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• W2043841973 abstract "The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10–50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+and Co2+ was decreased 3–10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2–5-fold. Projection of the third transmembrane segment as an α-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the α-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA. The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10–50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+and Co2+ was decreased 3–10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2–5-fold. Projection of the third transmembrane segment as an α-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the α-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA. third transmembrane segment (of CorA). CorA is a high capacity, constitutively expressed Mg2+transport system of Salmonella typhimurium (1Hmiel S.P. Snavely M.D. Miller C.G. Maguire M.E. J. Bacteriol. 1986; 168: 1444-1450Crossref PubMed Scopus (119) Google Scholar, 2Hmiel S.P. Snavely M.D. Florer J.B. Maguire M.E. Miller C.G. J. Bacteriol. 1989; 171: 4742-4751Crossref PubMed Google Scholar). ThecorA locus encodes a single polypeptide of 316 amino acids with a predicted molecular mass of 37 kDa which is sufficient by itself to mediate the uptake of Mg2+. CorA mediates the influx of Mg2+ with an affinity of about 20 μm and can also mediate the influx of Ni2+ and Co2+, albeit only at extracellular concentrations that are toxic to the cell. The amino acid sequence of CorA lacks homology to other known families of proteins (3Smith R.L. Banks J.L. Snavely M.D. Maguire M.E. J. Biol. Chem. 1993; 268: 14071-14080Abstract Full Text PDF PubMed Google Scholar). Studies of its phylogenetic distribution (4Smith R.L. Maguire M.E. J. Bacteriol. 1995; 177: 1638-1640Crossref PubMed Google Scholar) and the recent plethora of microbial genome sequences have demonstrated that CorA is virtually ubiquitous in bacteria and the Archaea and likely forms the major Mg2+ influx system for these kingdoms (5Kehres D. Lawyer C.H. Maguire M.E. Comp. Microb. Genom. 1998; 3: 151-169Crossref PubMed Scopus (99) Google Scholar). The membrane topology of CorA has been previously studied using C-terminal protein fusions to β-lactamase and β-galactosidase (3Smith R.L. Banks J.L. Snavely M.D. Maguire M.E. J. Biol. Chem. 1993; 268: 14071-14080Abstract Full Text PDF PubMed Google Scholar). Like its amino acid sequence, its membrane topology is also unlike that of other known transport proteins. The N-terminal 235 residues reside in the periplasm while the remaining 80 amino acids form three transmembrane segments, including a short 6 residue C-terminal sequence in the cytosol. Three transmembrane segments are unlikely to be sufficient to form a transport channel or pore; thus, CorA probably functions as a homoligomer. The mechanism of ion transport through CorA may also be unique. Unlike many ion transporters and channels, CorA contains only two charged residues, both within the first membrane domain; neither is conserved in other homologs (5Kehres D. Lawyer C.H. Maguire M.E. Comp. Microb. Genom. 1998; 3: 151-169Crossref PubMed Scopus (99) Google Scholar). This suggests that the most charge dense of the common biological cations passes through the membrane without involvement of electrostatic bonds. Mg2+ coordinates virtually exclusively with oxygen rather than nitrogen or sulfur (6Martin R.B. Metal Ions Biol. 1990; 26: 1-13Google Scholar) which suggests backbone carbonyl groups and hydroxyl bearing residues within the membrane environment would be important. Sequence alignment of the CorA homologs currently available suggest a high degree of conservation of such groups in the second and third transmembrane segments. In this study, conserved residues in the third transmembrane segment (TM3)1 of CorA were mutated. All but one mutation resulted in stable expression of protein and protein insertion into the membrane. Conserved residues Phe290, Tyr309, and Phe310 at the termini of TM3 could be substituted by other aromatic residues without significant change in transport properties suggesting a structural rather than a transport role. In contrast, mutations at Tyr292, Met299, and Tyr307 showed large decreases in transport capacity and/or changes in cation affinity. We suggest that these three residues form part of the Mg2+ transport pathway within CorA. All