Formate–nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport

Abstract

Microbial formate-nitrite transporter-type proteins (FNT) exhibit dual transport functionality. At neutral pH, electrogenic anion currents are detectable, whereas upon acidification transport of the neutral, protonated monoacid predominates. Physiologically, FNT-mediated proton co-transport is vital when monocarboxylic acid products of the energy metabolism, such as L-lactate, are released from the cell. Accordingly, Plasmodium falciparum malaria parasites can be killed by small-molecule inhibitors of PfFNT. Two opposing hypotheses on the site of substrate protonation are plausible. The proton relay mechanism postulates proton transfer from a highly conserved histidine centrally positioned in the transport path. The dielectric slide mechanism assumes decreasing acidity of substrates entering the lipophilic vestibules and protonation via the bulk water. Here, we defined the transport mechanism of the FNT from the amoebiasis parasite Entamoeba histolytica, EhFNT, and also show that BtFdhC from Bacillus thuringiensis is a functional formate transporter. Both FNTs carry a nonprotonatable amide amino acid, asparagine or glutamine, respectively, at the central histidine position. Despite having a nonprotonatable residue, EhFNT displayed the same substrate selectivity for larger monocarboxylates including L-lactate, a low substrate affinity as is typical for FNTs, and, strikingly, proton motive force-dependent transport as observed for PfFNT harboring a central histidine. These results argue against a proton relay mechanism, indicating that substrate protonation must occur outside of the central histidine region, most likely in the vestibules. Furthermore, EhFNT is the sole annotated FNT in the Entamoeba genome suggesting that it could be a putative new drug target with similar utility as that of the malarial PfFNT.

Transmembrane Facilitation of Lactate/H+ Instead of Lactic Acid Is Not a Question of Semantics but of Cell Viability

“…While AQPs act as lactic acid channels, the FNTs functionally represent lactate/H + co-transporters that are comparable in efficiency to the MCTs. Evolutionary, the FNT-type of monocarboxylate transport remained restricted to microbes [39][40][41], whereas the alternating-access mechanism of the MCTs with a more specific substrate binding site certainly constitutes a more modern principle.…”

Cell-Free and Yeast-Based Production of the Malarial Lactate Transporter, PfFNT, Delivers Comparable Yield and Protein Quality

“…We reconstituted purified P. pastoris cell-based and cell-free produced PfFNT into proteoliposomes following a protocol that we established earlier (Müller-Lucks et al, 2012; von Bülow et al, 2012; Golldack et al, 2017; Erler et al, 2018; Helmstetter et al, 2019). We evaluated PfFNT transport functionality by challenging the proteoliposomes with an inward directed, osmotic lactate gradient of 200 mM.…”

“…Many studies carried out cell-free membrane protein production even in precipitation mode, i.e., in the absence of a suitable detergent or lipid environment, and nevertheless successfully folded the precipitated protein in a subsequent renaturation step (Junge et al, 2010; Rath et al, 2011; Focke et al, 2016; Shinoda et al, 2016). Using these approaches, various classes of membrane proteins such as receptors [G-protein coupled (Junge et al, 2010; Chi et al, 2016; Shinoda et al, 2016; Jacobs et al, 2019); tyrosin kinase (Shinoda et al, 2016)], channels and transporters [aquaporins Shinoda et al, 2016; ion channels Shinoda et al, 2016; Vaish et al, 2018, ionotropic receptors Shinoda et al, 2016; mechanosensitive channels (Jacobs et al, 2019); bacterial porins (Junge et al, 2010); formate-nitrite transporters (Holm-Bertelsen et al, 2016; Helmstetter et al, 2019)], or cell adhesion proteins (claudins Shinoda et al, 2016) were successfully produced. Furthermore, quality assessment by functionality assays, ligand binding, and structure determination was positive in many cases.…”

“…In our hands, detergents of the Brij-family carrying polyoxyethylen polar head groups turned out to be most successful in facilitating cell-free membrane protein production. For stabilizing tetrameric aquaporins and pentameric FNTs, in particular Brij 58 and Brij 78, both carrying 20 ethylene glycol units, and C16 or C18 alkane chains, respectively, were most suitable (Müller-Lucks et al, 2012, 2013; Holm-Bertelsen et al, 2016; Helmstetter et al, 2019). We further successfully employed DDM with a di-saccharide polar head group (von Bülow et al, 2012).…”

Unraveling the substrate preference of an uncharacterized phylogenetic subgroup in Formate/Nitrite Transporter (FNT) family: Computational studies of anion transport inEscherichia coliFNT homolog

“…According to their hypothesis, the dielectric shift that causes the transfer of proton from the bulk water to the formate ion enables the substrate to permeate the channel. Recent reports also highlighted that FNT homologs in which central His is substituted by Asn or Gln also seem to exhibit the same selectivity indicating the important role of Lys-156 in protonating the substrate[29]. In the absence of Lys-156, such a possibility does not exist in EcYfdC.…”