Identification of key residues in the formate channel FocA that control import and export of formate

Abstract

The formate-nitrite transporter (FNT) family comprises pentameric channels that transport monovalent anions. The prototype of this family is the formate channel (FocA), which was originally identified as a formate channel in Escherichia coli. Each protomer in the channel has a pore with structural features that include periplasmic and cytoplasmic constriction sites, which are likely important for bi-directional gating of substrate passage. Highly conserved amino acid residues within FocA previously identified in structural studies are predicted to be important in the control of formate translocation. Here we present a first detailed in vivo analysis of these residues using a combined targeted amino acid exchange and formate-responsive lacZ fusion-based reporter approach. Sixteen exchanges were made and each variant was shown to be largely unaffected in its secondary and quaternary structure. The invariant H209 and T91 residues, which form part of the lower constriction site linking the Ω-loop with the pore cavity, proved to be important in governing the directionality of formate passage through the pore. A predicted salt-bridge triad of E208-K156-N213 along with the cytoplasmically-oriented N-terminal helix are also involved in pH-dependent gating of the channel. Together, our data are consistent with passive export and import of formate or formic acid through the channel.

The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity

“…PflB exists in both active and inactive forms ( Wagner et al, 1992 ) and results indicate that in its inactive form PflB impedes formate import ( Doberenz et al, 2014 ). A similar result was observed in mutants lacking PflB ( Doberenz et al, 2014 ; Hunger et al, 2014 ), suggesting that the active PflB enzyme is necessary to promote formate translocation in vivo .…”

“…Development of a reporter system based on a formate-responsive promoter ( Falke et al, 2010 ) has greatly facilitated the in vivo assessment of both import and export of the anion through FocA ( Hunger et al, 2014 ). This system has also helped to identify amino acids in the central substrate pore of the FocA protomer that are important for channel activity ( Hunger et al, 2014 ). However, no study has yet been carried out to examine the roles of the cytoplasmic N- or C-termini of these proteins and how they impact substrate transport and homopentamer formation.…”

A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum

“…For example, residues that have been implicated in the gating of the E. coli FocA transporter (T91, K156, E208, H209 and N213; ref. 16) are conserved in PfFNT.…”

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

“…We further isosterically changed Thr91 to valine (FocA T91V) eliminating a stabilizing hydrogen bond to the fixed water molecule (red sphere in Fig 1A), whereas a third, conservative Thr91-to-serine exchange (FocA T91S) should retain the water in the FocA protein. Hence, both the imidazole side chain of His209 and the fixed water molecule seem indispensible for FocA transport (Hunger et al, 2014). FocA H209N and T91V were non-functional, whereas FocA T91S transport rates were equal to wild-type FocA (Fig 2A).…”

“…FocA H209N and T91V were non-functional, whereas FocA T91S transport rates were equal to wild-type FocA (Fig 2A). Hence, both the imidazole side chain of His209 and the fixed water molecule seem indispensible for FocA transport (Hunger et al, 2014). The total loss of functionality, however, prevents further conclusions on their mechanistic contribution, which could be related to substrate protonation or purely structural.…”