media were obtained from Difco (Detroit, MI). All other reagents were from Sigma unless otherwise specified. Oligonucleotides were purchased from Oligos, Etc. (Wilsonville, OR) or Genosys (The Woodlands, TX). N-Minimal medium (7Nelson D.L. Kennedy E.P. J. Biol. Chem. 1971; 246: 3042-3049Abstract Full Text PDF PubMed Google Scholar) was modified to include 0.4% (w/v) glucose, 0.1% casamino acids and is referred to as supplemented N-minimal medium. When present, Mg2+ was added as MgSO4. In all media, ampicillin was used at 50 μg/ml and tetracycline at 20 μg/ml. CorA was subcloned from pRS117 (3Smith R.L. Banks J.L. Snavely M.D. Maguire M.E. J. Biol. Chem. 1993; 268: 14071-14080Abstract Full Text PDF PubMed Google Scholar) into the pAlter vector (Promega, Madison, WI) using EcoRI and BamHI sites to create pRS170. Mutants were created in pRS170 using site-directed mutagenesis with either the pAlter kit or with the Quik-Change kit (Stratagene, La Jolla, CA) and propagated in Escherichia coli DH5α. Mutations were verified by sequencing the plasmids with Sequenase (Amersham) or by automated sequencing at the Cleveland Clinic Foundation. Verified mutants were transferred by transformation to S. typhimurium JR501 for restriction modification before transformation into S. typhimurium MM281, a Mg2+ transport-deficient strain (2Hmiel S.P. Snavely M.D. Florer J.B. Maguire M.E. Miller C.G. J. Bacteriol. 1989; 171: 4742-4751Crossref PubMed Google Scholar) for transport assays. For determination of the ability of the various mutant strains to grow, each was streaked on Luria-Bertani (LB) plates containing the appropriate antibiotic overnight. Single colonies were then streaked out on N-minimal plates containing glucose and 0.1 mm leucine and incubated at 37 °C for 48 h. Overnight 25-ml cultures grown in LB broth with antibiotic were pelleted and resuspended in 1 ml of 10 mm Tris, 150 mm NaCl, pH 7.5, and passed through a French press at 12,000 psi. Cell debris and unlysed cells were pelleted by centrifugation at 16,000 × g for 20 min. The supernatant was centrifuged at 100,000 × gfor 60 min to pellet membranes. Samples resuspended in the same buffer were loaded on 10% SDS-polyacrylamide electrophoresis gels at 10 μg of protein/lane. Protein concentration was determined using the BCA assay (Pierce, Rockford, IL). A polyclonal antibody directed against a peptide of the N-terminal 16 residues of CorA was made in rabbits (Quality Controlled Biochem., Hopkinton, MA) and used for Western blot analysis. Protein was visualized by enhanced chemiluminescence (ECL, Amersham). Uptake of 63Ni2+ was performed as described previously (8Grubbs R.D. Snavely M.D. Hmiel S.P. Maguire M.E. Methods Enzymol. 1989; 173: 546-563Crossref PubMed Scopus (27) Google Scholar, 9Snavely M.D. Florer J.B. Miller C.G. Maguire M.E. J. Bacteriol. 1989; 171: 4761-4766Crossref PubMed Scopus (137) Google Scholar). Briefly, cultures of the appropriate mutant in MM281 were grown overnight in LB broth containing 100 mm Mg2+ and the appropriate antibiotic. Cells were washed twice with ice-cold N-minimal medium without added Mg2+, resuspended in supplemented N-minimal medium, and adjusted to an OD600 between 1.0 and 2.0 in the same medium. Cells could be kept on ice up to 1 h before initiation of the transport assay without loss of uptake capacity. Transport was initiated by adding 0.1-ml cells to 0.9 ml of supplemented N-minimal medium at 37 °C containing 200 μm Ni2+ (or as indicated), 0.1–1.5 μCi of 63Ni2+ per tube and the indicated Mg2+, Ni2+, or Co2+ concentration. Uptake was terminated after 5 min by addition of 5 ml of ice-cold transport wash buffer (N-minimal medium without glucose and casamino acids, with 5 mmMg2+ and 1 mm EDTA). Samples were filtered through BA85 (0.45 μ) nitrocellulose filters (Schleicher and Schuell, Keene, NH), and washed once with 5 ml of ice-cold wash buffer.63Ni2+ activity was determined by scintillation counting with an efficiency of >80%. Wild type uptake was about 1–1.5 nmol of Ni2+ min−1OD600−1. This corresponded to a range of 5 × 104 to 1.3 × 106 cpm uptake in each sample aliquot depending on the amount of63Ni2+ used. The nonspecific background level of 63Ni2+ binding to the filter and cells was 500–3000 cpm over the range of 63Ni2+used. Transport was generally measured by determining the ability of Mg2+, Ni2+, or Co2+ to inhibit the uptake of 63Ni2+. The wild type affinity of CorA for Mg2+, Ni2+, and Co2+ was about 20, 300, and 30 