Functional Analysis of Conserved Polar Residues in Vc-NhaD, Na+/H+ Antiporter of Vibrio cholerae

Vc-NhaD is a Na+/H+ antiporter from Vibrio cholerae with a sharp maximum of activity at pH ∼ 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na+-dependent H+ transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent Km values for alkali cations, with the Km for Li+ elevated more than that for Na+, indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na+(Li+)/H+ antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent Km for Na+ without affecting the Km for Li+. These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations. Vc-NhaD is a Na+/H+ antiporter from Vibrio cholerae with a sharp maximum of activity at pH ∼ 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na+-dependent H+ transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent Km values for alkali cations, with the Km for Li+ elevated more than that for Na+, indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na+(Li+)/H+ antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent Km for Na+ without affecting the Km for Li+. These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations. Sodium proton antiporters are universal secondary ion transporters in bacteria. Typically, they expel toxic Na+ and Li+ ions from the cytoplasm at the expense of the proton motive force, thus playing an important role in cytoplasmic Na+ and pH homeostasis and providing energy for Na+ symports (for review, see Refs. 1Busch W. Saier Jr., M.H. Crit. Rev. Biochem. Mol. Biol. 2002; 37: 287-337Crossref PubMed Scopus (221) Google Scholar, 2Padan E. Schuldiner S. Bakker E. Alkali Cation Transport Systems in Procaryotes. CRC Press, Inc., Boca Raton, FL1992: 3-24Google Scholar, 3Padan E. Schuldiner S. Biochim. Biophys. Acta. 1994; 1185: 129-151Crossref PubMed Scopus (143) Google Scholar, 4Padan E. Venturi M. Gerchman Y. Dover N. Biochim. Biophys. Acta. 2001; 1505: 144-157Crossref PubMed Scopus (289) Google Scholar). The Na+/H+ antiport in a bacterial cell is often mediated by antiporters of different types working in concert. Major enterobacterial antiporters, Ec-NhaA (5Goldberg B.G. Arbel T. Chen J. Karpel R. Mackie G.A. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2615-2619Crossref PubMed Scopus (183) Google Scholar) and Ec-NhaB (6Pinner E. Padan E. Schuldiner S. J. Biol. Chem. 1992; 267: 11064-11068Abstract Full Text PDF PubMed Google Scholar), have been extensively characterized in Escherichia coli (see Refs. 7Padan E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google Scholar, 8Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar, 9Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar, 10Pinner E. Padan E. Schuldiner S. J. Biol. Chem. 1994; 269: 26274-26279Abstract Full Text PDF PubMed Google Scholar, 11Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 5382-5387Abstract Full Text PDF PubMed Google Scholar, 12Dibrov P. Taglicht D. FEBS Lett. 1993; 336: 525-529Crossref PubMed Scopus (11) Google Scholar, 13Dibrov P. FEBS Lett. 1993; 336: 530-534Crossref PubMed Scopus (14) Google Scholar, 14Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (78) Google Scholar, 15Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar, 16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Olami Y. Rimon A. Gerchman Y. Rothman A. Padan E. J. Biol. Chem. 1997; 272: 1761-1768Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 18Inoue H. Noumi T. Tsuchiya T. Kanazawa H. FEBS Lett. 1995; 363: 264-268Crossref PubMed Scopus (116) Google Scholar, 19Noumi T. Inoue H. Sakurai T. Tsuchiya T. Kanazawa H. J. Biochem. 1997; 121: 661-670Crossref PubMed Scopus (47) Google Scholar; for review also see Ref. 4Padan E. Venturi M. Gerchman Y. Dover N. Biochim. Biophys. Acta. 2001; 1505: 144-157Crossref PubMed Scopus (289) Google Scholar, and for recent progress see Refs. 20Galili L. Rothman A. Kozachkov L. Rimon A. Padan E. Biochemistry. 