Unraveling Functional and Structural Interactions between Transmembrane Domains IV and XI of NhaA Na+/H+ Antiporter of Escherichia coli
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m400288200
ISSN1083-351X
AutoresLivnat Galili, Katia Herz, Orly Dym, Etana Padan,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoA functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N′-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA. A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N′-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA. Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals, and microorganisms. They are involved in cell energetics and play primary roles in the regulation of intracellular pH, cellular Na+ content, and cell volume (for a recent review, see Ref. 1Padan E. Rimon A. Tzuberi T. Muller M. Herz K. Galili L. Karmazyn N. Avkiran M. Fliegel L. The Sodium-Hydrogen Exchange, from Molecule to Its Role in Disease. Kluwer Academic Publishers, Boston, MA2003: 91-108Google Scholar). Escherichia coli has two antiporters, NhaA (2Padan E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google Scholar) and NhaB (3Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar), which specifically exchange Na+ or Li+ for H+. Only NhaA is indispensable for adaptation to high salinity, for challenging Li+ toxicity, and for growth at alkaline pH (in the presence of Na+ (4Padan E. Venturi M. Gerchman Y. Dover N. Biochim. Biophys. Acta. 2001; 1505: 144-157Crossref PubMed Scopus (276) Google Scholar)). NhaA is an electrogenic antiporter with a stoichiometry of 2H+/Na+ (5Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar, 6Venturi M. Padan E. Hunte C. Von Jagow G. Schagger H. A Practical Guide to Membrane Protein Purification. 2nd Ed. Academic Press, Amsterdam2002: 179-190Google Scholar). The first insight into the architecture of the NhaA protein was obtained by cryoelectron microscopy of two-dimensional crystals, revealing that NhaA is a dimer in which each monomer is composed of 12 TMSs 1The abbreviations used are: TMS, transmembrane segment; BMH, 1,6-bis-(maleimido)hexane; o-PDM, N,N′-o-phenylenedimaleimide; MT-S2-MTS, 1,2-ethanediyl bismethanethiosulfonate; ΔpH, pH difference across the membrane; DMF, dimethylformamide; CL-NhaA, His-tagged Cys-less NhaA.1The abbreviations used are: TMS, transmembrane segment; BMH, 1,6-bis-(maleimido)hexane; o-PDM, N,N′-o-phenylenedimaleimide; MT-S2-MTS, 1,2-ethanediyl bismethanethiosulfonate; ΔpH, pH difference across the membrane; DMF, dimethylformamide; CL-NhaA, His-tagged Cys-less NhaA. (7Williams K.A. Geldmacher-Kaufer U. Padan E. Schuldiner S. Kuhlbrandt W. EMBO J. 1999; 18: 3558-3563Crossref PubMed Scopus (110) Google Scholar, 8Williams K.A. Nature. 2000; 403: 112-115Crossref PubMed Scopus (201) Google Scholar). Similar to many other Na+/H+ antiporters, both prokaryotic (1Padan E. Rimon A. Tzuberi T. Muller M. Herz K. Galili L. Karmazyn N. Avkiran M. Fliegel L. The Sodium-Hydrogen Exchange, from Molecule to Its Role in Disease. Kluwer Academic Publishers, Boston, MA2003: 91-108Google Scholar) and eukaryotic (9Grinstein S. Woodside M. Sardet C. Pouyssegur J. Rotin D. J. Biol. Chem. 1992; 267: 23823-23828Abstract Full Text PDF PubMed Google Scholar, 10Putney L.K. Denker S.P. Barber D.L. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 527-552Crossref PubMed Scopus (415) Google Scholar, 11Wakabayashi S. Pang T. Hisamitsu T. Shigekawa M. Karmazyn N. Avkiran M. Fliegel L. The Sodium-Hydrogen Exchange, from Molecule to its Role in Disease. Kluwer Academic Publishers, Boston, MA2003: 35-39Google Scholar), one of the most interesting functional characteristics of NhaA is its dramatic dependence on pH. The rate of activity of NhaA changes over 3 orders of magnitude between pH 7 and 8 (4Padan E. Venturi M. Gerchman Y. Dover N. Biochim. Biophys. Acta. 2001; 1505: 144-157Crossref PubMed Scopus (276) Google Scholar, 5Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). This activation is accompanied by a conformational change that involves the N terminus as probed by a monoclonal antibody (1F6 (12Venturi M. Rimon A. Gerchman Y. Hunte C. Padan E. Michel H. J. Biol. Chem. 2000; 275: 4734-4742Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar)) and loop VIII-IX (Fig. 1) as probed by accessibility of NhaA to trypsin (13Gerchman Y. Rimon A. Padan E. J. Biol. Chem. 1999; 274: 24617-24624Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Amino acid residues that affect the transport activity can be identical to or different from those affecting the pH regulation of NhaA. They are clustered in various domains along the protein (14Galili L. Rothman A. Kozachkov L. Rimon A. Padan E. Biochemistry. 