Abstract
Amino acid transport into the cell is often coupled to the proton electrochemical gradient, as found in the solute carrier 36 family of proton-coupled amino acid transporters. Although no structure of a human proton-coupled amino acid transporter exists, the crystal structure of a related homolog from bacteria, GkApcT, has recently been solved in an inward-occluded state and allows an opportunity to examine how protons are coupled to amino acid transport. Our working hypothesis is that release of the amino acid substrate is facilitated by the deprotonation of a key glutamate residue (E115) located at the bottom of the binding pocket, which forms part of the intracellular gate, allowing the protein to transition from an inward-occluded to an inward-open conformation. During unbiased molecular dynamics simulations, we observed a transition from the inward-occluded state captured in the crystal structure to a much more open state, which we consider likely to be representative of the inward-open state associated with substrate release. To explore this and the role of protons in these transitions, we have used umbrella sampling to demonstrate that the transition from inward occluded to inward open is more energetically favorable when E115 is deprotonated. That E115 is likely to be protonated in the inward-occluded state and deprotonated in the inward-open state is further confirmed via the use of absolute binding free energies. Finally, we also show, via the use of absolute binding free energy calculations, that the affinity of the protein for alanine is very similar regardless of either the conformational state or the protonation of E115, presumably reflecting the fact that all the key interactions are deep within the binding cavity. Together, our results give a detailed picture of the role of protons in driving one of the major transitions in this transporter.
Highlights
The intracellular concentrations of amino acids are regulated by various transporters that utilize different mechanisms
Given the location of E115 on TM3, and its unusual pKa of 8.2 compared to 4.38 for D237 (calculated by PROPKA3 [12]), we postulated the conformation of the intracellular gate is likely dependent on the protonation state of this side chain [9]
During one of the simulations with E115 deprotonated, we observed a significant expansion of the binding cavity (Fig. 2, B and C) along with an increase in the distance between TM3 and TM6 at the intracellular end of the helices, resulting in a state that would be consistent with an inward-open conformation
Summary
The intracellular concentrations of amino acids are regulated by various transporters that utilize different mechanisms. In some cases, be coupled to another electrochemical gradient, such as sodium [1] or protons [2], which are used to drive the concentrative uptake of the amino acid into the cell. Some transporters can act as facilitators, whereby the substrate is exchanged for its metabolized product [5] or another amino acid [6]. This exchange activity can be regulated by protons (pH) in which the transporter is only active in acidic pH [7]. There have been many hypotheses put forward concerning the overall conformational cycle of secondary active transport proteins [8], the precise details of how the structure of these membrane proteins and their associated transport activity is regulated by protons remain incomplete
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