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J Biol Chem, Vol. 273, Issue 44, 28663-28669, October 30, 1998
From the Department of Pharmacology, School of Medicine, Case
Western Reserve University, Cleveland, Ohio 44106-4965
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 CorA is a high capacity, constitutively expressed Mg2+
transport system of Salmonella typhimurium (1, 2). The
corA 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 (3). Studies of its phylogenetic distribution (4) 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
(5).
The membrane topology of CorA has been previously studied using
C-terminal protein fusions to 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 (5). 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 (6)
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 (7) 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.
Mutant Construction--
CorA was subcloned from pRS117 (3) 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 DH5 Western Blot Analysis--
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 × g for 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).
Transport--
Uptake of 63Ni2+ was
performed as described previously (8, 9). 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 mM
Mg2+ 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
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 the
Ka for Ni2+ uptake by the wild type CorA
transporter and is roughly comparable to the Km for
an enzyme. The maximal transport capacity of the system will be
referred to as Vmax and is roughly comparable to
this same parameter for an enzyme. If Ka for
Ni2+ is not changed by a mutation, then the maximal uptake
measured is directly proportional to the Vmax of
the transport system when 63Ni2+ is used as the
test radioisotope. If the Ka for Ni2+ is
changed by a mutation, then the Vmax or maximal
uptake capacity cannot be directly compared with that of the wild type
transporter. Conversely, however, regardless of whether a mutation
changes Vmax, 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 apparent
Ki 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 (5). 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 (5). 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.
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 sequence
291GYP293, 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
The CorA Mg2+ Transport Protein of Salmonella
typhimurium
MUTAGENESIS OF CONSERVED RESIDUES IN THE THIRD MEMBRANE DOMAIN
IDENTIFIES A Mg2+ PORE*
,
,
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-helix suggests that Tyr292,
Met299, and Tyr307 all reside on
the same face of the -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.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-lactamase and -galactosidase (3).
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.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
. 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
(2) 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.
1
OD6001. This corresponded to a range of
5 × 104 to 1.3 × 106 cpm uptake in
each sample aliquot depending on the amount of 63Ni2+ used. The nonspecific background level
of 63Ni2+ binding to the filter and cells was
500-3000 cpm over the range of 63Ni2+
used.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
Properties of mutant CorA transport systems
-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.
View larger version (45K):
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Fig. 1.
Conserved residues in the third transmembrane
segment of CorA. Currently available genomic sequence data shows
>23 genes encoding CorA-like proteins in bacteria and the Archaea (5).
An alignment of the third transmembrane domain of CorA is shown for a
representative selection. The numbering is that of the S. typhimurium CorA and differs slightly for the other sequences
shown. Abbreviations for the different bacterial CorA proteins are:
ST, S. typhimurium; EC, E. coli; HI, Hemophilus influenzae;
MT, Mycobacterium tuberculosis; ML,
Mycobacterium leprae; PA, Pseudomonas
aeruginosa; SY, Synechocystis sp. 6803; and
TM, Thermatoga maritima. AF denotes
the Archaeal Archeoglobus fulgidus CorA sequence. Residues
conserved in most CorA family members are in bold-face type
(5). E. coli, P. aeruginosa, and
Synechocystis sp. 6803 contain one or more CorA-like
proteins more distantly related that are not shown.
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.
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Mutations of Aromatic Residues at the Membrane Interface-- 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.
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Mutation of Gly291, Tyr292, and Pro293-- 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 (5) 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).
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Mutations at Met299-- Alteration of Met299 to Cys or Ala resulted in a significant shift in cation affinity. Measurement of uptake capacity at the wild type Ka 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 Ki 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).
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Mutations at Tyr307-- 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.
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Growth on Minimal Media--
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 (2, 10).
