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

Abstract

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.

Locking Two Rigid-body Bundles in an Outward-Facing Conformation: The Ion-coupling Mechanism in a LeuT-fold Transporter

“…Whether this thermodynamic impact is a general effect within the LeuT-fold superfamily is still controversial. The studies on LeuT consistently suggest Na + binding stabilizes the OF conformation 36,50,54,58–60 , whereas experimental measurements on Mhp1 show that Na + has little effect on the conformational equilibrium 45,47 . Different from previous experimental studies on Mhp1, our computational results indicate that Mhp1 might still benefit from a similar Na + -binding effect as LeuT by stabilization of the OF conformation.…”

“…Probably the contribution made by Na + binding to the OF state is still much less than the free energy difference between IF and OF states, thus Na + binding cannot significantly shift Mhp1 conformational equilibrium. Above all, to clarify this discrepancy between LeuT and Mhp1 reports 36,45,47,50,54,58,59 , characterization of the free energy profiles along the OF ↔ IF transition pathway in the presence/absence of Na + would be required for multiple LeuT-fold transporters.…”

“…The structural commonalities among LeuT-fold transporters, i.e., same 5-helix inverted repeats, similar substrate-binding sites, and conserved Na + -binding sites, strongly suggest a similar mechanism of transport 5,6,11 . However, some of the recent studies have proposed completely different ion-coupling mechanisms for different transporter families within the LeuT-fold superfamily 36,44,45,47,50,54,58–60 . A number of studies on LeuT (NSS family) using smFRET, EPR, cysteine accessibility measurements, or hydrogen/deuterium exchange mass spectrometry indicate that Na + binding stabilizes the OF conformation 36,50,54,58–60 , and that such an effect requires binding of Na + to the Na2 site 36,58 , a site corresponding to the only Na + -binding site in Mhp1.…”

“…However, some of the recent studies have proposed completely different ion-coupling mechanisms for different transporter families within the LeuT-fold superfamily 36,44,45,47,50,54,58–60 . A number of studies on LeuT (NSS family) using smFRET, EPR, cysteine accessibility measurements, or hydrogen/deuterium exchange mass spectrometry indicate that Na + binding stabilizes the OF conformation 36,50,54,58–60 , and that such an effect requires binding of Na + to the Na2 site 36,58 , a site corresponding to the only Na + -binding site in Mhp1. Albeit a computational study on Mhp1 (NCS1 family) proposes a similar Na + -binding effect as LeuT 44 , recent experimental studies using EPR, cysteine accessibility and mass spectrometry provide a conflicting picture, in which Mhp1 adopts predominantly IF conformations and Na + has little effect on the conformational equilibrium of Mhp1 45,47 .…”

Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

“…The addition of Na + to LeuT induces opening of the extracellular vestibule suggesting formation of an outward-open state, which is altered upon interactions with substrates like alanine or leucine that induce an occluded state (Zhang et al, 2018).…”