Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Erlendsson, Simon; Gotfryd, Kamil; Larsen, Flemming H.; Mortensen, Jonas S.; Geiger, Michel-Andreas; Rossum, Barth‐Jan van; Oschkinat, Hartmut; Gether, Ulrik; Teilum, Kaare; Løland, Claus J.

doi:10.7554/elife.19314

“…3D). Overall, our conclusions are consistent with other recent results that argue against substrate binding at the S2 site (26,27).…”

Section: Resultssupporting

confidence: 93%

“…Mutation of residues proposed to contribute to the S2 site, Ile111 and Leu400, inactivated transport, highlighting the importance of this region (19), and a Leu400 mutant was unable to respond to substrate-induced conformational changes (15). However, crystallographic studies have not detected substrate binding in that site (23)(24)(25) and more recent studies using MD simulation (26) and solid-state NMR (27) have argued against substrate binding in the S2 site.…”

Section: Introductionmentioning

confidence: 99%

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

Zhang

¹

,

Tavoulari

²

,

Sinning

³

et al. 2018

Preprint

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The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na + stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na + transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na + , allowing intracellular substrate and Na + release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na + on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456(JBC 291: , 2016. Here, we used cysteine accessibility to measure conformational changes of LeuT in E. coli membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na + , these results identify mechanistic components underlying ion-substrate coupling in NSS transporters. Significance StatementMembrane transport proteins are responsible for moving substrates such as nutrients, vitamins, drugs and signaling molecules across cellular membranes. A subset of these proteins, the ion-coupled transporters, use a transmembrane ion gradient to drive energetically unfavorable substrate movement from a lower concentration on one side of the membrane to a higher concentration on the other side. They do this by coupling the movement of substrate and ions in the same or the opposite direction. Coupled transport requires conformational changes that occur exclusively or predominantly when specific conditions of ion and substrate binding are met. This work identifies, for a family of Na + -coupled neurotransmitter transporters, how the rules controlling these conformational changes are encoded in the transporter structure.

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“…3C). Overall, our conclusions are consistent with other recent results that argue against substrate binding at the S2 site (34,35).…”

Section: Discussionsupporting

confidence: 93%

“…In addition, a Leu400 mutant was unable to undergo substrateinduced conformational changes (22). However, crystallographic studies have not detected substrate binding in that site (31)(32)(33), and more recent studies using MD simulation (34) and solid-state NMR (35) have argued against substrate binding in the S2 site.…”

Section: Significancementioning

confidence: 99%

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

Zhang

¹

,

Tavoulari

²

,

Sinning

³

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the neurotransmitter:sodium symporter (NSS) family, Na stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na, allowing intracellular substrate and Na release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein. Here, we used cysteine accessibility to measure conformational changes of LeuT in membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

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“…The dynamics of LeuT has been studied by electron paramagnetic resonance (EPR) [9,10], solid state NMR [11], fluorescence resonance energy transfer microscopy (FRET) [12][13][14][15][16], lanthanide resonance energy transfer (LRET) [17] and also by molecular dynamics (MD) simulations [15][16][17][18][19][20][21][22][23][24]. The human monoamine transporters have been extensively studied with respect to efflux of substrate [25][26][27][28].…”

Section: Introductionmentioning

confidence: 99%

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

Khan

¹

,

Sohail

²

,

Jayaraman

³

et al. 2019

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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.

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Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Cited by 16 publications

References 23 publications

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

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

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