2002; 41: 609-617Crossref PubMed Scopus (66) Google Scholar, 15Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (77) Google Scholar, 16Inoue H. Noumi T. Tsuchiya T. Kanazawa H. FEBS Lett. 1995; 363: 264-268Crossref PubMed Scopus (113) Google Scholar, 17Rimon A. Gerchman Y. Kariv Z. Padan E. J. Biol. Chem. 1998; 273: 26470-26476Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), implying the need of atomic structure for understanding the mechanism of activity and pH regulation of NhaA. Since atomic resolution of NhaA is not yet available, indirect approaches for structure analysis have been applied. Second site suppressor mutations were isolated to mutation G338S, a pH-conditional lethal mutant that is located in TMS XI and grows on high Na+-selective medium at neutral pH but not at alkaline pH (17Rimon A. Gerchman Y. Kariv Z. Padan E. J. Biol. Chem. 1998; 273: 26470-26476Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The suppressor mutations were found clustered in helix IV (A127T, P129L, and A127V) (Fig. 1) (17Rimon A. Gerchman Y. Kariv Z. Padan E. J. Biol. Chem. 1998; 273: 26470-26476Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These results have suggested that residues in TMSs IV and XI are in close proximity and functionally and/or structurally interact (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar). We therefore scanned genetically by Cys replacements conserved residues in TMSs IV and XI and identified new residues that participate in the activity and/or pH regulation of the antiporter. We then constructed double Cys replacements, one in TMS IV and the other in TMS XI, and found that many of the pairs of the Cys replacements exhibited a negative interaction, suggesting functional and/or structural interactions between TMSs IV and XI. However, long range effects can explain interaction between pairs of mutants. Site-directed thiol cross-linking studies have provided considerable insight into the structure of membrane proteins by providing estimates of distances between certain positions in proteins (19Falke J.J. Koshland Jr., D.E. Science. 1987; 237: 1596-1600Crossref PubMed Scopus (228) Google Scholar, 20Yu H. Kono M. McKee T.D. Oprian D.D. Biochemistry. 1995; 34: 14963-14969Crossref PubMed Scopus (119) Google Scholar, 21Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar, 22Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). We have used this approach to probe proximities between periplasmic and cytoplasmic loops of NhaA and found very strong cross-linking within the pairs of Cys replacements S146C/L316C and A118C/S352C (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar) (Fig. 1), suggesting close proximity between the neighboring TMSs IV and XI. However, since loops can be flexible, the distances obtained are not conclusive. Therefore, in the present work, in addition to the genetic approach, we measured distances between the Cys replacements in TMSs IV and XI by intramolecular site-directed cross-linking and identified several double mutants that strongly cross-linked. Together with the phenotypes of the double mutants, this cross-linking defined an interface between TMSs IV and XI with functional implications. Remarkably, cross-linking of the lethal double mutant T132C/G338C revived both the Na+ and Li+ antiporter activities of the mutant and even its regulation by pH, as would be expected if the cross-linking restored an active conformation of the antiporter. Bacterial Strains and Culture Conditions—EP432 is an Escherichia coli K-12 derivative, which is melBLid, ΔnhaA1::kan, ΔnhaB1::cat, ΔlacZY, thr1 (3Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar). TA16 is nhaA+nhaB+lacIQ (TA15lacIQ) and otherwise isogenic to EP432 (5Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). Cells were grown either in L broth (LB) or in modified L broth (LBK (2Padan E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google Scholar)). Where indicated, the medium was buffered with 60 mm 1,3-bis[tris(hydroxymethyl)methyl]amino]propane. Cells were also grown in minimal medium A without sodium citrate (24Davies B. Mingioli E. J. Bacteriol. 