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J. Biol. Chem., Vol. 275, Issue 31, 23834-23840, August 4, 2000
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From the Department of Biochemistry, Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Received for publication, February 17, 2000, and in revised form, May 10, 2000
The lactose transport protein (LacS) of
Streptococcus thermophilus belongs to a family of
transporters in which putative The lactose transport protein, LacS, of Streptococcus
thermophilus is a secondary transport system that belongs to the
family of the galactosides-pentosides-hexuronides
(GPH)1 transporters (1).
In vivo, LacS catalyzes a lactose/galactose exchange
reaction, which is driven by the concentration gradients of both sugars
across the membrane (2, 3). The LacS protein also catalyzes
solute-H+ symport, which is the proton motive force
( When catalyzing exchange transport, sugar binding occurs in an
alternating manner at the cytoplasmic and extracellular binding site.
Specificity studies have revealed that the sugar binding site has a
different architecture when exposed to the cytoplasmic or the
extracellular face of the membrane (6). The intracellularly facing
binding site has a high affinity for galactose through interactions
with the C-2 and C-6 hydroxyl groups of the sugar; these hydroxyl
groups do not participate in the binding of the sugar to the
extracellularly facing binding site. Both binding sites and the
translocation pathway are spacious in the C-1 to C-4 axes of the
galactose moiety, because they are able to accommodate galactosides
with large substitutions, especially the galactose C-1, e.g.
trisaccharides or galactosides substituted with an aromatic ring are
bound and transported.
In an effort to understand where the sites for substrate and cation
binding are located in the members of the GPH family, several
approaches, ranging from mutant isolation/selection to biophysical
methods (e.g. 7, 8, 9, 10), have been used. The
transmembrane or carrier domain of the proteins from the GPH family
comprises 12 transmembrane-spanning Obviously, proton and sugar transport by LacS are not separate events.
Conformational coupling between sugar and proton binding/translocation will occur, and possibly the two ligands are transported through the
same translocation pathway. By isolating and characterizing site-directed and second-site suppressor mutants, and by assessing site-directed modification of LacS in the presence and absence of
sugar, we have obtained further information on the localization of
regions and residues that are important for sugar and proton binding/translocation. An extended helix packing model is presented that brings together the catalytically important regions, that is,
Materials
D-[glucose-1-14C]lactose (2.11 teslabecquerels/mol) was obtained from the Radiochemical Center,
Amersham Pharmacia Biotech. Restriction enzymes, Pwo DNA polymerase,
Triton X-100, and streptavidin-alkaline phosphatase conjugate were from
Roche Molecular Biochemicals. Bacteriological media were from Difco.
Hydroxylamine, 3-(N-maleimidopropinyl)biocytin (biotin-maleimide), and N-ethylmaleimide (NEM) were
purchased from Sigma. Ni-NTA resin was from Qiagen, Inc. All other
materials were of reagent grade and obtained from commercial sources.
Bacterial Strains and Plasmids
E. coli strains HB101 (15) and DW2 (16) were grown
anaerobically in Luria broth (LB) at 37 °C or on MacConkey plates
supplemented with 0.5% lactose. When appropriate, the medium was
supplemented with 50 µmg/ml ampicillin and/or 1 mM
isopropyl-1-thio- Hydroxylamine Mutagenesis and Selection of Second-site Suppressor
Mutants
Plasmid DNA of pSKE8his(C320A, R64C) and pSKE8his(C320A,
D71C), 2.75 µg, in 55 µl of water, was incubated for 45 min at
65 °C with 65 µl of a fresh solution of NH2OH in 100 mM sodium phosphate, pH 7.0, 200 mM NaCl plus 4 mM EDTA. To stop the reaction, a 20-µl sample was diluted
into 15 µl of ice-cold 2.35 M NH4Ac, and the DNA was precipitated with ethanol and dissolved in 4 µl of water. 2.5 µl was used to transform E. coli HB101, and cells were
plated on MacConkey agar with 1% lactose. Red colonies appeared after overnight incubation. Notice that HB101/pSKE8his(C320A, R64C) and
HB101/pSKE8his(C320A, D71C) gave rise to white colonies due to an
inactive LacS protein (see "Results"). DNA was isolated from 50 red
colonies, and used to retransform the HB101 cells. About 70% of the
clones had retained the red phenotype on lactose-MacConkey agar, and
DNA was isolated and subjected to DNA sequencing from 18 of these.
