Structural and Functional Analysis of Bacterial Formate Channels
Zusammenfassung der Projektergebnisse
The pentameric channels of the formate/nitrite transporter family (FNT) represent a conserved module for the selective transport of a series of monovalent anions across biological membranes. An important member of this protein family is the formate channel/transporter FocA that plays a central role in the bacterial metabolic pathway of mixed-acid fermentation. The extraordinary feature of FocA is its ability to switch its transport mode and direction in response to the physiological conditions. The formate anion, reaction product of cytoplasmic pyruvate:formate lyase, is initially exported passively for periplasmic oxidation to CO2. With increasing acidification of the medium through mixed-acid fermentation, however, the channel responds at a distinct threshold pH value by switching to active import of formic acid, which is then disproportionated in the cytoplasm to yield CO2 and H2. The FocA protein investigated in the present project originates from the enteropathogenic Salmonella typhimurium and was characterized by X-ray crystallography at a resolution of 2.2 Å. Other than in previous structures of FocA orthologs, the S. typhimurium protein was crystallized at low pH, presumably representing the active transporter configuration. The structure revealed conformational changes at the N-termini of the five protomers, and electrophysiological studies showed a sharply defined, pH-dependent gating behavior of the channel. In subsequent electrophysiological experiments on planar lipid bilayer membranes we showed that FocA exhibits a rather low substrate selectivity, which is not untypical for anion channels. It was, however, unexpected that three anions were clearly preferred by FocA among all those tested and were transported with almost identical maximal conductances. Beside the expected formate anion these were lactate and acetate, so that FocA by itself is capable to export all anionic products of the pathway of mixed-acid fermentation. The structure of the substrate channel suggests a mechanism for this: The selectivity filter of the system is located almost exactly in the middle of the membrane and consists of a hydrophobic vestibule that is bordered towards both sides by constrictions consisting of the side chains of hydrophobic amino acids. Three protonable positions are present in the center of the membrane that raise the possibility that an anion is transiently protonated for passing the selectivity filter, while the proton itself remains in the membrane after completing the transport cycle. For FocA, this process changes with an increasing acidification of the medium, when uncharged formic acid is present in the external medium and can then be re-imported through a pmf-driven H+/HCOO–symport. Its unusual architecture enables FocA to serve both transporter modes without major structural rearrangements, and the conformational changes of the N-termini hereby mainly act as a barrier for further export of formate. In a second part of the project we additionally characterized the structure and function of a further member of the FNT family, the nitrite channel NirC, also originating from S. typhimurium. NirC is structurally similar to FocA, but does not switch its functionality depending on the pH value, as this is not required for its physiological role, the assimilation of nitrite as a nitrogen source. Nevertheless, the substrate channel of NirC is nearly identical to the one of FocA, although the relative conductivities for different anions vary characteristically: While FocA preferred formate over nitrite, NirC shows the opposite behavior.
Projektbezogene Publikationen (Auswahl)
- (2011) pH- dependent gating in a FocA formate channel. Science, 332, 352-354
Lü W, Du J, Wacker T, Gerbig-Smentek E, Andrade SLA & Einsle O
(Siehe online unter https://doi.org/10.1126/science.1199098) - (2012) Structure and mechanism of the nitrite channel NirC from Salmonella typhimurium. Proc Natl Acad Sci USA, 109, 18395-18400
Lü W, Schwarzer NJ, Du J, Gerbig-Smentek E, Andrade SLA & Einsle, O
(Siehe online unter https://doi.org/10.1073/pnas.1210793109) - (2012) The formate channel FocA exports the products of mixed-acid fermentation. Proc Natl Acad Sci USA, 109, 13254-13259
Lü W, Du J, Schwarzer NJ, Gerbig-Smentek E, Einsle O & Andrade SLA
(Siehe online unter https://doi.org/10.1073/pnas.1204201109) - (2013) The Formate/Nitrite Transporter family of anion channels. Biol Chem, 394, 715-727
Lü W, Du J, Schwarzer NJ, Wacker T, Andrade SLA & Einsle O
(Siehe online unter https://doi.org/10.1515/hsz-2012-0339) - (2013) The tricky task of nitrate/nitrite antiport. Angew Chem Intl Ed, 52, 10422-10424
Andrade SLA & Einsle O
(Siehe online unter https://doi.org/10.1002/anie.201305421) - (2015) Structural and Functional Studies of NirC from Salmonella typhimurium. Meth. Enzymol., 556, 475-497
Rycovska-Blume A, Lü W, Andrade SLA, Fendler K & Einsle O
(Siehe online unter https://doi.org/10.1016/bs.mie.2014.12.034)
