Proton-control of transitions in an amino-acid transporter

Abstract Amino acid transport into the cell is often coupled to the proton electrochemical gradient, as found in the solute carrier (SLC) 36 family of proton coupled amino acid transporters (PATs). Although no structure of a human PAT exists, the crystal structure of a related homolog, GkApcT, from bacteria 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 deprotonation of a key glutamate residue (E115), located at the bottom of the binding pocket and 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, 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 substrate release state. 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 favourable 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 state or the protonation of E115, presumably reflecting key interactions 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. Significance Statement For transporter proteins that utilize the proton gradient to drive the uptake of solutes, the precise mechanistic details of proton-coupling remain poorly understood. Structures can only infer the position of protons. All-atom molecular dynamics simulations however, are the ideal complementary tool. Here, we report extensive MD simulations on GkApcT, a proton-coupled transporter. We observe a spontaneous transition from the crystallographically derived inward-occluded state, to an inward-open state, which we then characterise with umbrella sampling and absolute binding free energy calculations. The results suggest that a conserved glutamate is protonated in the inward-occluded state and subsequent deprotonation of this glutamate allows the transporter to move into the inward-open state, thus facilitating substrate release into the cell.