Transport Assays
Transport of [14C]lactose was assayed at 30 °C
using the rapid filtration technique (17). The transport reactions were
stopped at different time intervals by dilution into 2 ml of ice-cold 0.1 M LiCl, rapid filtering on 0.45-µm cellulose nitrate
filters (Schleicher & Schuell), and washing with another 2 ml 0.1 M LiCl.
Downhill Uptake--
Lactose transport down the concentration
gradient was measured in E. coli HB101 cells that were grown
overnight in LB with 1 mM
isopropyl-1-thio- Proton Motive Force ( Exchange and Efflux Down the Concentration
Gradient--
E. coli DW2 cells, grown overnight on LB and
washed twice with KPM, were preloaded with [14C]lactose
by overnight incubation with 2.75 mM
[14C]lactose in KPM. Cells were de-energized by
incubation with 30 mM sodium azide and 50 µM SF6847 for
2 h at room temperature. The uptake was started by dilution of 2 µl of 50 mg/ml cell suspension into KPM or KPM with 100 µM lactose
for efflux and exchange, respectively.
Substrate Protection of NEM Inactivation of Single Cys LacS
Mutants
Overnight cultures of E. coli HB101 were washed three
times and resuspended in KPM to 30-40 mg/ml. To aliquots of 100-µl
cell suspensions, 10 µl of 100 mM
methyl- Substrate Protection of Biotin-maleimide Labeling of LacS Mutants
Single Lys
The labeling with biotin-maleimide was performed as described in
the previous paragraph, except that 950 µl of cell suspension, 400 µl of 250 mM lactose or buffer, and 45 µl of 100 mM biotin-maleimide in Me2SO were used. The
reaction was stopped by diluting the cells 40 times into KPM with 4 mM DTT. After washing with KPM, the cells were resuspended
in 2 ml of KPM and disrupted by sonication. The cell debris was removed
by centrifugation at 9000 × g for 10 min, after which
the membranes were collected at 250,000 × g for 15 min. The membranes were solubilized in 0.5% Triton X-100, and LacS was purified using Ni+-affinity chromatography as
described previously, except that the column was washed with double the
volume of wash buffer (4). The purified fractions were analyzed by
immunodetection with antibodies raised against LacS and
streptavidin-alkaline phosphatase conjugate.
Immunodetection of LacS
The amount of wild type and mutant LacS protein was estimated by
immunodetection of LacS with antibodies raised against the IIA domain
(18). Whole cell samples, prepared by boiling washed cell suspensions
for 5 min in SDS-polyacrylamide gel electrophoresis sample buffer, or
inside-out membrane vesicle samples (17) in SDS-polyacrylamide gel
electrophoresis sample buffer were separated on a 12.5%
SDS-polyacrylamide gel and transferred to polyvinylidene difluoride
membranes by semi-dry electroblotting. Detection, using the
Western-Light chemiluminescence detection kit with
3-(4-methoxyspiro{1,2-dioxetan-3.2'-(5'chloro)tricyclo[3.3.1.13.7]decan}-4-yl)phenylphosphate,
disodium salt as substrate, was performed as described by
Tropix, Inc.
