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

Wiechert, Marie; Beitz, Eric

doi:10.15252/embj.201695776

Cited by 57 publications

(118 citation statements)

References 43 publications

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“…We found that eukaryotic PfFNT and BbFNT transport formate and acetate at the same rate, and L-lactate at about half the rate. The prokaryotic FocA equally transported formate and acetate, yet excluded L-lactate confirming earlier data [22]. NirC turned out to be selective for the smallest substrate formate, which is bioisosteric to its genuine substrate nitrite.…”

Section: Eukaryotic Fnts Conduct Larger Substrates Than Prokaryotic Fntssupporting

confidence: 85%

“…This is in accordance with experimental evidence from electrophysiological single channel recordings with FocA that revealed rapid opening and closing events at the millisecond scale indicating flickering side‐chain movements . As part of the transport mechanism, the flexible side chains give way to the neutral substrate species that originates from a protonation event in the vestibule . Therefore, the selective function of the lipophilic constrictions seems to reside in posing energetic barriers against the passage of charged substrates .…”

Section: Discussionmentioning

confidence: 99%

“…The imidazole side chain of an indispensable histidine (position 209 in FocA; Fig. A) is sandwiched between both constrictions .…”

Section: Introductionmentioning

confidence: 99%

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A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

et al. 2017

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Bacterial formate-nitrite transporters (FNT) regulate the metabolic flow of small weak mono-acids derived from anaerobic mixed-acid fermentation, such as formate, and further transport nitrite and hydrosulfide. The eukaryotic Plasmodium falciparumFNT is vital for the malaria parasite by its ability to release the larger l-lactate substrate as the metabolic end product of anaerobic glycolysis in symport with protons preventing cytosolic acidification. However, the molecular basis for substrate discrimination by FNTs has remained unclear. Here, we identified a size-selective FNT substrate filter region around an invariant lysine at the bottom of the periplasmic/extracellular vestibule. The selectivity filter is reminiscent of the aromatic/arginine constriction of aquaporin water and solute channels regarding composition, location in the protein, and the size-selection principle. Bioinformatics support an adaptation of the eukaryotic FNT selectivity filter to accommodate larger physiologically relevant substrates. Mutations that affect the diameter at the filter site predictably modulated substrate selectivity. The shape of the vestibule immediately above the filter region further affects selectivity. This study indicates that eukaryotic FNTs evolved to transport larger mono-acid substrates, especially l-lactic acid as a product of energy metabolism.

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Section: Eukaryotic Fnts Conduct Larger Substrates Than Prokaryotic Fntssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

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A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

et al. 2017

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“…To detect not only the electrogenic anion transport of FocA and PfFNT but additionally capture the transport of the neutral acid substrate species, we used radiolabeled substrates in our assays. 3,7 Strikingly and contrary to the electrophysiology studies, we observed increasing transport rates with increasing acidity of the assay media. Apparently, neither FocA nor PfFNT exhibited closure or gating.…”

contrasting

confidence: 99%

“…Further, the alkalization of the media during transport indicated that protons are co-transported with the substrate and disruption of the transmembrane proton gradient revealed that transport is driven by a proton-motive force. 7 Together, our data show that FNTs act as non-gated anion/proton co-transporters and the major portion of the substrate is transported as the protonated, neutral monoacid.…”

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confidence: 59%

Formate-nitrite transporters: Monoacids ride the dielectric slide

Wiechert

Beitz

2017

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The discovery that the Escherichia coli focA gene product facilitates the transmembrane exchange of formate 1 sparked research on proteins that turned out to be functionally situated between classical transporters and channels. 2,3 The strictly microbial protein family was named formate-nitrite transporters (FNT) according to the physiologic substrates of the 2 initially characterized members. However, subsequently, the substrate spectrum was extended and comprises several weak monoacids up to the size of lactic acid. On the cellular level, FNTs regulate the metabolic substrate flow and fulfill vital functions, for instance the Plasmodium falciparum PfFNT that we and others identified as the malaria parasite's long-sought lactic acid transporter.3,4 Blockade of PfFNT by small druglike molecules kills the parasites rendering PfFNT a novel antimalarial drug target. 5,6 Their physiologic relevance as well as structural peculiarities merit a deeper look into the inner workings of the FNTs.High resolution crystal data revealed a common homopentameric structure.2 The protomers consist of 6 transmembrane spans with both termini at the cytoplasmic side. Surprisingly, FNTs almost perfectly mimic the fold of the aquaporin water and solute channels despite unrelated amino acid sequences. The substrate path through each FNT protomer harbors 2 hydrophobic constrictions, which sandwich the imidazole sidechain of a highly-conserved histidine. The transport mechanism, however, remained unclear regarding to the question whether the substrate anion, e.g. formate, is required to be neutralized by protonation to pass the lipophilic constriction sites.Initially, electrophysiology was used to determine substrate selectivity and pH dependent transport of FocA.2 These studies showed electrogenic, i.e. anion, transport at neutral pH and very low, i.e., channellike, substrate affinity in the high millimolar range. Further, a sudden loss of anion transport was observed when the buffer pH was slightly shifted toward acidic conditions suggesting a gating mechanism for which different conformations of the N-terminal domain were made responsible.2 Together, FocA seemed to behave as a pH-gated anion channel.Due to their weak acidity, e.g., pK a/formate 3.8 and pK a/acetate 4.8, the FNT substrates interconvert between the negatively charged anion and the protonated, neutral acid in a chemical equilibrium. To detect not only the electrogenic anion transport of FocA and PfFNT but additionally capture the transport of the neutral acid substrate species, we used radiolabeled substrates in our assays.3,7 Strikingly and contrary to the electrophysiology studies, we observed increasing transport rates with increasing acidity of the assay media. Apparently, neither FocA nor PfFNT exhibited closure or gating. Further, the alkalization of the media during transport indicated that protons are co-transported with the substrate and disruption of the transmembrane proton gradient revealed that transport is driven by a proton-motive force.7 Together, our da...

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Pentafluoro‐3‐hydroxy‐pent‐2‐en‐1‐ones Potently Inhibit FNT‐Type Lactate Transporters from all Five Human‐Pathogenic Plasmodium Species

et al. 2021

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The protozoan parasite Plasmodium falciparum causes the most severe and prevailing form of malaria in sub-Saharan Africa. Previously, we identified the plasmodial lactate transporter, PfFNT, a member of the microbial formate-nitrite transporter family, as a novel antimalarial drug target. With the pentafluoro-3-hydroxy-pent-2-en-1-ones, we discovered PfFNT inhibitors that potently kill P. falciparum parasites in vitro. Four additional human-pathogenic Plasmodium species require attention, that is, P. vivax, most prevalent outside of Africa, and the regional P. malariae, P. ovale and P. knowlesi. Herein, we show that the plasmodial FNT variants are highly similar in terms of protein sequence and functionality. The FNTs from all human-pathogenic plasmodia and the rodent malaria parasite were efficiently inhibited by pentafluoro-3-hydroxy-pent-2-en-1-ones. We further established a phenotypic yeast-based FNT inhibitor screen, and found very low compound cytotoxicity and monocarboxylate transporter 1 off-target activity on human cells, particularly of the most potent FNT inhibitor BH267.meta, allowing these compounds to proceed towards animal model malaria studies.

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

Cited by 57 publications

References 43 publications

A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

Formate-nitrite transporters: Monoacids ride the dielectric slide

Pentafluoro‐3‐hydroxy‐pent‐2‐en‐1‐ones Potently Inhibit FNT‐Type Lactate Transporters from all Five Human‐Pathogenic Plasmodium Species

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