2002; 41: 609-617Crossref PubMed Scopus (67) Google Scholar, 21Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar, 22Hunte C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (523) Google Scholar, 23Tzuberi T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The genome of pathogenic Vibrio cholerae encodes as many as six putative structural genes of Na+/H+ antiporters (24Heidelberg J.F. et al.Nature. 2000; 406: 477-483Crossref PubMed Scopus (1447) Google Scholar). The physiological basis for such apparent redundancy could be in part explained by recent findings of additional, predominantly regulatory functions of certain bacterial Na+/H+ antiporters affecting processes such as antibiotic efflux (25Krulwich T.A. Jin J. Guffanti A.A. Bechhofer D.H. J. Mol. Microbiol. Biotechnol. 2001; 3: 237-246PubMed Google Scholar), the transcription of the Pho operon (26Pragai Z. Eschevins C. Bron S. Harwood C.R. J. Bacteriol. 2001; 183: 2505-2515Crossref PubMed Scopus (27) Google Scholar), the initiation of sporulation (27Kosono S. Ohashi Y. Kawamura F. Kitada M. Kudo T. J. Bacteriol. 2000; 182: 898-904Crossref PubMed Scopus (39) Google Scholar), and endospore germination (28Southworth T.W. Guffanti A.A. Moir A. Krulwich T.A. J. Bacteriol. 2001; 183: 5896-5903Crossref PubMed Scopus (48) Google Scholar). Studying Na+/H+ antiport in V. cholerae, we have cloned a new Na+/H+ antiporter from V. cholerae, Vc-NhaD, and expressed it in its functional form in the ΔNhaAΔNhaB strain of E. coli (29Dzioba J. Ostroumov E. Winogrodzki A. Dibrov P. Mol. Cell. Biochem. 2001; 229: 119-124Crossref Scopus (31) Google Scholar). A distinctive feature of Vc-NhaD is its pH dependence with a sharp maximum of activity at pH ∼ 8.0 (29Dzioba J. Ostroumov E. Winogrodzki A. Dibrov P. Mol. Cell. Biochem. 2001; 229: 119-124Crossref Scopus (31) Google Scholar, 30Ostroumov E. Dzioba J. Loewen P.C. Dibrov P. Biochim. Biophys. Acta. 2002; 1564: 99-106Crossref PubMed Scopus (15) Google Scholar). This distinguishes Vc-NhaD from other major enterobacterial antiporters. Indeed, Ec-NhaB from E. coli is pH-independent, whereas the activity of Ec-NhaA gradually increases upon pH shift from 7.0 to 8.0, reaching a plateau (9Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). Curiously, homologous NhaD from Vibrio parahaemolyticus exhibits pH dependence similar to Ec-NhaA rather than Vc-NhaD (31Nozaki K. Kuroda T. Mizushima T. Tsuchiya T. Biochim. Biophys. Acta. 1998; 1369: 213-220Crossref PubMed Scopus (59) Google Scholar). The Na+/H+ antiporters of NhaD type are widely distributed in nature, being found in genomes of pathogenic vibrios, nitrogen-fixing symbionts, magnetotactic cocci and photosynthetic bacteria as well as in higher plants (Fig. 1). In obligate intracellular parasites of Chlamydia genus, NhaD serves as a sole Na+/H+ antiporter (32Häse C. Fedorova N. Galperin M.Y. Dibrov P. Microbiol. Mol. Biol. Rev. 2001; 65: 353-370Crossref PubMed Scopus (196) Google Scholar, 33Dibrov P. Dibrov E. Pierce G.N. Galperin M.Y. J. Mol. Microbiol. Biotechnol. 2004; 8: 1-6Crossref PubMed Scopus (17) Google Scholar). However, very little is known about the molecular mechanisms of cation exchange mediated by these proteins. In the absence of a detailed crystal structure, identification of functionally important residues in antiporters by site-directed mutagenesis remains one of the most informative approaches. Because Na+/H+ antiporters are exchanging cations, negatively charged residues are obvious primary targets for mutagenesis (see for example Refs. 18Inoue H. Noumi T. Tsuchiya T. Kanazawa H. FEBS Lett. 1995; 363: 264-268Crossref PubMed Scopus (116) Google Scholar and 23Tzuberi T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In our previous work we found that mutation of three polar residues, Asp-344, Thr-345 (TMS 3The abbreviations used are:TMStransmembrane segmentEc-NhaA and Ec-NhaBNa+/H+ antiporters of NhaA type and NhaB type from E. coli, respectivelyVc-NhaDNa+/H+ antiporter of NhaD type from V. choleraeΔpHpH difference across the membranePBSphosphate-buffered saline X and loop IX-X), and Asp-393 (within TMS XI) severely affects the Na+-dependent proton transfer mediated by Vc-NhaD (30Ostroumov E. Dzioba J. Loewen P.C. Dibrov P. Biochim. Biophys. Acta. 