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.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The CorA family of Mg2+ transporters lacks similarity in sequence and in apparent structure to other known transport systems (3, 5). 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 (11, 12) 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 argue strongly that both the hydroxyl group and the aromatic ring at this position are essential. A conservative change to Phe almost abolished uptake and appeared to decrease cation affinity at least 10-fold, thus implicating the hydroxyl residue. Substitution of a Ser also abolished uptake, suggesting that the phenolic ring is required. The severe phenotype of the Y292C and Y292I mutants is fully consistent with this interpretation. The relative lack of transport by the Tyr292 mutants is presumably not due to misfolding of the protein as all of the mutants appear to insert into the membrane properly (data not shown).
The effect of mutations at Gly291 and Pro293
are relevant here. The sequence at this site in CorA consists of a
large aromatic residue, Phe290, followed by
Gly291, lacking any side chain, followed by two bulky
residues, Tyr292 and Pro293. A Gly may be
present at position 291 primarily because of the need for a small
residue. In agreement with this hypothesis, the G291A mutation is not
severely affected but is diminished by at least half in uptake
capacity. The decrease in capacity may be even greater since the G291A
CorA mutant appears to be somewhat overexpressed relative to wild type
(Fig. 2). A proline or a bulky hydrophobic residue is conserved at
position 293 in CorA (5). Proline is known to bend or kink an -helix
but also has significant side chain bulk. Thus its presence at residue
293 may be to position Tyr292 appropriately, and the
relatively mild phenotypes of the Gly291 and
Pro293 mutations may be indirect, resulting from improper
positioning of Tyr292.
Mutations at Met299 also have major effects. Cation accumulation in the M299C mutant is decreased by 90-95% at a substrate concentration of 200 µM Ni2+, and cation affinity is decreased 10-30-fold. The decrease in uptake capacity is largely due to the decrease in cation affinity. If transport is measured at increased extracellular Ni2+ concentrations, accumulation is greatly increased, consistent with a decrease in cation affinity. Similar results were seen with the M299A mutation which is somewhat more severely affected. Thus Met299 also likely interacts with Mg2+ during passage through the membrane. The involvement of a methionine residue is surprising. Mg2+ normally coordinates with oxygen via its free electrons. It rarely exhibits coordination with free electrons on nitrogen with the obvious exception of chlorophyll and is not known to coordinate with sulfur in biological systems (6, 13). Since substitution of an Ala at this site had a major effect, Mg2+ is presumably not coordinating with the backbone carbonyl oxygen. Moreover, interaction is apparently through the alkylated thiol since substitution of a free thiol at this position, represented by the M299C mutant, results in a marked cation affinity change. This suggests that the relatively hydrophobic nature of the methionine side chain may be required rather than direct coordination via a thiol group. This residue is highly conserved in CorA homologs in a wide variety of bacterial and archaeal species (5). Since a side chain oxygen atom, via a Ser or Thr substitution, is not seen at this position in other CorA homologs, the apparent requirement for an alkylated sulfur atom at this location would appear to be of some importance.
The Tyr307 mutant is also of interest because of the slight but significant alterations in cation selectivity with the different mutants. Mutations at this site do not significantly alter uptake capacity, and absolute Mg2+ affinity is decreased 3-10-fold among the various mutants, less than effects of mutation at some other sites. However, affinity changes for Ni2+ and Co2+ are different than with Mg2+. Y307A, Y307F, and Y307S CorA mutants show significant decreases in affinity for Mg2+. In contrast, the Y307A and Y307F mutants show no change in affinity for Ni2+, whereas the Y307S mutant shows a slight decrease in affinity for Ni2+. Changes in Co2+ affinity are intermediate in effect. The molecular basis for this selectivity is unclear and is presumably related to the precise microenvironment within the membrane. Nonetheless, the observation that alterations in cation selectivity occur with mutation at this site suggests strongly that Tyr307 is an additional residue with which Mg2+ interacts during transport.
A Pathway for Mg2+ through the Membrane--
The
transport results discussed above suggest that residues
Tyr292, Met299, and Tyr307 within
TM3 directly interact with Mg2+ during the transport
process. A simple model of TM3 as an -helix is supportive of a role
for these three residues in interaction with Mg2+ (Fig.