1950; 60: 17-28Crossref PubMed Google Scholar) with 0.5% glycerol, 0.01% MgSO4·7H2O, and thiamine (2.5 μg/ml). For plates, 1.5% agar was used. Antibiotics were 100 μg/ml ampicillin and/or 50 μg/ml kanamycin. The resistance to Li+ and Na+ was tested as described previously (2Padan E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google Scholar). Plasmids—Plasmid pAXH (previously called pYG10) is a pET20b (Novagen) derivative encoding His-tagged NhaA (25Olami 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). pCL-AXH is a derivative of pAXH encoding a His-tagged Cys-less NhaA (CL-NhaA (25Olami 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)). pCL-GMAR100 is a derivative of pGM36 (26Karpel R. Olami Y. Taglicht D. Schuldiner S. Padan E. J. Biol. Chem. 1988; 263: 10408-10414Abstract Full Text PDF PubMed Google Scholar) encoding CL-NhaA (15Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (77) Google Scholar). pCL-BstX, a derivative of pECO (14Galili L. Rothman A. Kozachkov L. Rimon A. Padan E. Biochemistry. 2002; 41: 609-617Crossref PubMed Scopus (66) Google Scholar), carries a silent mutation that introduces a BstXI site at codon 248 of the nhaA gene and encodes CL-NhaA. pCL-HAH4 bears the silent BstXI mutation and encodes His-tagged CL-NhaA as described in Ref. 18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar. Site-directed Mutagenesis—Site-directed mutagenesis was conducted following a polymerase chain reaction-based protocol (27Ho S. Hunt H. Horton R. Pullen J. Pease L. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar). All mutations were verified by DNA sequencing of the entire gene, through the ligation junctions with the vector plasmid. For Cys replacement of Gly125, Phe136, and Ala137, pCL-HAH4 was used as a template with the mutagenic primers described in Table I. The resulting plasmids were pCL-HAH4-G125C, pCL-HAH4-F136C, and pCL-HAH4-A137C. For Cys replacement of Leu334 and Ser342, pCL-BstX was used as a template with the mutagenic primers described in Table I. The resulting plasmids were pCL-BstX-L334C and pCL-BstX-S342C. To generate L334C and S342C mutation in pCL-HAH4, the MunI-MluI fragment (571 bp) of pCL-BstX-L334C or pCL-BstX-S342C was ligated with the MunI-MluI fragment (4.17 kb) of pCL-HAH4, yielding pCL-HAH4-L334C and pCL-HAH4-S342C. For Cys replacement of Gly338, pCL-GMAR100 was used as a template with the mutagenic primers described in Table I. The resulting plasmid was pCL-GMAR100-G338C. For construction of this mutation in pCL-AXH (pCL-AXH-G338C), a NheI-MluI fragment (879 bp) of the resulting PCR product was ligated to NheI-MluI (4.1-kb) fragment of pAXH.Table IOligonucleotides used for constructing the Cys mutations in nhaA The mutated bases are shown in boldface type. Additional substitutions (*) have been introduced to create silent mutations in Gly-125, Phe-136, Ala-137, Leu-334, Gly-338, and Ser-342 that generate unique restriction sites BsmI, NsbI, ScaI, NdeI, SspI, and BsaBI, respectively, in the sequence of nhaA.MutationDNA sequence of mutagenic oligonucleotideCodon change observedG125CCCCGCGAATGCT*GGGCGATCCCGGG → TGCF136CGCTACTGACATTGCTTGC*GCACTTGGTTT → TGCA137CGCTTTTTGTCTTGGAGT*ACTGGCGCA → TGTL334CGGGCA*TATGTTCCGGTATCGGCTG → TGTG338CCGGTATCTGTTTTACTATGTCAAT*GGT → TGTS342CCGGTTTTACGATGT*GTATCTTTATTGCCTCT → TGTEnd primers for mutations G125C, F136C, A137C, and G338CTTTAACGATGATTCGTGGCGG (sense primer)NoneGCTCATTTCTCTCCCTGATAAC (antisense primer)End primers for mutations L334C and S342CGTGTGGTTGTCGACGCACGGGCG (sense primer)NoneGTGGAGTTAAATAAAGCGCC (antisense primer) Open table in a new tab To generate all double mutants, the MunI-MluI fragment (571 bp) of single Cys NhaA mutants in Helix XI, on C-less background, were ligated with MunI-MluI fragment (4.17 kb) of plasmid encoding the single Cys NhaA mutants in Helix IV. Isolation of Membrane Vesicles, Assay of Na+/H+ Antiporter Activity—EP432 cells transformed with the respective plasmids were grown and everted vesicles were prepared and used to determine the Na+/H+ or Li+/H+ antiporter activity as described (28Rosen B. Annu. Rev. Microbiol. 