An Unusual Twin-His Arrangement in the Pore of Ammonia Channels Is Essential for Substrate Conductance

Abstract

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine. Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine. Ammonium is the preferred nitrogen source for many organisms, and its transport across cellular membranes is a fundamental biological process. (In this report, we use the term ammonium to refer to both the protonated (NH4+) and the unprotonated (NH3) forms and ammonia to refer specifically to NH3.) Ammonium transport is mediated by a family of ubiquitous membrane proteins (designated Amt), found in bacteria, archaea, fungi, and plants, whose homologues in animals are the Rhesus (Rh) 3The abbreviations used are: Rh, Rhesus; GS, glutamine synthetase; r.m.s., root mean square. 3The abbreviations used are: Rh, Rhesus; GS, glutamine synthetase; r.m.s., root mean square. proteins (1von Wirén N. Merrick M. Trends Curr. Genet. 2004; 9: 95-120Crossref Google Scholar, 2Marini A.-M. Matassi G. Raynal V. Andre B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (296) Google Scholar). Evidence is accumulating that Rh proteins also facilitate the transport of ammonium (3Westhoff C.M. Ferreri-Jacobia M. Mak D.O. Foskett J.K. J. Biol. Chem. 2002; 277: 12499-12502Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 4Winkler F.K. Pfluegers Arch. Eur. J. Physiol. 2006; 451: 701-707Crossref PubMed Scopus (70) Google Scholar), but the mechanism of action of both the Amt and Rh proteins is not yet understood. Escherichia coli encodes a single Amt protein, AmtB, which is a stable trimer in the cytoplasmic membrane and retains this structure when purified and reconstituted in the presence of lipids (5Blakey D. Leech A. Thomas G.H. Coutts G. Findlay K. Merrick M. Biochem. J. 2002; 364: 527-535Crossref PubMed Scopus (74) Google Scholar, 6Conroy M.J. Jamieson S.J. Blakey D. Kaufmann T. Engel A. Fotiadis D. Merrick M. Bullough P.A. EMBO Rep. 2004; 5: 1153-1158Crossref PubMed Scopus (41) Google Scholar). X-ray crystallographic structures of E. coli AmtB and of the orthologous Archaeoglobus fulgidus Amt-1 have revealed a narrow channel within each monomer (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, 8Zheng L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar, 9Andrade S.L. Dickmanns A. Ficner R. Einsle O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14994-14999Crossref PubMed Scopus (185) Google Scholar). At its periplasmic end, a binding site for NH4+ has been identified, but entry from this site into the channel appears sterically hindered by the side chains of two conserved phenylalanines (Phe-107 and Phe-215 in AmtB). The structure analysis of E. coli AmtB (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, 8Zheng L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) led to the conclusion that the protein is a channel that conducts uncharged NH3. Stopped-flow experiments with purified AmtB reconstituted into proteoliposomes (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar) and in vivo functional characterization of E. coli AmtB are consistent with this view (10Javelle A. Thomas G. Marini A.M. Kramer R. Merrick M. Biochem. J. 2005; 390: 215-222Crossref PubMed Scopus (81) Google Scholar). A variety of studies on Rh proteins also support an ammonia channel model (11Ripoche P. Bertrand O. Gane P. Birkenmeier C. Colin Y. Cartron J.-P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17222-17227Crossref PubMed Scopus (141) Google Scholar, 12Zidi-Yahiaoui N. Mouro-Chanteloup I. D'Ambrosio A.M. Lopez C. Gane P. Le Van K.C. Cartron J.P. Colin Y. Ripoche P. Biochem. J. 2005; 391: 33-40Crossref PubMed Scopus (73) Google Scholar, 13Mayer M. Schaaf G. Mouro I. Lopez C. Colin Y. Neumann P. Cartron J.P. Ludewig U. J. Gen. Physiol. 2006; 127: 133-144Crossref PubMed Scopus (84) Google Scholar). Almost all Amt/Rh proteins contain two conserved histidines, which in AmtB are located within transmembrane helices M5 (His-168) and M10 (His-318) such that their side chains protrude into the channel (14Conroy M.J. Bullough P.A. Merrick M. Avent N.D. Br. J. Haematol. 2005; 131: 543-551Crossref PubMed Scopus (80) Google Scholar). They are related by the 2-fold quasi-symmetry of AmtB, and their adjacent imidazole rings are arranged such that an unusual lateral hydrogen bond is formed between their two δ1 nitrogens. It has been proposed that these histidines might have a role in the deprotonation of ammonium ions before they traverse the channel as uncharged NH3 (4Winkler F.K. Pfluegers Arch. Eur. J. Physiol. 2006; 451: 701-707Crossref PubMed Scopus (70) Google Scholar, 7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, 8Zheng L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar). Alternatively, it has been suggested that deprotonation of NH4+ occurs at the periplasmic binding site through some general base present in the periplasm (4Winkler F.K. Pfluegers Arch. Eur. J. Physiol. 