Transport by Single Cysteine Mutants in Helices II and IV
The lactose transport protein (LacS) of S. thermophilus
has several charged amino acids in the second and fourth transmembrane
Close Approximation of Putative
-Helices II, IV, VII, X, and
XI in the Translocation Pathway of the Lactose Transport Protein of
Streptococcus thermophilus*
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-helices II and IV have been
implicated in cation binding and the coupled transport of the substrate
and the cation. Here, the analysis of site-directed mutants shows that
a positive and negative charge at positions 64 and 71 in helix II are
essential for transport, but not for lactose binding. The conservation
of charge/side-chain properties is less critical for Glu-67 and Ile-70
in helix II, and Asp-133 and Lys-139 in helix IV, but these residues
are important for the coupled transport of lactose together with a
proton. The analysis of second-site suppressor mutants indicates an ion
pair exists between helices II and IV, and thus a close approximation of these helices can be made. The second-site suppressor
analysis also suggests ion pairing between helix II and the
intracellular loops 6-7 and 10-11. Because the C-terminal region of
the transmembrane domain, especially helix XI and loop 10-11, is
important for substrate binding in this family of proteins, we propose
that sugar and proton binding and translocation are performed by the
joint action of these regions in the protein. Indeed, substrate
protection of maleimide labeling of single cysteine mutants confirms
that -helices II and IV are directly interacting or at least
conformationally involved in sugar binding and/or translocation. On the
basis of new and published data, we reason that the helices II, IV,
VII, X, and XI and the intracellular loops 6-7 and 10-11 are in close proximity and form the binding sites and/or the translocation pathway
in the transporters of the galactosides-pentosides-hexuronides family.
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p)-driven solute accumulation, but this transport mode is an order
of magnitude slower than the exchange reaction (4). Kinetic analysis
has shown that, not only in the solute-H+ symport but also
in the exchange mode of transport, protons participate in the
translocation process (5).
-helices. Comparison of the
primary sequence of members of the GPH family has identified some
general features. First, the putative -helices II, IV, and IX have
an amphipathic character suggesting interactions with both
apolar/hydrophobic and polar/hydrophilic surfaces (1). The strongly
hydrophilic character of one side of the transmembrane helices II and
IV results from the presence of a number of conserved positively and
negatively charged residues (see Fig. 1). These residues are thought to
coordinate cation binding in the melibiose carrier (MelB) from
Escherichia coli (1, 11, 12, 13). Second, the GPH family is
characterized by a high sequence conservation in the loop between
helices X and XI (see Fig. 1) (1). Electron spin resonance (ESR)
studies have indicated that this region is not nearly as flexible as
would be expected for such a large loop, and thus possibly it is
located in the core of the protein (14). Moreover, a conserved residue Glu-379 in this loop is essential in coupling the transport of protons
to the transport of sugar, because neutral substitution renders LacS
unable to catalyze lactose-H+ symport, whereas equilibrium
transport is still catalyzed with wild type rates (5). Approximation of
residue 373 in loop 10-11 within 15 Å from the C-1 atom of a
galactose molecule in the binding site of LacS is apparent from
solid-state nuclear magnetic resonance studies (14).
-helices II and IV, loop region 10-11, and -helix IX.
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-D-galactopyranoside. pSKE8His-derived
plasmids, encoding single cysteine mutants of LacS with a C-terminal
6-histidine tag, are listed in Table I. pSKE8his(C320A) was constructed by exchange of the
NcoI/PstI fragment from pGKhis(C320A) (4) to
pSKE8his (4). Site-directed mutagenesis was performed by PCR using
mutagenic primers and pSKE8his(C320A) DNA as template. Mutants are
indicated by a letter-number-letter code, in which the first letter
corresponds to the amino acid that is substituted and the second one
indicates the substitution; the number corresponds to the position of
the residue in wild type LacS. Mutations were verified by DNA
sequencing of the entire genes.
LacS mutants specified by the corresponding alleles in pSKE8hisC320A
-D-galactopyranoside for maximal
expression of -galactosidase. The cells were washed twice in KPM (50 mM potassium phosphate, pH 7.0, with 2 mM
MgSO4) and resuspended to 30-40 mg/ml. Aliquots of 6 µl
were diluted into 200 µl of KPM with 10 mM
D-lithium lactate, and after 2 min of aeration, 50 µM [14C]lactose was added to initiate the reaction.
p)-driven Uptake--
p-driven
accumulation of lactose was measured in E. coli DW2 cells
that were grown overnight on LB and washed twice with KPM. Aliquots of
6 µl cells (30-40 mg/ml) were diluted into 200 µl of KPM with 10 mM D-lithium lactate, and after 2 min of
aeration, the uptake was started by the addition of 50 µmM
[14C]lactose.
-D-thiogalactoside (TMG) or 10 µl of buffer
(control) was added. After 10 min of equilibration at 37 °C, freshly
prepared NEM was added to a final concentration of 3 mM.