8). All three residues are on a continual
face of the presumed -helix. In this context, it makes sense that
mutations at Tyr309 do not alter transport, as opposed to
those at Tyr307, because Tyr309 would reside
about halfway around the -helix. Such a model for a pathway
involving TM3 also suggests additional experiments. For example,
Ala295 and 302AG303 are all small
residues roughly one turn around the -helix from the
Tyr292-Met299-Tyr307 axis.
Substitution of a large hydrophobic residue at one or more of these
positions might not provoke gross changes in the overall structure of
CorA since the side chain of the substitution might simply protrude
into an already formed transport channel; however, such a protrusion
would likely afford some degree of steric hindrance to cation
passage.
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The Remainder of the Mg2+ Pathway-- Even in the context of an oligomeric CorA transporter, residues within TM3 are unlikely to form the entire transport pathway. Although some multimeric ion channels appar to form a transport pore using similar or identical transmembrane segments from 5 or more monomers, formation of a Mg2+ pore using only TM3 seems less likely with CorA as it would necessitate, by analogy with ion channels, at least a pentamer of 15 transmembrane segements. Likewise, the three transmembrane segments of the monomer form of CorA seem too few to form a functional pore. Thus, a homoligomer of some order is the likely functional form of CorA. Preliminary evidence suggests that CorA is indeed an oligomer.2 This would provide 6 or more total membrane segments. TM2 also has a large number of hydroxyl-bearing residues, and we have preliminary evidence that Thr270 is important.3 This suggests that TM2 and TM3 are involved in forming a transport pathway. The involvement of TM1 is more problematic. We have shown previously that the E. coli and S. typhimurium CorA proteins possess three transmembrane segments (3). Nevertheless, phylogenetic and sequence analysis suggests that some members of the family may contain only 2 transmembrane segments, corresponding to TM2 and TM3 in the S. typhimurium CorA (5). In certain CorA family members, the TM1 domain contains multiple charged residues, as many as 9, making it most unlikely that this segment would be resident within the membrane. Thus, while the most parsimonious model for CorA would involve a homodimer with all three transmembrane segments of each monomer participating in forming the transport pathway, additional models involving only TM2 and TM3 must be considered. These will require not only additional mutagenesis studies as described in this report, but also structural studies to resolve the oligomeric structure of CorA and the arrangement of its transmembrane segments.
Comparison with Other Systems-- S. typhimurium contains two other Mg2+ transporters, MgtA and MgtB, both of which are P-type ATPases (14-16) and both of which are highly homologous with Ca2+- and H+-transporting P-type ATPases of eukaryotic cells (14, 16-19). Mutational studies have suggested that several conserved charged residues within the transmembrane segments of the Ca2+- and Na+,K+-ATPases form part of the cation pathway (20-25). These residues are largely conserved in the two Mg2+-transporting P-type ATPases. Mutagenic studies suggest that some, although not all, of these conserved charged residues are involved in Mg2+ transport.4 Thus, Mg2+ movement through the membrane via the P-type ATPases is likely to involve different mechanisms than movement via CorA.
The lack of involvement of charged residues within the transmembrane
domains in transport proteins mediating the movement of cations is
unusual. Most transport systems that mediate ion movement,
e.g. the proton/sugar symporter lactose permease (26), have
charged residues within transmembrane domains. However, although unusual, the lack of charged residues within the transmembrane domains
of a transporter is not unique to CorA. For example, the movement of
cation through the K+ channel of Streptomyces
lividans involves coordination of two cations within the channel
with closely spaced backbone carbonyl oxygen atoms rather than
negatively charged residues or free electrons on hydroxyls or nitrogen
atoms (27). Nonetheless, movement of cation through a membrane without
interaction with charged residues within the transmembrane segments is
relatively unusual. The Mg2+ cation is unique among the
common biological cations with its small ionic radius and large
hydrated radius, and we have previously hypothesized that because of
these unique properties of the Mg2+ ion that
Mg2+ transport systems would be highly unusual or even
unique members of the transport protein family. These mutational
studies on the CorA Mg2+ transporter strongly support this
hypothesis (5, 10, 28, 29).