2002; 1564: 99-106Crossref PubMed Scopus (15) Google Scholar). In the present study we extended these observations by mutating Vc-NhaD residues that are conserved in NhaD homologues from different organisms (see Fig. 1). In particular, the following questions polar residues and Asp-393 in TMS functionally important as the conserved polar residues associated with TMS and the of of the conserved His residues involved in the pH of Vc-NhaD, such as in Ec-NhaA Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar, 16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google the of residues by the transmembrane Na+/H+ antiporters of NhaA type and NhaB type from E. coli, Na+/H+ antiporter of NhaD type from V. cholerae pH difference across the membrane saline we the identification of three distinct of functionally important polar residues the is associated with TMS at the side of the is at the opposite side cytoplasm (TMS and in TMS is located in the middle of the transmembrane with residues His-93 and the pH of the antiport and its to alkali cations, these form a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported ions in a to the of Ec-NhaA C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (523) Google Scholar). from from and Na+/H+ strain of E. coli, by E. Padan of and used as the at in in by E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google Scholar). in with mutagenesis with the site-directed mutagenesis as by the The to in the a for the the with the and as a used (29Dzioba J. Ostroumov E. Winogrodzki A. Dibrov P. Mol. Cell. Biochem. 2001; 229: 119-124Crossref Scopus (31) Google Scholar). from and by to for the of The the mutation and used to the in the of of in by of and of membrane by a bacterial a and for Na+/H+ antiport activity as (29Dzioba J. Ostroumov E. Winogrodzki A. Dibrov P. Mol. Cell. Biochem. 2001; 229: 119-124Crossref Scopus (31) Google Scholar, 30Ostroumov E. Dzioba J. Loewen P.C. Dibrov P. Biochim. Biophys. Acta. 2002; 1564: 99-106Crossref PubMed Scopus (15) Google Scholar). in membrane by the The Na+/H+ antiporter activity by the of of in of a and to the of the by the of and the resulting of with the at and at Na+/H+ antiporter activity its to the upon the of the of in the for the of The antiport are expressed as of the In of homologues in organisms a S. T. A. J. Z. W. D. 1997; PubMed Scopus Google Scholar) the A the conserved polar residues located associated with putative transmembrane segments as well as conserved His and residues is in Fig. membrane of NhaD from V. cholerae (Fig. the M. 1994; Google Scholar) and J. Mol. Biol. 1998; PubMed Scopus Google Scholar) The the of Ec-NhaA C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (523) Google Scholar) in to the functionally important residues of Vc-NhaD W. A. K. J. Mol. PubMed Scopus Google Scholar). of Vc-NhaD in by the following from a putative loop of for The the the to The by the with a the in of and of pH to to the of membrane of membrane by the for 15 in of The by at in for The membrane in of a and at for with of in and a membrane The at with in with for the membrane with the at a of in in The for with and for with at a of in in The for with the as by the A used to the resulting of the for the for of from different in Fig. be three with higher of The one from bacteria that are have a in their in Fig. obligate intracellular three and in Fig. the The third NhaD from nitrogen-fixing and higher residues are conserved in and by in Fig. 1). In Vc-NhaD these residues are and this we Asp-393 (30Ostroumov E. Dzioba J. Loewen P.C. Dibrov P. Biochim. Biophys. Acta. 2002; 1564: 99-106Crossref PubMed Scopus (15) Google Scholar) in a study that also Asp-344, and are conserved in antiporters to as well as the conserved The residues in this a and Because our to charged side chains in of in two the of by In to the conserved residues, six His residues, and are conserved at in to (Fig. could have a role in the pH of the antiporter of their Indeed, mutation of conserved in Ec-NhaA the pH of the antiporter Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar, 