2006; 451: 701-707Crossref PubMed Scopus (70) Google Scholar) or, based on molecular dynamics simulations, through a highly conserved Asp residue (Asp-160 in E. coli AmtB) (15Lin Y. Cao Z. Mo Y. J. Am. Chem. Soc. 2006; 128: 10876-10884Crossref PubMed Scopus (65) Google Scholar). In a number of fungal Amt proteins, a Glu residue replaces the His-168 orthologue, and these proteins can also facilitate ammonium transport (16Smith D.G. Garcia-Pedrajas M.D. Gold S.E. Perlin M.H. Mol. Microbiol. 2003; 50: 259-275Crossref PubMed Scopus (85) Google Scholar, 17Biswas K. Morschhauser J. Mol. Microbiol. 2005; 56: 649-669Crossref PubMed Scopus (149) Google Scholar, 18Marini A.-M. Soussi-Boudekou S. Vissers S. Andre B. Mol. Cell. Biol. 1997; 17: 4282-4293Crossref PubMed Scopus (451) Google Scholar). Site-directed mutants offer the possibility of testing some of these predictions. In this study, we report the first such analysis and confirm that the unusual twin-His structure in the pore of AmtB is essential for optimum substrate conductance. Strains, Plasmids, and Culture Conditions—Strains and plasmids are listed in Table 1. Mutagenesis primers are listed in Supplemental Table SI. Growth of E. coli was as described previously (10Javelle A. Thomas G. Marini A.M. Kramer R. Merrick M. Biochem. J. 2005; 390: 215-222Crossref PubMed Scopus (81) Google Scholar). To construct pJT6E (the template for all PCR experiments), the PvuI-BamHI fragment from pGC2 was cloned into PvuI-BamHI-cut pJT6. pAJ2011 and pAJ2012 were constructed using the QuikChange™ mutagenesis kit (Stratagene). pAJ2014, pAJ2015, pAJ2017, and pAJ2036-pAJ2045 were constructed according to a published method (19Zheng L. Baumann U. Reymond J.L. Nucleic Acids Res. 2004; 32: e115Crossref PubMed Scopus (835) Google Scholar). The vector for pAJ2045 was pET22b.TABLE 1E. coli strain and plasmids used in this studyStrain/plasmidGenotypeRelevant phenotypeReference/sourceStrainGT1000rbs lacZ::IS gyrA hutCK ΔglnKamtBΔAmtB20Coutts G. Thomas G. Blakey D. Merrick M. EMBO J. 2002; 21: 536-545Crossref PubMed Scopus (187) Google ScholarPlasmidspAJ2011glnK amtB19AmtBH318AThis workpAJ2012glnK amtB17AmtBH168EThis workpAJ2014glnK amtB16AmtBH168AThis workpAJ2015glnK amtB18AmtBH168FThis workpAJ2017glnK amtB20AmtBH318FThis workpAJ2036glnK amtB21AmtBH168QThis workpAJ2037glnK amtB22AmtBH168NThis workpAJ2038glnK amtB23AmtBH168KThis workpAJ2039glnK amtB24AmtBH168DThis workpAJ2040glnK amtB25AmtBH168TThis workpAJ2041glnK amtB26AmtBH318EThis workpAJ2042glnK amtB27AmtBH318TThis workpAJ2043glnK amtB28AmtBH168E/H318EThis workpAJ2044glnK amtB29AmtBH168T/H318TThis workpAJ2045glnKamtB30AmtBH168A/H318AThis workpJT6EglnKamtB15AmtBThis workpJT6glnKamtB9AmtB21Javelle A. Severi E. Thornton J. Merrick M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google A. Severi E. Thornton J. Merrick M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google in a AmtB and coli AmtB was purified as described (5Blakey D. Leech A. Thomas G.H. Coutts G. Findlay K. Merrick M. Biochem. J. 2002; 364: 527-535Crossref PubMed Scopus (74) Google Scholar). and were as described G. Thomas G. Blakey D. Merrick M. EMBO J. 2002; 21: 536-545Crossref PubMed Scopus (187) Google Scholar) using E. coli proteins, AmtB A. Severi E. Thornton J. Merrick M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), each AmtB of of strain a were to using each and of AmtB were for X-ray were and using and were as described (10Javelle A. Thomas G. Marini A.M. Kramer R. Merrick M. Biochem. J. 2005; 390: 215-222Crossref PubMed Scopus (81) Google Scholar). and of and and were as described L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) that we used strain B. J. Mol. Biol. PubMed Scopus Google Scholar). was used for AmtB and the and the 2005; PubMed Scopus Google Scholar) was used in all The AmtB proteins for and were from a at was using A was used in and the were used to were by of protein with an of in at imidazole and in their when with those L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar). The in the imidazole was and in the and and and and and and and and and and all in are for the is for the and for the in are for the is for the and for the in are for the is for the and for the the the and in are for the is for the and for the bond bond of structures using and as at at at at and and in are for the is for the and for the the the and of structures using and L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) as structure in a were on and was at using a and a of at the protein at the of the in were with the J. 26: Scopus Google Scholar). A was used a we used the structure structure L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) in which His-168 and were to and all at and in the as as those with were Biol. 50: PubMed Scopus Google Scholar), an with to was by a of and with to the Table the residues at and of were using the previously and between and the In this was used to in the and the was using a of At this for the analysis of pore were some imidazole at site and at a and in the to the led to the and of the His-168 and the proposed of His-168 and we constructed 14 variants of E. coli AmtB, the histidines by non-polar and polar residues were into a and in the strain of the variants AmtB in to the in as a with an molecular of on and in their of the polar mutants and the mutants and were and but were also to a as on and not for the of their and that they might be variant in from AmtB were using an of cytoplasmic and membrane as in A but using of the all and archaea, is to which encodes a protein G. Coutts G. Merrick M. Trends Genet. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. Merrick M. Microbiol. Mol. Biol. PubMed Scopus Google Scholar). is a of the proteins that as of cellular nitrogen T. R. Merrick M. Microbiol. Mol. Biol. PubMed Scopus Google Scholar). of is by in that are to nitrogen and this is in nitrogen P. PubMed Scopus Google Scholar). A in ammonium to with AmtB, to the of the channel in to the nitrogen A. Severi E. Thornton J. Merrick M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In a study, we that a highly conserved AmtB was essential for as an AmtB but not affect the of protein A. Severi E. Thornton J. Merrick M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). also that AmtB was in an ammonium A. Severi E. Thornton J. Merrick M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of AmtB that which is on the periplasmic has a role (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar), and we that in AmtB its with through by the structure on the cytoplasmic this can be as of a and we the variants of AmtB for their to and a not of an ammonium of for that with one they a structure. the variants that stable on were to a with of the variant AmtB proteins was in the cytoplasmic that they were all to the these that with the of the variants are all and present as in the The of the of some variants does not that their structure is X-ray of of the variants and the variant be in the previously which their structure to a of at L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) with AmtB, the were to the residue with in the of the histidine in some not of the of the residues this In the of the the side chain is and to for the a in side chains are that the pore structure is The glutamate at which be present in its protonated appears as a of the It the and its is with the imidazole of and a hydrogen bond to its δ1 In its protonated it also as hydrogen bond for in the on the of the two side His-168 and for of the two pore histidines led to the that they a role in the substrate to the of the channel (4Winkler F.K. Pfluegers Arch. Eur. J. Physiol. 2006; 451: 701-707Crossref PubMed Scopus (70) Google Scholar, 7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, 8Zheng L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar, 9Andrade S.L. Dickmanns A. Ficner R. Einsle O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14994-14999Crossref PubMed Scopus (185) Google Scholar). to as for an ammonium to the pore and its has an and were to confirm their proposed essential the transport of each of the variants and AmtB with of when using as substrate described in previously (10Javelle A. Thomas G. Marini A.M. Kramer R. Merrick M. Biochem. J. 2005; 390: 215-222Crossref PubMed Scopus (81) Google Scholar), in these we both of by the and its by glutamine The variant a but when with in of of to was in the of protein and its within the cell to that of in all and have previously that is to AmtB (10Javelle A. Thomas G. Marini A.M. Kramer R. Merrick M. Biochem. J. 2005; 390: 215-222Crossref PubMed Scopus (81) Google Scholar), the mechanism of this is that the of a AmtB for is to an between the and also in a using and At a of when with the a strain and the variant a of that has was of at are To the role of the conserved residues we the and of in and at the pore of AmtB for the and all variant that this might be a of in the pore structure and of its and have been as an ammonium binding site at the periplasmic pore (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, 8Zheng L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) and as ammonia in the pore (designated and (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar), as L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar). between and and when and binding it be that both of to the The that the were with from of the and of the of the not and that they from of the side chains that they from the of in the The variants show at site the protein and the show and The and in these structures show of to in not At a of be and using the of The at is similar to that at with the of a and is the non-polar at this site can some that is not at In the is in the pore whose appears to be The non-polar mutants show and a of preferred The polar mutants have to and have only been for the the only that between and at and and a at site the presence of two many the presence of a with an ammonium The presence of be to pore and the of all when with the between site and present at also in the protein the of a of in this of the A number of the of imidazole in the In all these the site is and appears that imidazole can this site imidazole to the model with that those of the protein side that the be to ammonium binding ammonium is also present at in the a protonated imidazole appears to with ammonium these The for structure of E. coli AmtB (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, 8Zheng L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) revealed the of two conserved histidine side