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grant GM39447 (to M. E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Metabolism Training Grant DK07319. Current address:
Dept. of Biology, University of Texas at Arlington, Arlington, TX
76019.
§ Supported by Cell and Molecular Biology Training Grant GM08056 during this work.
¶ Supported by a Summer Undergraduate Research Fellowship from the American Society of Pharmacology and Experimental Therapeutics.
Supported by a Summer Undergraduate Research Fellowship from
the American Society of Pharmacology and Experimental Therapeutics.
** To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4965. Tel.: 216-368-6186; Fax: 216-368-3395; E-mail: mem6{at}po.cwru.edu.
The abbreviation used is: TM3, third transmembrane segment (of CorA).
2 P. F. Grulich, M. A. Szegedy, and M. E. Maguire, manuscript in preparation.
3 M. A. Szegedy and M. E. Maguire, unpublished data.
4 D. G. Kehres and M. E. Maguire, unpublished observations.
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K. M. Papp-Wallace, M. Nartea, D. G. Kehres, S. Porwollik, M. McClelland, S. J. Libby, F. C. Fang, and M. E. Maguire The CorA Mg2+ Channel Is Required for the Virulence of Salmonella enterica Serovar Typhimurium J. Bacteriol., October 1, 2008; 190(19): 6517 - 6523. [Abstract] [Full Text] [PDF]
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 S.-Z. Wang, Y. Chen, Z.-H. Sun, Q. Zhou, and S.-F. Sui Escherichia coli CorA Periplasmic Domain Functions as a Homotetramer to Bind Substrate J. Biol. Chem., September 15, 2006; 281(37): 26813 - 26820. [Abstract] [Full Text] [PDF]
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 S. Eshaghi, D. Niegowski, A. Kohl, D. M. Molina, S. A. Lesley, and P. Nordlund Crystal Structure of a Divalent Metal Ion Transporter CorA at 2.9 Angstrom Resolution. Science, July 21, 2006; 313(5785): 354 - 357. [Abstract] [Full Text] [PDF]
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K. M. Papp and M. E. Maguire The CorA Mg2+ Transporter Does Not Transport Fe2+ J. Bacteriol., November 15, 2004; 186(22): 7653 - 7658. [Abstract] [Full Text] [PDF]
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M. A. Warren, L. M. Kucharski, A. Veenstra, L. Shi, P. F. Grulich, and M. E. Maguire The CorA Mg2+ Transporter Is a Homotetramer J. Bacteriol., July 15, 2004; 186(14): 4605 - 4612. [Abstract] [Full Text] [PDF]
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F. Sebbane, M.-A. Mandrand-Berthelot, and M. Simonet Genes Encoding Specific Nickel Transport Systems Flank the Chromosomal Urease Locus of Pathogenic Yersiniae J. Bacteriol., October 15, 2002; 184(20): 5706 - 5713. [Abstract] [Full Text] [PDF]
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L. Wolfram and P. Bauerfeind Conserved Low-Affinity Nickel-Binding Amino Acids Are Essential for the Function of the Nickel Permease NixA of Helicobacter pylori J. Bacteriol., March 1, 2002; 184(5): 1438 - 1443. [Abstract] [Full Text] [PDF]
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M. A. Szegedy and M. E. Maguire The CorA Mg2+ Transport Protein of Salmonella typhimurium. MUTAGENESIS OF CONSERVED RESIDUES IN THE SECOND MEMBRANE DOMAIN J. Biol. Chem., December 24, 1999; 274(52): 36973 - 36979. [Abstract] [Full Text] [PDF]
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L. M. Kucharski, W. J. Lubbe, and M. E. Maguire Cation Hexaammines Are Selective and Potent Inhibitors of the CorA Magnesium Transport System J. Biol. Chem., May 26, 2000; 275(22): 16767 - 16773. [Abstract] [Full Text] [PDF]
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