After 30 min of incubation at room temperature, the reaction was
stopped by the addition of 12 mM dithiothreitol (DTT). The cells were washed four times with 15 volumes of KPM. Lactose uptake down the concentration gradient was measured as described above.
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-helix that are highly conserved within the GPH family (Fig. 1). To investigate the role of these
charged amino acids in catalysis, each of these residues was replaced
by cysteines or a charge-conserving amino acid, and the effect of the
mutation on lactose transport was determined. Each of the mutants was
made in the lacS(C320A) allele; the activity of LacS(C320A)
was comparable to that of wild type LacS (Fig. 3A). Wild
type LacS and the different mutants were expressed to similar levels as
was determined by immunodetection of LacS in whole cell samples;
typical examples are shown in Fig. 2.
Because LacS(R64C) and LacS(D71C) were inactive, it was possible that
these mutants were not assembled in the cytoplasmic membrane. Immunoblotting of inside-out membrane vesicles isolated from HB101 cells showed that LacS(R64C) and LacS(D71C) were present in the membrane at comparable levels as the wild type protein. Next, each of
the mutants was characterized by assaying four modes of transport:
[14C]lactose uptake down the concentration gradient
(downhill uptake), p-driven [14C]lactose uptake,
[14C]lactose efflux down the concentration gradient, and
[14C]lactose/lactose exchange. Transport rates are
presented as percentage of the transport rates catalyzed by wild type
LacS, as expressed from pSKE8his (Fig.
3A).
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Fig. 1.
Sequence alignment of putative
-helices II, IV, VII, and XI and the loop regions
2-3, 4-5, 6-7, and 10-11 of LacS and MelB. Charged residues
within the putative -helices are underlined and in
boldface (33, 34); residues that were altered in LacS(R64C)
(this study) or MelB(R52S) second-site suppressors (32) are in
pink; residues that were altered in LacS(D71C) second-site
suppressors are in green; residues important for
sugar-H+ symport in LacS are in red (this study;
Refs. 5, 17); residues of which mutation resulted in an altered sugar
or cation specificity in MelB are in blue (12, 25, 26);
residue 373 of LacS, which is less than 15 Å from bound lactose, is in
light blue (14). Similar (colons) or identical
(asterisks) residues throughout the GPH family (1) or
between MelB from E. coli and LacS from S. thermophilus are indicated in the upper and lower
lines, respectively.
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Fig. 2.
Expression levels of LacS in E. coli HB101. The expression levels of various mutants was
analyzed by immunoblotting of total cell lysates. Lane 1,
LacS(D71C, R377H); lane 2, LacS(D71C, R377D); lane
3, LacS(D71C, R377C); lane 4, LacS(R64C, D133C);
lane 5, LacS(K139C); lane 6, LacS(D71C, R230C);
lane 7, LacS(D71C, R230D); lane 8, LacS(C320A);
lane 9, LacS(D378C); lane 10, LacS(R64C);
lane 11, LacS(D71C, L357F); lane 12, LacS(D71C,
D378D); lane 13, LacS(D71C); lane 14,
LacSwt.
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Fig. 3.
Transport activities of cysteine-less and
single cysteine mutants of LacS. A, the initial rates
of transport are shown as a percentage of the initial transport rate
catalyzed by wild type LacS; 100% corresponds to 4, 2.5, 50, and 7.5 nmol/min × mg of protein for downhill uptake, efflux down the
concentration gradient, exchange, and p-driven uptake, respectively.
Plasmid pSKE8his with the indicated mutations was used to transform
E. coli HB101 or DW2. Downhill [14C]lactose
uptake (white bars) was measured in E. coli HB101
cells in which the intracellular concentration of lactose remains low
due to the expression of -galactosidase. Efflux down the
concentration gradient (gray bars), exchange (striped
bars), and p-driven uptake (black bars) were
measured in E. coli DW2, which has a chromosomal deletion in
the lacZ gene allowing lactose to accumulate inside the
cell. Downhill and p-driven uptake of [14C]lactose
were assayed for different time intervals in pre-energized cells; the
reaction was started by the addition of 50 µM
[14C]lactose. Efflux and exchange were measured in
de-energized cells that were preloaded with 2.75 mM
[14C]lactose. The exit of [14C]lactose was
assayed in the absence (efflux) and presence (exchange) of 100 µM lactose in the external buffer. B, exit of
[14C]lactose from E. coli cells expressing
LacS(C320A) (closed symbols) and LacS(K139C) (open
symbols) under conditions of [14C]lactose/lactose
exchange (squares) and efflux of [14C]lactose
down the concentration gradient (triangles).
