Exploring the pH-Dependent Substrate Transport Mechanism of FocA Using Molecular Dynamics Simulation

Abstract

FocA belongs to the formate-nitrate transporter family and plays an essential role in the export and uptake of formate in organisms. According to the available crystal structures, the N-terminal residues of FocA are structurally featureless at physiological conditions but at reduced pH form helices to harbor the cytoplasmic entrance of the substrate permeation pathway, which apparently explains the cessation of electrical signal observed in electrophysiological experiments. In this work, we found by structural analysis and molecular dynamics simulations that those N-terminal helices cannot effectively preclude the substrate permeation. Equilibrium simulations and thermodynamic calculations suggest that FocA is permeable to both formate and formic acid, the latter of which is transparent to electrophysiological studies as an electrically neutral species. Hence, the cease of electrical current at acidic pH may be caused by the change of the transported substrate from formate to formic acid. In addition, the mechanism of formate export at physiological pH is discussed.

Mechanism of formate–nitrite transporters by dielectric shift of substrate acidity

“…Earlier studies suggested a pH-gating mechanism of FocA (Lü et al, 2011). It showed that, independent of the conformation, the FocA N-terminus cannot sufficiently narrow the pore entry to prevent formate from passing (Lv et al, 2013). Yet, we found a steady increase in FocA transport rates from neutral to acidic assay conditions, that is, a permanently open conformation.…”

“…Despite these substrate-derived differences, the overall layout of the individual transport units in pentameric FNTs and trimeric ammonium transporters is quite similar. Yet, due to the observed low positive activation energy that matches earlier molecular dynamic simulations of FocA with a neutral His209 (Lv et al, 2013), we prefer to think of the histidine as non-protonated. The histidines in ammonium transporters are assumed be uncharged (Javelle et al, 2006); we cannot conclude on the debated protonation state of His209 in FocA by this study.…”

“…It is energetically highly favorable when substrate neutralization occurs before passing the lipophilic FocA constrictions (Lv et al, 2013). Therefore, it is questionable whether the central His209 is suited for the protonation of formate, because the imidazole side chain is positioned in the hydrophobic space between the lipophilic constrictions ( Fig 1A).…”

“…Despite ample structure information, key questions remain unresolved and debated concerning FNT selectivity, gating, and transport (Waight et al, 2010(Waight et al, , 2013Lü et al, 2013;Lv et al, 2013). A substrate discriminating selectivity filter site has not been described, probably because the preference of bacterial FNTs for formate, nitrate, or hydrosulfide was evident from the genetic context of the transporter-encoding genes (Lü et al, 2013).…”

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

“…It should be noted, however, that recent molecular dynamics simulations [34] suggest that formate can still enter and exit the channel in FocA even when the N-terminal helix covers the pore entrance. This finding further underscores the likely importance of the interaction with PflB for controlled movement of formate through FocA.…”