Is the E. coli Homolog of the Formate/Nitrite Transporter Family an Anion Channel? A Computational Study

Abstract

Formate/nitrite transporters (FNTs) selectively transport monovalent anions and are found in prokaryotes and lower eukaryotes. They play a significant role in bacterial growth and act against the defense mechanism of infected hosts. Because FNTs do not occur in higher animals, they are attractive drug targets for many bacterial diseases. Phylogenetic analysis revealed that they can be classified into eight subgroups, two of which belong to the uncharacterized YfdC-α and YfdC-β groups. Experimentally determined structures of FNTs belonging to different phylogenetic groups adopt the unique aquaporin-like hourglass helical fold. We considered the formate channel from Vibrio cholerae, the hydrosulphide channel from Clostridium difficile, and the uncharacterized channel from Escherichia coli (EcYfdC) to investigate the mechanism of transport and selectivity. Using equilibrium molecular dynamics and umbrella sampling studies, we determined temporal channel radius profiles, permeation events, and potential of mean force profiles of different substrates with the conserved central histidine residue in protonated or neutral form. Unlike the formate channel from V. cholerae and the hydrosulphide channel from C. difficile, molecular dynamics studies showed that the formate substrate was unable to enter the vestibule region of EcYfdC. Absence of a conserved basic residue and presence of acidic residues in the vestibule regions, conserved only in YfdC-α, were found to be responsible for high energy barriers for the anions to enter EcYfdC. Potential of mean force profiles generated for ammonia and ammonium ion revealed that EcYfdC can transport neutral solutes and could possibly be involved in the transport of cations analogous to the mechanism proposed for ammonium transporters. Although YfdC members belong to the FNT family, our studies strongly suggest that EcYfdC is not an anion channel. Absence or presence of specific charged residues at particular positions makes EcYfdC selective for neutral or possibly cationic substrates. Further experimental studies are needed to get a definitive answer to the question of the substrate selectivity of EcYfdC. This provides an example of membrane proteins from the same family transporting substrates of different chemical nature.

Figure 1

Temporal channel radius profiles are plotted for 500 ns simulation time for the VcFocA, CdHSC, and EcYfdC channels. (A–C) The central His is protonated, and the substrate is a formate ion; (D–F) the central His is protonated, and the substrate is formic acid; and (G–I) the central His is neutral, and the substrate is formic acid. Position “0” in the y axis corresponds to the central His residue. The positive and negative y axis values correspond to the periplasmic and cytoplasmic sides, respectively. HOLE (44) profile data from all five monomers in each time frame have been combined to create the time evolution of channel radius profiles. See Materials and Methods for further details. To see this figure in color, go online.

Figure 2

Evolution of z coordinates of the center of mass of individual substrate molecules (formate ion or formic acid) as a function of time is plotted for the entire 500 ns production run for all nine systems. Each color represents an individual substrate molecule. Analysis is shown for the VcFocA, CdHSC, and EcYfdC systems. (A–C) The substrate is a formate ion and the central His protonated; (D–F) the substrate is formic acid and the central His protonated; (G–I) the substrate is formic acid and the central His neutral. Position “0” in the y axis corresponds to the central His residue. The positive and negative y axis values correspond to the periplasmic and cytoplasmic sides, respectively. To see this figure in color, go online.

Figure 3

Location and conservation of the Lys-156 residue and its interaction with neighboring residues. (A) The formate channel structure (PDB: 3KCU), showing the location of Lys-156 residue in the P-helix and its interacting partners Glu-208 from the S-loop and Asn-213 from the TM5b helix, is given. (B) The salt bridge and hydrogen bond interactions of Lys-156 with the Glu-208 and Asn-213 residues are shown. (C) The sequence logo of the P-helix region is shown for the FocA, HSC, and YfdC-α subfamilies. The position of Lys-156 in the FocA and HSC subfamilies and the equivalent position in the YfdC-α subfamily are highlighted within a red rectangular box. It is clear that Lys-156 exhibits absolute conservation in FocA and HSC subfamilies, whereas the same position in YfdC-α shows enormous variation. To see this figure in color, go online.

Figure 4

Sequence logo generated for the S-loop/TM5b region for the FocA, HSC, and YfdC-α subfamilies. Residues interacting with Lys-156 of P-helix show near-absolute conservation in the FocA and HSC subgroups, and these positions are highlighted within red rectangular boxes. To see this figure in color, go online.

Figure 5

P-helix stability shown for (A) the original homology model and (B) the revised homology model of the EcYfdC channel after a 500 ns production run. In the original homology model, a Lys residue faced the periplasmic entrance, whereas in the revised model, a Val residue is at the periplasmic entrance. The P-helix is shown as opaque, and the rest of the protein is shown as transparent. The position of the Lys/Val residue occupying the equivalent Lys-156 position is shown in a space-filling representation. To see this figure in color, go online.

Figure 6

The potential of mean force (PMF) profiles of formate ion or formic acid substrates through the EcYfdC channel. PMF profiles shown were calculated for substrate permeation in EcYfdC channel, and the different colors correspond to different simulation conditions (red: substrate—formate ion, central His protonated; orange: substrate—formic acid, central His protonated; brown: substrate—formic acid, central His neutral). Each point in the profile is shown with the standard deviation. The gray bands depict the central constriction toward the periplasmic side and the cytoplasmic slit. Locations of Glu residues at the vestibular regions are shown by dotted lines. To see this figure in color, go online.

Figure 7

Glu residues at the channel entrance in EcYfdC. (A) The location of two Glu residues in the vestibular regions is shown for the EcYfdC channel. Glu-237 and Glu-115 residues are located in TM5b and TM2b and correspond to Met-216 and Ser-92, respectively, in PDB: 3KCU numbering. (B) A sequence logo showing the conservation of Glu-115 in YfdC-α is given, whereas this is substituted with small residues in the FocA and HSC subfamilies. To see this figure in color, go online.

Figure 8

PMF profiles for the permeation of ammonium ions when the central His was considered (A) in protonated and (B) in neutral form for the EcYfdC channel. (C) A comparison of the PMF profiles of formate ion and ammonium ion permeation through the EcYfdC channel with the central His protonated is shown. The two gray bands represent the positions of the central constriction and the cytoplasmic slit. The locations of two conserved Glu residues at the cytoplasmic and periplasmic vestibule regions are shown in dotted lines. To see this figure in color, go online.

Figure 9

Ammonia PMF profiles calculated with the central His in (A) protonated and (B) neutral forms. (C) A comparison of the PMF profiles of ammonium ion and ammonia permeation through the EcYfdC channel with central His considered in both protonated and neutral forms is shown. The two gray bands depict the locations of the cytoplasmic slit and the central constriction region. The dotted lines represent the positions of the two conserved Glu residues toward the periplasmic and cytoplasmic vestibule regions. To see this figure in color, go online.