1986; 40: 263-286Crossref PubMed Scopus (52) Google Scholar, 29Goldberg E.B. 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 (181) Google Scholar, 30Rosen B. Methods Enzymol. 1986; 125: 328-336Crossref PubMed Scopus (113) Google Scholar). The assay of antiport activity was based upon the measurement of Na+- or Li+-induced changes in the ΔpH as measured by acridine orange, a fluorescent probe of ΔpH. The fluorescence assay was performed with 2.5 ml of reaction mixture containing 50-100 μg of membrane protein, 0.5 μm acridine orange, 150 mm KCl, 50 mm 1,3-bis[tris(hydroxymethyl)methyl-]amino]propane, 5 mm MgCl2, and the pH was titrated with HCl. Where indicated, 10 mm β-mercaptoethanol or 20 mm diamide (Sigma) was added to the reaction mixture. After energization with either ATP (2 mm) or d-lactate (2 mm), quenching of the fluorescence was allowed to achieve a steady state, and then either Na+ or Li+ (10 or 100 mm each) was added. A reversal of the fluorescence level (dequenching) indicates that protons are exiting the vesicles in antiport with either Na+ or Li+ as indicated (see Figs. 4A and 6A for data of typical experiments). As shown previously, the end level of dequenching is a good estimate of the antiporter activity (31Schuldiner S. Fishkes H. Biochemistry. 1978; 17: 706-710Crossref PubMed Scopus (103) Google Scholar), and the concentration of the ion that gives half-maximal dequenching is a good estimate of the apparent Km of the antiporter (31Schuldiner S. Fishkes H. Biochemistry. 1978; 17: 706-710Crossref PubMed Scopus (103) Google Scholar, 32Tsuboi Y. Inoue H. Nakamura N. Kanazawa H. J. Biol. Chem. 2003; 278: 21467-21473Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The concentration range of the cations tested was 0.01-100 mm at the indicated pH values, and the apparent Km values were calculated by linear regression of a Lineweaver-Burk plot.Fig. 6Cross-linking rescues the Li+/H+ and Na+/H+ antiporter activity of the double mutant T132C/G338C. Everted membrane vesicles were prepared from EP432 cells expressing CL-NhaA-His6 or mutant T132C/G338C and either treated or not with the indicated cross-linking reagents as described in Fig. 5. The Na+/H+ or Li+/H+ antiporter activity was measured with the acridine orange assay as described in the legend to Fig. 2. A and B, results of typical experiments. C, isolated membrane vesicles of mutant T132C/G338C were treated with MTS-2-MTS (2 mm). The antiporter activity (end level of dequenching as a percentage) was measured either with 100 mm NaCl (open circles) or 100 mm LiCl (closed circles) at the indicated pH values as described above. For comparison, the pH dependence of CL-NhaA is also shown. All experiments were repeated at least three times with practically identical results.View Large Image Figure ViewerDownload (PPT) Membranes treated with cross-linking reagents lost the lactate-dependent respiration. Therefore, when such membranes were used in the antiport assay, the membranes were energized by ATP. Detection and Quantitation of NhaA and Its Mutated Proteins in the Membrane—NhaA and its mutated derivatives were quantitated by Western analysis using an anti-NhaA monoclonal antibody 1F6 (33Padan E. Venturi M. Michel H. Hunte C. FEBS Lett. 1998; 441: 53-58Crossref PubMed Scopus (41) Google Scholar) as described previously (34Gerchman 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). Total membrane protein was determined according to Ref. 35Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar. In Situ Site-directed Inter- and Intramolecular Cross-linking—Site-directed intramolecular cross-linking was conducted in situ on membrane vesicles isolated from