μm, respectively. In these assays, unless otherwise indicated, the Ni2+ concentration was set at 200 μm. This is approximately equal to theK a for Ni2+ uptake by the wild type CorA transporter and is roughly comparable to the K m for an enzyme. The maximal transport capacity of the system will be referred to as V max and is roughly comparable to this same parameter for an enzyme. If K a for Ni2+ is not changed by a mutation, then the maximal uptake measured is directly proportional to the V max of the transport system when 63Ni2+ is used as the test radioisotope. If the K a for Ni2+ is changed by a mutation, then the V max or maximal uptake capacity cannot be directly compared with that of the wild type transporter. Conversely, however, regardless of whether a mutation changes V max, the apparent inhibition constant measured in this manner directly reflects the apparent affinity of the cation for the system as a whole as long as the dose-response curves are normalized to the maximal uptake of the individual mutant within each individual experiment. Thus if the dose-response curves are plotted as a percent of the maximal uptake of that particular mutant protein within a given experiment, then the apparentK i values may be directly compared with those determined for the wild type protein. In practice, transport was performed on several strains carrying mutant CorA alleles at the same time. Control dose-response curves measuring cation inhibition of uptake by the wild type CorA allele were performed within each experiment; unless otherwise stated, comparisons of cation inhibition between strains are always within rather than between experiments. Apparent cation affinities between experiments did not vary more than 3-fold for the same mutant allele. Recent genomic sequence data has provided almost 30 examples of presumed CorA homologs in both bacteria and Archaea (5Kehres D. Lawyer C.H. Maguire M.E. Comp. Microb. Genom. 1998; 3: 151-169Crossref PubMed Scopus (99) Google Scholar). Since Mg2+ is the most charge dense of biological cations, one might expect it to interact with negatively charged residues during passage through the membrane domains of the transporter. However, among the three transmembrane segments of all the CorA homologs, only the first transmembrane segment carries charge. In the S. typhimurium CorA, Glu251 is the only negative charge within the membrane, but a negative charge at this or nearby positions is not conserved in the other CorA homologs (5Kehres D. Lawyer C.H. Maguire M.E. Comp. Microb. Genom. 1998; 3: 151-169Crossref PubMed Scopus (99) Google Scholar). Moreover, mutation of Glu251 to Ala had minimal effect on transport capacity (Table I) or apparent Mg2+affinity (data not shown), suggesting that electrostatic interactions within the membrane domains of CorA are not required for Mg2+ uptake.Table IProperties of mutant CorA transport systemsResidueMutationProtein expression at wild type level1-aSee Fig. 2 for representative examples. “Y” means that the mutant allele was expressed at levels approximately that of wild type. “N” means that little or no protein was detected on Western blot (<5% of wild type). Intermediate levels of expression were not seen for any of the mutants discussed.Fold decrease in Mg2+affinity1-bCation inhibition curves measured at 200 μm63Ni2+ were plotted for each mutant as shown in Figs. 3-7. The number shown is an average from multiple experiments with each mutant allele, estimated by taking the concentration of cation required to give 50% inhibition of the mutant allele and dividing by the concentration of cation required to give 50% inhibition of the wild type allele measured in the same experiment. A value greater than 1 indicates that the mutant allele exhibited an apparent decrease in cation affinity compared to wild type while a value of 1 indicates no change in cation affinity compared to wild type. No mutant tested exhibited an increase in affinity.Percent of wild type transport1-cThe value given is the percent of uptake of the mutant allele compared to the uptake of a wild type allele measured in the same experiment at a single extracellular Ni2+ concentration of 200 μm, approximating the K a of the CorA transporter for Ni2+ (see “Materials and Methods”). Values are the average of at least two (± range) and as many as nine (± S.D.) independent experiments with each mutant. A level of 2% uptake would correspond in most experiments to 1000–3000 cpm uptake over a