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.
Acknowledgments
We thank Lydia Beyer for discussion. This work was carried out in the Graduate Training Group (GRK 1026) ‘Conformational transitions in macromolecular interactions’ and was funded by the Deutsche Forschungsgemeinschaft.
References
Beckham, K.S., Potter, J.A., and Unkles, S.E. (2010). Formate-nitrite transporters: optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives. Protein Expr. Purif. 71, 184–189.10.1016/j.pep.2009.12.005Search in Google Scholar
Begg, Y., Whyte, J., and Haddock, B. (1977). The identification of mutants of Escherichia coli deficient in formate dehydrogenase and nitrate reductase activities using dye indicator plates. FEMS Microbiol. Lett. 2, 47–50.10.1111/j.1574-6968.1977.tb00905.xSearch in Google Scholar
Beyer, L., Doberenz, C., Falke, D., Hunger, D., Suppmann, B., and Sawers, R.G. (2013). Coordinating FocA and pyruvate formate-lyase synthesis in Escherichia coli: preferential translocation of formate over other mixed-acid fermentation products. J. Bacteriol. 195, 1428–1435.10.1128/JB.02166-12Search in Google Scholar
Casadaban, M.J. (1976). Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104, 541–555.10.1016/0022-2836(76)90119-4Search in Google Scholar
Czyzewski, B.K. and Wang, D.N. (2012). Identification and characterization of a bacterial hydrosulphide ion channel. Nature 483, 494–497.10.1038/nature10881Search in Google Scholar
Falke, D., Schulz, K., Doberenz, C., Beyer, L., Lilie, H., Thiemer, B., and Sawers, R.G. (2010). Unexpected oligomeric structure of the FocA formate channel of Escherichia coli: a paradigm for the formate-nitrite transporter family of integral membrane proteins. FEMS Microbiol. Lett. 303, 69–75.10.1111/j.1574-6968.2009.01862.xSearch in Google Scholar
Feng, Z., Hou, T., and Li, Y. (2012). Concerted movement in pH-dependent gating of FocA from molecular dynamics simulations. J. Chem. Inf. Model. 52, 2119–2131.10.1021/ci300250qSearch in Google Scholar
Jia, W.J., Tovell, N., Clegg, S., Trimmer, M., and Cole, J. (2009). A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake. Biochem. J. 417, 297–304.10.1042/BJ20080746Search in Google Scholar
Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.10.1038/227680a0Search in Google Scholar
Leonhartsberger, S., Korsa, I., and Böck, A. (2002). The molecular biology of formate metabolism in enterobacteria. J. Mol. Microb. Biotechnol. 4, 269–276.Search in Google Scholar
Lowry, O., Rosebrough, N., Farr, A., and Randall R. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.10.1016/S0021-9258(19)52451-6Search in Google Scholar
Lü, W., Du, J., Wacker, T., Gerbig-Smentek, E., Andrade, S.L., and Einsle, O. (2011). pH-dependent gating in a FocA formate channel. Science 332, 352–354.10.1126/science.1199098Search in Google Scholar
Lü, W., Du, J., Schwarzer, N.J., Gerbig-Smentek, E., Einsle, O., and Andrade, S.L. (2012a). The formate channel FocA exports the products of mixed-acid fermentation. Proc. Natl. Acad. Sci. USA 109, 13254–13259.10.1073/pnas.1204201109Search in Google Scholar
Lü, W., Schwarzer, N.J., Du, J., Gerbig-Smentek, E., Andrade, S.L., and Einsle, O. (2012b). Structural and functional characterization of the nitrite channel NirC from Salmonella Typhimurium. Proc. Natl. Acad. Sci. USA 109, 18395–18400.10.1073/pnas.1210793109Search in Google Scholar
Lü, W., Du, J., Schwarzer, N.J., Wacker, T., Andrade, S.L., and Einsle, O. (2013). The formate/nitrite transporter family of anion channnels. Biol. Chem. 394, 715–727.10.1515/hsz-2012-0339Search in Google Scholar
Lutz, S., Jacobi, A., Schlensog, V., Böhm, R., Sawers, G., and Böck, A. (1991). Molecular characterisation of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli. Mol. Microbiol. 5, 123–135.10.1111/j.1365-2958.1991.tb01833.xSearch in Google Scholar
