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. 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.

Inhibitor binding mode and allosteric regulation of Na+-glucose symporters

“…In addition, mutation of Na2 site residues blocks the voltage-dependent opening and closing of the outer gates 31 . As already discussed, vSGLT 29 and Mhp1 28 , both of which have 1-to-1 stoichiometries, fail to fully open their outer gates in high Na + , while cysteine accessibility studies of LeuT and SERT, which have 2-to-1 stoichiometries, both require Na + binding to open the outer gate 34 . Similarly, double electron–electron resonance studies on LeuT reveal that high Na + levels cause the outer gate to open 35 .…”

Serotonin transport in the 21st century

“…The γ-carboxyl of Asp98 is located between the Na1 site and a tyrosine residue (Tyr176) corresponding to Tyr108 in LeuT. Moreover, mutation of Tyr176 in SERT blocks the ability of substrate (5-HT) to activate the conformational transition of SERT from outward-to inward-facing conformations, similar to the way that mutating Tyr108 blocked the corresponding substrate-induced conformational change in LeuT (Zhang et al, 2018). We still do not understand how 5-HT binding in SERT activates the interactions among Tyr176, Asp98, and Na + , and this activation is a focus of our current research.…”

Structure and Gating Dynamics of Na+/Cl– Coupled Neurotransmitter Transporters

“…Unlike the major facilitator superfamily transporters that function as rocking switches to allow vectorial movement of substrate, the LeuT family transporters employ a rocking bundle of two helices (Drew and Boudker, 2016). The transition of NSS transporters from O occ to I o state is triggered by the binding of substrate in the primary binding site of transporters like LeuT and tyrosine transporter Tyt1 (Quick et al, 2006; Zhang et al, 2018). While the inward movement of the TMs 1b and 6a close the extracellular gate, the opening of TM1a facilitates solvent access to the binding site.…”