background of 2500–3000 cpm, but replicate variability is very high at such levels of uptake because of the extremely high amount of radioisotope added; thus, any value less than 5% of wild type should be considered as an estimate. See the discussion under “Materials and Methods” and comments on specific mutations in the text.Glu251AY187 ± 2Phe290WY199 ± 6AN3<2Gly291AY148 ± 14Tyr292CY>10<2FY>10<2IY>10<2SY>10<2Pro293AY528 ± 6Met299CY507 ± 2AY503 ± 1Tyr307AY1075 ± 10SY357 ± 4FY3102 ± 21Tyr309AY184 ± 2FY1102 ± 40Phe310AY10<2YY169 ± 24WY140 ± 71-a See Fig. 2 for representative examples. “Y” means that the mutant allele was expressed at levels approximately that of wild type. “N” means that little or no protein was detected on Western blot (<5% of wild type). Intermediate levels of expression were not seen for any of the mutants discussed.1-b Cation inhibition curves measured at 200 μm63Ni2+ were plotted for each mutant as shown in Figs. Figure 3, Figure 4, Figure 5, Figure 6, Figure 7. The number shown is an average from multiple experiments with each mutant allele, estimated by taking the concentration of cation required to give 50% inhibition of the mutant allele and dividing by the concentration of cation required to give 50% inhibition of the wild type allele measured in the same experiment. A value greater than 1 indicates that the mutant allele exhibited an apparent decrease in cation affinity compared to wild type while a value of 1 indicates no change in cation affinity compared to wild type. No mutant tested exhibited an increase in affinity.1-c The value given is the percent of uptake of the mutant allele compared to the uptake of a wild type allele measured in the same experiment at a single extracellular Ni2+ concentration of 200 μm, approximating the K a of the CorA transporter for Ni2+ (see “Materials and Methods”). Values are the average of at least two (± range) and as many as nine (± S.D.) independent experiments with each mutant. A level of 2% uptake would correspond in most experiments to 1000–3000 cpm uptake over a background of 2500–3000 cpm, but replicate variability is very high at such levels of uptake because of the extremely high amount of radioisotope added; thus, any value less than 5% of wild type should be considered as an estimate. See the discussion under “Materials and Methods” and comments on specific mutations in the text. Open table in a new tab We therefore turned our attention to more conserved domains within the CorA family of which the most highly conserved is the third transmembrane segment (TM3). An alignment through the TM3 regions of a selection of currently available CorA homologs is shown in Fig. 1. Assuming that the TM3 domain is bounded by the charged residues at each end, it is about 21 residues in length. Within this segment, there is a high degree of conservation for an aromatic residue at position 290, the sequence291GYP293, and a methionine at position 299, and more moderate conservation for a tyrosine at position 307 and aromatic residues at positions 309 and 310. We therefore mutated each of these positions and measured the resulting effect on formation of the CorA protein and its activity. Most positions were changed to alanine since this is a relatively benign structural substitution and would not be expected to disrupt the presumed transmembrane α-helix. Additional substitutions were chosen to conserve size or functionality or both as far as was possible. The mutants made and a summary of their properties is listed in Table I. Mutation of virtually all of these conserved residues had little effect on formation and membrane association of CorA (Fig. 2). Only the F290A mutant could not be detected in significant quantity; extended exposure indicated a small amount of F290A CorA present, roughly estimated at ≈3% of the wild type level (not shown). The fact that each mutant was expressed at about the same level as the wild type CorA indicated that a direct comparison of wild type and mutant transport activity was largely reflective of the actual transport capacity of each CorA molecule and did not reflect any significant contribution from varying levels of protein expression. Since each mutant was being expressed from a multicopy plasmid, we also checked for the presence of CorA in inclusion bodies. In all cases, including the wild type CorA expressed from pRS170, some immunoreactive material was found in inclusion bodies; however, the amount did not appear to vary