chains in the pore and suggested that they a role in substrate conductance. In this mutagenesis study, we present the first of this and confirm their essential for the of the substrate and also for to to this conclusion is the that with one the variants are at the and to the E. coli they a trimer as in vivo by their with by of some of the these we that the in AmtB are to a at the substitution site and not to a in the pore protein structure. for the partially which we show to be a of a histidine in this of the variants polar and non-polar of were suggests a functional role for the two histidines for which two of their might as for ammonium ions that traverse the of the channel as ammonia and on the Alternatively, substrate might such a narrow pore with a hydrogen bond for interactions with that The non-polar of the AmtB channel when with might some for ammonia as the is to be in studies between these two a role in substrate it can be that it is not the be from His-168 across the and Phe-215 side chains to the cytoplasmic (4Winkler F.K. Pfluegers Arch. Eur. J. Physiol. 2006; 451: 701-707Crossref PubMed Scopus (70) Google Scholar). on molecular dynamics such a mechanism has also been in of deprotonation at the periplasmic ammonium binding site (15Lin Y. Cao Z. Mo Y. J. Am. Chem. Soc. 2006; 128: 10876-10884Crossref PubMed Scopus (65) Google Scholar). for the its transport through the to ammonium and which is not in with studies (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, A. Thomas G. Marini A.M. Kramer R. Merrick M. Biochem. J. 2005; 390: 215-222Crossref PubMed Scopus (81) Google Scholar). The that the is partially active, show that on a of and of the pore and that the two histidines are not equivalent the crystallographic analysis of in the which we as ammonium that only the a The of the glutamate is but the of pore be of a the is to the side chain in the The with is it be that the presence of such a is of an AmtB in a L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar) and Amt-1 from A. fulgidus S.L. Dickmanns A. Ficner R. Einsle O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14994-14999Crossref PubMed Scopus (185) Google Scholar) not show in the that also pore In both those the pore is on the cytoplasmic side of of a at the of transmembrane (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar). In a and their might to we are that within the that we have in this study, the can be in of in pore Amt/Rh the E. coli AmtB as a we have that the two conserved histidine residues that many of the Amt/Rh family are required for a functional might that these two histidines are a for ammonia conductance. of those of the Amt/Rh fungal and the proteins, that not contain the twin-His In some fungal Amt proteins, a Glu residue replaces the first conserved have that substitution of His-168 by Glu in E. coli AmtB does not the structure and that the variant protein retains an of when with previously proposed (4Winkler F.K. Pfluegers Arch. Eur. J. Physiol. 2006; 451: 701-707Crossref PubMed Scopus (70) Google Scholar), it that the Glu residue the and can make similar interactions as the equivalent It is of to that all the fungal for which are Amt proteins, at one of which retains the twin-His those fungal Amt proteins the Glu substitution have a In the Amt homologues are the Rh proteins that have been in The Rh proteins and in contain both conserved residues and have been by a variety of to be of ammonium transport Colin Y. Cartron J.P. 2006; PubMed Scopus Google Scholar). By in the proteins, which the and proteins J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar), the twin-His is not conserved (14Conroy M.J. Bullough P.A. Merrick M. Avent N.D. Br. J. Haematol. 2005; 131: 543-551Crossref PubMed Scopus (80) Google Scholar). The equivalent residues to AmtB His-168 and are replaced by and and they hydrogen bond and a for polar interactions with substrate (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar), their be by a hydrogen bond between as occurs between the residues in AmtB (7Khademi S. O'Connell J. II I Remis J. Robles-Colmenares Y. Miercke L.J. Stroud R.M. Science. 2004; 305: 1587-1594Crossref PubMed Scopus (553) Google Scholar, 8Zheng L. Kostrewa D. Bernèche S. Winkler F.K. Li X.-D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17090-17095Crossref PubMed Scopus (281) Google Scholar). The role of and is and these proteins have not been in in the presence of Colin Y. Cartron J.P. 2006; PubMed Scopus Google Scholar). studies on from variants with of the Rh show by has on ammonium transport (11Ripoche P. Bertrand O. Gane P. Birkenmeier C. Colin Y. Cartron J.-P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17222-17227Crossref PubMed Scopus (141) Google Scholar). on these and with E. coli AmtB, it that proteins not as ammonium but have a Colin Y. Cartron J.P. 2006; PubMed Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar). In not only the of E. coli AmtB as a model for of the of the Amt protein family but also the essential role of the twin-His a for Amt protein and for on the Severi for of Thornton for for and Kostrewa for with crystallographic were at the and we the from and with