16Rimon A. Gerchman Y. Olami Y. Schuldiner S. Padan E. J. Biol. Chem. 1995; 270: 26813-26817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Vc-NhaD only three residues, and are conserved in (Fig. Because the of Ec-NhaA is as as type Y. Rimon A. Gerchman Y. Rothman A. Padan E. J. Biol. Chem. 1997; 272: 1761-1768Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 21Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar), the residues of Vc-NhaD also for of Vc-NhaD of the and the the the Na+/H+ of E. The to in with indicating that the Vc-NhaD the of activity of the Vc-NhaD in inside-out membrane from that it is to that of the type with Na+ and Li+ (Fig. as in the of Ec-NhaA Y. Rimon A. Gerchman Y. Rothman A. Padan E. J. Biol. Chem. 1997; 272: 1761-1768Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 21Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar), in Vc-NhaD be without of of His the of the conserved His residues in Vc-NhaD with In inside-out and the Na+(Li+)/H+ antiport activity H93A and an acidic shift of the pH of the antiport for alkali (Fig. The shift more in the H93A also to Na+ and Li+ higher activity with Li+ (Fig. the of H93A and to alkali cations, the of Na+ and Li+ for Km at pH in the that apparent Km is only to the Km of the this is used to antiporter to substrate ions (for see Ref. 14Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (78) Google Scholar). the apparent Km of H93A for Na+ higher than in the whereas its apparent Km for Li+ only a increase in the apparent Km of for Li+ of different Vc-NhaD to alkali Km in a new with IV, V, and of the Vc-NhaD membrane suggests that the conserved residues and form a the side of the (see Fig. S150A, D154G, N155A, D199A, T201A, T202A, and to the in and of activity in that they to the and H+ at pH (Fig. and data that this to the of (Fig. the T157A in the of The T157A also activity in (Fig. and a for alkali the of apparent Km values a difference in of the antiporter to alkali with Na+ the substrate of different Vc-NhaD in the with at D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, in a new of different of Vc-NhaD in the of mutation the and of the The of the of very similar to that of with as of in TMS and we found that Asp-393 in TMS as well as and Thr-345 at the cytoplasmic of TMS X are functionally important (30Ostroumov E. 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Biochemistry. 2002; 41: 609-617Crossref PubMed Scopus (67) Google Scholar and E. Tzubery T. K. Kozachkov L. Rimon A. Galili L. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar). This the residues that the pH of an antiporter and involved in the translocation of cations. The residues are to have a in the physiological pH the pH of antiport without an the Km of antiport, and the pH of a at of substrate alkali cations. In of such residues could form an pH L. Rothman A. Kozachkov L. Rimon A. Padan E. Biochemistry. 2002; 41: 609-617Crossref PubMed Scopus (67) Google Scholar, E. Tzubery T. K. Kozachkov L. Rimon A. Galili L. Biochim. Biophys. 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In particular, a negatively charged with and at its from the cytoplasm to a putative catalytic site in the middle of the membrane the charged/polar residues and the opposite side of the a negatively charged with at its the to the site C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (523) Google Scholar). The for the access of substrate ions the to the catalytic could in the C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. 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The of in and in residues of are in with the that of with form a with that substrate the putative catalytic site associated with The distribution of functionally important residues in the Vc-NhaD molecule suggests that two of transmembrane and be located close to one Because the of Vc-NhaD is (Fig. the of Vc-NhaD as well as the membrane of the be by In particular, of residues at and different one the of This work is are to T. A. Krulwich for of the and with

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