between the various CorA proteins expressed (not shown). We concluded that comparison of total uptake normalized to OD600 between mutants was therefore valid as a measure of relative uptake capacity. Phe290, on the periplasmic side of TM3, was mutated to another aromatic residue, tryptophan, with little effect on transport capacity (Table I) or cation affinity (Fig. 3). In contrast, mutation of Phe290 to alanine resulted in loss of expression, the only mutant to show such a result. This position therefore appears to need a bulky, presumably aromatic residue. The lack of change in cation affinity further suggests that this position plays a largely structural role in CorA. On the cytosolic side of TM3, mutations at Tyr309 and Phe310 also had relatively little effect on cation uptake. Cation affinity was unchanged by mutation to Phe or Ala at Tyr309 (Fig. 4), and uptake capacity was unaffected (Table I). Likewise, the F310W (Fig. 4) and F310Y (not shown) mutants exhibited no change in cation affinity and maintained 40–70% of wild type uptake capacity. The F310A mutant exhibited properties similar to those seen with the F290A mutation, greater than a 95% decrease in transport capacity and a 10-fold decrease in apparent cation affinity (not shown). These results again suggest a requirement for a bulky, probably aromatic residue at position 310. Position 309 appears more flexible since the Y309A mutation had little effect.Figure 4Cation inhibition of63Ni2+ uptake with mutations at residue Tyr309 and Phe310. Transport was performed as described under “Materials and Methods.” Final Ni2+concentration was 200 μm. Within each experiment uptake is normalized to the maximal uptake of each CorA allele as 100%. See Table I for the total uptake of each CorA mutant. The data are the average of duplicate samples in one experiment representative of at least two similar experiments for all mutants shown. In the experiments shown, Co2+ was used to inhibit uptake with the mutants at residue Tyr309 while Mg2+ was used for the F301W mutant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The sequence GYP in the periplasmic half of TM3 is highly conserved in CorA, although in some homologs the proline is replaced by a bulky hydrophobic residue. A G291A mutation had no effect on cation affinity but decreased uptake capacity about 50% (Table I). A glycine may be conserved at this position in most CorA homologs (5Kehres D. Lawyer C.H. Maguire M.E. Comp. Microb. Genom. 1998; 3: 151-169Crossref PubMed Scopus (99) Google Scholar) because of its small size, positioned as it is among three amino acids with large side chains. Substitution of the tyrosine at position 292 had dramatic effects on CorA function (Fig. 5 and Table I). The sterically conservative substitution from Tyr to Phe reduced uptake to less than 2% that of wild type CorA. Mutation to Ser, another hydroxyl-bearing residue, was similarly deleterious as were mutations to Cys or Ile (Fig. 5). Because of the extremely low levels of uptake in these mutants, shifts in apparent cation affinity could not be accurately measured but appeared to be at least 10-fold for Mg2+ (Fig. 5), Co2+, and Ni2+ (data not shown). Mutation of the adjacent Pro293 residue to Ala decreased uptake to about 30% of wild type and also decreased affinity for Mg2+ about 10-fold (Table I and Fig. 3). Alteration of Met299 to Cys or Ala resulted in a significant shift in cation affinity. Measurement of uptake capacity at the wild typeK a for Ni2+ showed that the M299A mutant had about 7% of the uptake of the wild type allele while the M299C mutant had about 3% wild type uptake (Table I). With both mutants the apparent K i for Mg2+ was shifted 30-fold to the right (Fig. 6). The effect of these mutations is largely due, however, to an altered affinity for cation rather than a lessened transport capacity. Measurement of uptake at higher (1 mm) extracellular Ni2+ indicated a significant increase in total uptake with a decrease in the degree of the shift in the cation dose-response curve (not shown). Mutations at Tyr307 had more subtle effects on transport than mutations at Tyr292 and Met299 (Table I and Fig. 7). Transport capacity was not markedly affected by mutation of Tyr307 to Ser, Phe, or Ala; capacities of each of these mutants were all >50% of wild type uptake at 200 μm Ni2+ (Table I). The Y307A and Y307S mutants showed a 5–10-fold decrease in Mg2+ affinity while the Y307F mutant showed about a 3-fold decrease