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 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 inwardoccluded 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 inwardopen 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.

The KDEL trafficking receptor exploits pH to tune the strength of an unusual short hydrogen bond

“…(d) KDEL binding creates a zone of ordered waters, where the largest cavity is occupied by two waters surrounded by a hydrophobic pocket formed from I124, W166 and A180. www.nature.com/scientificreports/ proton binding, water network rearrangements and structural stability are similar themes in secondary active transporters [24][25][26] with which the KDELR shares the same topology and suggested evolutionary ancestry 27 . We summarize our results in Fig.…”

“…The KDEL-bound KDEL receptor (KDELR) and the Apo KDEL receptor were retrieved from the PDB (Apo: 6I6B; KDEL-bound: 6I6H) and embedded in a DMPC bilayer following a previously described protocol 28 . The QM region was defined as the three key residues (H12, Y158 and E127) and was described at the level of B97-3c 29 , whereas the qmcut was set to 3 nm to cover the whole protein.…”

The KDEL Trafficking Receptor Exploits pH to Tune the Strength of an Unusual Short Hydrogen Bond

“…Clearly much more work is required to properly address this hypothesis. However, a mechanistic link between proton binding, water network rearrangements and structural stability are similar themes in proton coupled secondary active transporters [25][26][27] with which the KDELR shares the same topology and suggested evolutionary ancestry 28 . We summarize our results in Fig.…”

“…The KDEL-bound KDEL receptor (KDELR) and the Apo KDEL receptor were retrieved from the PDB (Apo: 6I6B; KDEL-bound: 6I6H) and embedded in a DMPC bilayer following a previously described protocol 29 . The QM region was defined as the three key residues (H12, Y158 and E127) and was described at the level of B97-3c, 30 whereas the qmcut was set to 3 nm to cover the whole protein.…”

Sequence analysis and spatiotemporal developmental distribution of the Cat-1-type transporter slc7a1a in zebrafish (Danio rerio)

“…White arrows denote those proteins for which low substrate affinity has been ascertained (human CAT-2A) (see, e.g., Closs et al 1997;Habermeier et al 2003) or can be hypothesized (zebrafish Slc7a1a isoform X2, zebrafish Slc7a1b isoform X2 and zebrafish Cat-2a). MgtS indicates a small protein (31 amino acids; a single transmembrane domain) that complexes with GkApcT (Jungnickel et al 2018;Wu et al 2019) amino acid lysine also forms the majority of collagen (Wittmann et al 2005;Barbul 2008). In this regard, the "putative-to-date" transport of arginine and lysine via Slc7a1a would act as a rate-limiting step in providing precursors for collagen synthesis.…”