Lv, X., Liu, H., Ke, M., and Gong, H. (2013). Expolring the pH-dependent substrate transport mechanism of FocA using molecular dynamics simulation. Biophys. J. 105, 2714–2723.10.1016/j.bpj.2013.11.006Search in Google Scholar
Miller J. (1972). Experiments in Molecular Genetics. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).Search in Google Scholar
Rossmann, R., Sawers, G., and Böck, A. (1991). Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon. Mol. Microbiol. 5, 2807–2814.10.1111/j.1365-2958.1991.tb01989.xSearch in Google Scholar
Saier, M.H. Jr., Eng, B.H., Fard, S., Garg, J., Haggerty, D.A., Hutchinson, W.J., Jack, D.L., Lai, E.C., Liu, H.J., Nusinew, D.P., et al. (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422, 1–56.10.1016/S0304-4157(98)00023-9Search in Google Scholar
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. 2nd edition. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).Search in Google Scholar
Sawers, G. (1994). The hydrogenases and formate dehydrogenases of Escherichia coli. Antonie van Leeuwenhoek 66, 57–88.10.1007/BF00871633Search in Google Scholar PubMed
Sawers, R.G. (2005). Formate and its role in hydrogen production in E. coli. Biochem. Soc. Trans. 33, 42–46.10.1042/BST0330042Search in Google Scholar PubMed
Sawers, G. and Böck, A. (1988). Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12. J. Bacteriol. 170, 5330–5336.10.1128/jb.170.11.5330-5336.1988Search in Google Scholar
Sawers, G. and Clark, D.P. (2004). Fermentative pyruvate and acetyl CoA metabolism. In: Curtiss, R. III (Editor in Chief), EcoSal–Escherichia coli and Salmonella: Cellular and Molecular Biology. (Washington, D.C: ASM Press), Available from: http://www.ecosal.org.Search in Google Scholar
Schägger, H. and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223–231.10.1016/0003-2697(91)90094-ASearch in Google Scholar
Schmidt, T.G. and Skerra, A. (2007). The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat. Protoc. 2, 1528–1535.10.1038/nprot.2007.209Search in Google Scholar PubMed
Suppmann, B. and Sawers, G. (1994). Isolation and characterisation of hypophosphite-resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter. Mol. Microbiol. 11, 965–982.10.1111/j.1365-2958.1994.tb00375.xSearch in Google Scholar PubMed
Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.10.1073/pnas.76.9.4350Search in Google Scholar PubMed PubMed Central
von Heijne, G. and Gavel, Y. (1988). Topogenic signals in integral membrane proteins. Eur. J. Biochem. 174, 671–678.10.1111/j.1432-1033.1988.tb14150.xSearch in Google Scholar PubMed
Waight, A.B., Love, J., and Wang, D.N. (2010). Structure and mechanism of a pentameric formate channel. Nat. Struct. Mol. Biol. 17, 31–37.10.1038/nsmb.1740Search in Google Scholar PubMed PubMed Central
Waight, A.B., Czyzewski, B.K., and Wang, D.N. (2013). Ion selectivity and gating mechanisms of FNT channels. Curr. Opin. Struct. Biol. 23, 499–506.10.1016/j.sbi.2013.05.007Search in Google Scholar PubMed PubMed Central
Wang, Y., Huang, Y., Wang, J., Cheng, C., Huang, W., Lu, P., Xu, Y.N., Wang, P., Yan, N., and Shi, Y. (2009). Structure of the formate transporter FocA reveals a pentameric aquaporin-like channel. Nature 462, 467–472.10.1038/nature08610Search in Google Scholar PubMed
White, W.B. and Ferry, J.G. (1992). Identification of formate dehydrogenase-specific messenger-RNA species and nucleotide-sequence of the fdhC gene of Methanobacteriumformicicum. J. Bacteriol. 174, 4997–5004.10.1128/jb.174.15.4997-5004.1992Search in Google Scholar PubMed PubMed Central
Supplemental Material: The online version of this article (DOI 10.1515/hsz-2014-0154) offers supplementary material, available to authorized users.
©2014 by Walter de Gruyter Berlin/Boston