compared with wild type (Fig. 7). With Co2+ (not shown), apparent affinity changes from wild type were apparent for all three mutations but were less than for Mg2+. In contrast, neither the Y307F nor the Y307A mutant showed any change in Ni2+ affinity (Fig. 7), whereas Ni2+ affinity remained slightly decreased for the Y307S mutant. Thus, we conclude that Tyr307 also apparently interacts with cation as it moves through the membrane and to at least a small degree determines cation selectivity. Growth on N-minimal medium plates containing 0.4% glucose as carbon source roughly reflected the ability of the various mutants to transport magnesium. The F290A mutant, as would be expected from its lack of production of protein, was unable to grow. Likewise, the Y292F, Y292S, Y292C, and Y292I mutants grew only on media supplemented with a high concentration of Mg2+. Other mutations were able to grow normally, despite, in some cases, a significant decrease in capacity and/or cation affinity. This latter result is not surprising since CorA has a very high capacity, one which certainly exceeds the needs for normal growth (2Hmiel S.P. Snavely M.D. Florer J.B. Maguire M.E. Miller C.G. J. Bacteriol. 1989; 171: 4742-4751Crossref PubMed Google Scholar, 10Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (147) Google Scholar). Therefore, for a mutation in CorA to prevent growth, either capacity must be completely abolished or both capacity and cation affinity must be markedly decreased, as appears to be the case with most of the Tyr292 mutations. The CorA family of Mg2+ transporters lacks similarity in sequence and in apparent structure to other known transport systems (3Smith R.L. Banks J.L. Snavely M.D. Maguire M.E. J. Biol. Chem. 1993; 268: 14071-14080Abstract Full Text PDF PubMed Google Scholar, 5Kehres D. Lawyer C.H. Maguire M.E. Comp. Microb. Genom. 1998; 3: 151-169Crossref PubMed Scopus (99) Google Scholar). The most charge dense of the biological cations, Mg2+ might be expected to interact with negatively charged amino acids in passing through the membrane. Despite this expectation, the sequence and determined membrane topology of the S. typhimurium CorA put only a single non-conserved glutamate within the membrane. Mutation of this residue had no effect on transport. Therefore, as part of a systematic effort to determine membrane residues involved in passage of Mg2+ through the membrane, we have mutated highly conserved amino acids within the third transmembrane segment (Fig. 1). Mutation of conserved aromatic residues at each end of TM3 suggests that these residues play largely a structural role, likely stabilizing the interaction of the membrane segment at the interface of the bilayer and the aqueous solution. Alteration of Phe290, Tyr309, and Phe310 to alternative aromatic residues had little effect on either cation affinity or transport capacity. In contrast, mutation to alanine had significant deleterious effects with Phe290 and Phe310. Substitution of a small, non-aromatic residue at the membrane interface provides little stabilization within this energetically difficult region. Since other aromatic residues can substitute, this suggests that the specific residue is not particularly important as long as an aromatic ring is present. This result is consistent with results from other membrane proteins. In the photosynthetic reaction center, intramembrane aromatic residues are markedly concentrated at the membrane interfaces (11Roth M. Lewit-Bentley A. Michel H. Deisenhofer J. Huber R. Oesterhelt D. Nature. 1989; 340: 659-662Crossref Scopus (185) Google Scholar, 12Deisenhofer J. Michel H. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 247-266Crossref PubMed Scopus (92) Google Scholar) and appear to be involved in the structural transition from the hydrophobic to the hydrophilic environment. This is presumably because the electron cloud of the aromatic ring can accommodate both types of environment. In contrast to the results with the aromatic residues at the ends of TM3, mutagenesis of Tyr292 and Met299 had profound effects on function while mutations at Tyr307 had differential effects on cation affinity. Changes at the first two of these positions greatly decreased cation affinity. Results from mutations at Tyr292" @default.
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• W2043841973 title "The CorA Mg2+ Transport Protein of Salmonella typhimurium" @default.
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