TA16 cells expressing the various NhaA double mutants. Membranes (3 mg of protein) were resuspended in a buffer (4.5 ml) containing 100 mm potassium phosphate, 5 mm MgSO4 (pH 7.4), and one of the freshly prepared homobifunctional cross-linkers: 2 mm BMH (Pierce), 1 mm o-PDM (Sigma), or 2 mm MTS-2-MTS (Toronto Research Chemicals). The stock solutions of the cross-linkers were prepared in DMF at 200 mm, so that the amount of DMF in the reaction mixture does not exceed 1%, a concentration that does not affect the antiporter activity. The suspension was incubated at 26 °C with gentle rotation for 60 min. The reaction with BMH or o-PDM was terminated by the addition of 10 mm β-mercaptoethanol and that with MTS-2-MTS, by centrifugation (Beckman; TLA 100.4, 265,000 × g, 20 min, 4 °C). The protein was Ni2+-NTA affinity-purified, treated with trypsin at alkaline pH, and separated on SDS-PAGE (nonreducing conditions in the case of treatment with MTS-2-MTS) to identify the proteolytic products according to Ref. 18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar. Since there is only one trypsin cleavage site at Lys249, located between TMSs IV and XI (Fig. 1), trypsinolysis results in two tryptic peptides of mobility faster than the intact protein. On the other hand, intramolecular cross-linking results in one fragment of mobility equal to that of the intact protein (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar). To measure the effect of the cross-linking agents on the activity of the antiporter, everted membrane vesicles were isolated from EP432 expressing the mutations (300 μg of protein); treated with one of the homobifunctional cross-linkers as described above; washed in 1.5 ml containing 140 mm choline chloride, 250 mm sucrose, and 10 mm Tris-HCl (pH 7.5); resuspended in 75 μl of the same solution; and used to test antiporter activity. The degree of cross-linking in vesicles derived from EP432 and TA16 was indistinguishable. In situ site-directed intermolecular cross-linking was tested as above but without treatment with trypsin. When intermolecular cross-linking takes place, a band, corresponding in mobility to that of NhaA dimer, appears in SDS-PAGE (15Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (77) Google Scholar, 18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar). Accessibility of the Cys Replacements to the Cross-linking Reagents— The procedure was essentially as described in Ref. 36Tzubery T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar. Following treatment with the cross-linking reagents, the Ni2+-NTA affinity-purified protein was left bound to the Ni2+-NTA beads, washed with binding buffer (5 mm imidazol, 500 mm NaCl, 20 mm Tris, and 0.1% n-dodecyl-β-d-maltoside, pH 7.4) and exposed to 0.2 mm fluorescein 5-maleimide (Molecular Probes, Inc.; dissolved in Me2SO) to titrate any Cys residue left free. The incubation was for 30 min at 25 °C with gentle tilting. Then the protein was washed and eluted as described in Ref. 36Tzubery T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar. The degree of fluorescence labeling of the protein resolved in SDS-PAGE was determined under UV light (260 nm) as described (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar, 36Tzubery T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Modeling of TMSs IV and XI—Initially, two ideal poly-Ala α-helices were built using the program Moleman (37Kleywegt G.J. Zou J.Y. Kjeldgaard M. Jones T.A. Rossmann M.G. Arnold E. Around O: International Tables for Crystallography, Crystallography of Biological Macromolecules. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 353-356Google Scholar). Then the side chains were altered to those of the respective TMS, according to the putative topology model of NhaA (Fig. 1A) (38Rothman A. Padan E. Schuldiner S. J. Biol. Chem. 1996; 271: 32288-32292Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) by using the rotamer library in the O software (39Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M.W. