming the TM1a-TM12 pair (Fig 1A).…”

“…Single-molecule Förster Resonance Energy Transfer (smFRET) (Ha et al, 1996;Lerner et al, 2018;Hellenkamp et al, 2018;Lerner et al, 2021;Agam et al, 2023) for example, has been successfully used to study the dynamics of membrane transporters at the molecular level (Bartels et al, 2021;Gouridis et al, 2015Gouridis et al, , 2015Dyla et al, 2017;Husada et al, 2018;Yang et al, 2018;de Boer et al, 2019;Lasitza-Male et al, 2020;Debruycker et al, 2020). Relevant to this work, smFRET could help to show that the LeuT transporter, a bacterial homolog of the neurotransmitter sodium symporter (NSS) family with APC fold (Yamashita et al, 2005), undergoes a rocking motion and switch from an outward-facing to an inward-facing conformation (Zhao et al, 2010(Zhao et al, , 2011Tavoulari et al, 2016;Malinauskaite et al, 2014;Terry et al, 2018;Khan et al, 2020).…”

“…Moreover, smFRET studies of LeuT seeking to track the inner gate movement used a similar position in TM1a (Zhao et al, 2010;Tavoulari et al, 2016;Terry et al, 2018;Khan et al, 2020). In BasC, Ile7 in TM1a was combined with two other residues in the protein: Thr120 in TM4, forming the TM1a-TM4 pair, and Cys427, the only natural cysteine in BasC, in TM12, forming the TM1a-TM12 pair (Fig 1A).…”

“…Single-molecule Förster Resonance Energy Transfer (smFRET) (Ha et al , 1996; Lerner et al , 2018; Hellenkamp et al , 2018; Lerner et al , 2021; Agam et al , 2023) for example, has been successfully used to study the dynamics of membrane transporters at the molecular level (Bartels et al , 2021; Gouridis et al , 2015, 2015; Dyla et al , 2017; Husada et al , 2018; Yang et al , 2018; de Boer et al , 2019; Lasitza-Male et al , 2020; Debruycker et al , 2020). Relevant to this work, smFRET could help to show that the LeuT transporter, a bacterial homolog of the neurotransmitter sodium symporter (NSS) family with APC fold(Yamashita et al , 2005), undergoes a rocking motion and switch from an outward-facing to an inward-facing conformation (Zhao et al , 2010, 2011; Tavoulari et al , 2016; Malinauskaite et al , 2014; Terry et al , 2018; Khan et al , 2020). The proposed rocking motion of the bundle domain (TM1, TM2, TM6 and TM7) versus the scaffold domain (TM3, TM4, TM8 and TM9) between outward-facing to inward-facing conformations is consistent with the general mechanism observed in other members of the APC transporter superfamily, including LAT structures (Errasti-Murugarren et al , 2019; Yan et al , 2019; Lee et al , 2019; Rodriguez et al , 2021; Parker et al , 2021; Yan et al , 2020a; Wu et al , 2020; Lee et al , 2022; Jeckelmann et al , 2022; Yan et al , 2020b, 2021; Lee et al , 2023).…”

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure–Function Relationships in Mammalian SLC6 Transporters

“…Since 2005 the structure of the bacterial amino acid transporter LeuTAa has been the main source of information regarding the architecture and structure-based mechanism of transport in a SLC6-type protein [19,58,[70][71][72]. All these data represent a milestone to understand the functionality of different mammalian neurotransmitter transporters and model their different states [73][74][75]. Different conformations of LeuTAa: substrate-free state [58,76,77], inward-open state [58,[78][79][80] and competitive [81] and non-competitive inhibitor- [58] bound states [18] have revealed many mechanistic structures for the transport and transport inhibition of neurotransmitters transport proteins [72].…”