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Next, the distances estimated by cross-linking of double Cys replacement mutants located in TMSs IV and XI (see "Results") and on neighboring loops (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar) were used as constraints to determine the proximity between the two helices. Alternate orientations of side chains were tested, and those with geometric clashes with neighboring residues were ruled out. Energy minimization of the putative model was carried out using the program CNS (40Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16918) Google Scholar) to eliminate further clashes between neighboring residues. Construction of Single and Pairs of Cys Replacements in TMSs IV and/or XI of NhaA—In this study, site-directed Cys replacements and site-directed thiol cross-linking were used to identify functional interactions and determine proximities between TMSs IV and XI of NhaA. For this purpose, we used the single Cys replacements mutants (D133C, T132C, P129C, and A127C (14Galili L. Rothman A. Kozachkov L. Rimon A. Padan E. Biochemistry. 2002; 41: 609-617Crossref PubMed Scopus (66) Google Scholar)) that had already been constructed in TMS IV and constructed new single Cys replacement mutants: G125C, F136C, and A137C in TMS IV and L334C, G338C, and S342C in TMS XI (Tables I and II and Fig. 1). All old and new mutations replace conserved amino acid residues (Fig. 1). We then constructed double mutants, each containing one replacement in TMS IV and one in TMS XI (Table II and Fig. 1). All mutants, both single and double, were constructed in plasmid-encoded CL-NhaA that is very similar to the wild type protein in transport activity and pH regulation (25Olami 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) (Table II). To determine the level of expression, growth phenotype, and antiporter activity, the plasmids encoding the mutants were transformed into EP432, an E. coli strain devoid of both Na+-specific antiporters NhaA and NhaB (3Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar). This strain can grow in the presence of high Na+ or Li+ only when transformed with a plasmid expressing active NhaA (3Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar) and Table II (compare EP432/pCL-HAH4 with EP432/pBR322). The results summarized in Table II show that the single and double mutants were expressed to a level that ranges between 10 and 100% of the control level (EP432/pCL-HAH4). It should be stressed that because all mutants are expressed from multicopy plasmids, even the lowest level of expression observed here is significant, readily detected by Western analysis, and way above the level expressed from a single chromosomal gene that is hardly detected by Western analysis (17Rimon A. Gerchman Y. Kariv Z. Padan E. J. Biol. Chem. 1998; 273: 26470-26476Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar).Table IISingle and double Cys replacements in TMSs IV and XI of nhaA For characterization of the mutations, EP432 cells transformed with the respective plasmids were used. The expression level was expressed as a percentage of the control cells (EP432/pCL-HAH4). Growth experiments were conducted on agar plates with high Na+ (0.6 m) or high Li+ (0.2 m) at the pH values indicated in parentheses. +++, the number and size of the colonies after 24 h of incubation at 37 °C was identical to that of the wild type; ++ and +, the number of colonies was similar but size was slightly or much smaller than that of the wild type, respectively; -, no growth. Na+/H+ and Li+/H+ antiporter activity at pH 8.5 was determined with 10 mm NaCl or LiCl.MutationExpressionGrowthActivityaThe activity (end level of dequenching) is expressed as a percentage of the positive control, EP432/pCL-HAH4. EP432/pBR322 served as a negative control. The apparent Km for the ions was determined at pH 8.5, as described under "Experimental Procedures"Apparent KmNa+ (pH 7)Na+ (pH 8.3)Li+ (pH 7)Na+Li+Na+Li+%mmHelix IVG125C30+++-+++1679120.56A127C92+++++++++1001000.240.02P129C99+++++++++1001000.40.028T132C95+++++++++549112.40.7D133C10+++++++++87703.61.24F136C100+--10102A137C100+++++++++42847.20.8Helix XIL334C70+++++++++100830.840.16G338C100+++--39790.680.5S342C80++++++
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