After substitution of the residues Arg-64 and Asp-71 by cysteines,
transport of lactose was no longer observed (Fig. 3A). The
charge-conserving mutations R64K and D71E rendered the proteins partially active in all four modes of transport (not shown), from which
we conclude that a positively charged residue at position 64 and a
negatively charged one at position 71 are essential for transport
activity. Cysteine substitution of the residues Glu-67 and Asp-133
abolished p-driven uptake completely but did not effect the exchange
and downhill uptake. Strikingly, the E67C and D133C mutants showed a
more than 4-fold increase in the rate of lactose efflux. The K139C
mutant displayed transport properties similar to those of E67C and
D133C, although the downhill uptake and exchange reactions were
significantly reduced; the efflux and exchange transport of the parent
allele and K139C are shown in somewhat more detail in Fig.
3B. Cysteine substitution of Ile-70 did not affect exchange
and efflux and uptake down the concentration gradient, but largely
impaired p-driven uptake. Contrary to the substitutions of the
heretofore described highly conserved residues, cysteine substitution
of residues on the hydrophobic faces of helices II and IV,
e.g. Ile-65, Val-134, and Phe-135 did not have an effect on
any of the transport modes. Altogether, we conclude that the residues
Arg-64 and Asp-71 in helix II are essential for transport, whereas
Glu-67, Ile-70, Asp-133, and Lys-139 are needed for the coupled
transport of a sugar molecule together with a proton.
Second-site Suppressor Mutants
Second-site suppressor mutations can yield information about close
approximation of residues within the protein, because the defect of the
primary mutation can be restored by substitution of one or more
neighboring residues (19, 20). This genetic technique can be
particularly powerful when functional ion pairs within the protein are
involved (21-23). Two mutants in helix II, i.e.
LacShis(R64C) and LacShis(D71C), were used for selection of second-site
suppressors, because these displayed a white phenotype on
lactose-MacConkey plates. Consistent with the transport data (Fig.
3A), the wild type and all other mutants had a red (or pink, K139C) colony phenotype. Transport positive colonies turn red on the
indicator plates as a result of acidification of the medium after
lactose fermentation. Hydroxylamine-mutagenized plasmid DNA
(pSKE8his(C320A, R64C) or pSKEhis(C320A, D71C)) was
transformed to HB101, and cells were plated on lactose-MacConkey agar.
DNA was isolated from 50 red colonies and used to retransform HB101 cells. About 70% of the clones had retained the red phenotype and from
18 highly red colonies, 8 suppressors from pSKE8his(R64C) and 10 from
pSKE8his(D71C), DNA was isolated and the entire lacS genes
were sequenced. The second-site suppressors had on average two
mutations of which some were silent (Table
II). All mutations are C 
T or G A
substitutions as is expected from the mechanism of hydroxylamine
mutagenesis (24).
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Second-site Suppressors of LacS(R64C)--
Mutations suppressing
the defect in R64C were found in the portion of the gene that
corresponds to the N-terminal region of the protein, i.e.
-helix II (S61F, P72L), cytoplasmic loop 2-3 (G75S), and -helix
IV (D133N). Although clearly red on lactose-MacConkey agar, downhill
lactose uptake catalyzed by these suppressors was less than 15% of the
wild type activity, indicating a poor Km and/or
Vmax (Fig. 4; data
not shown). Neither of the suppressor mutations restored the
p-driven lactose accumulation capacity of the wild type (not shown).
Regained activity in LacS(R64C, D133N) suggests that in the wild type
LacS the opposite charges at positions 64 and 133 are stabilized by the
formation of an ion pair, indicating a close approximation of helices
II and IV. Consistent with this suggestion is the observation that the
residues 133 and 64 are located at approximately the same height in the membrane. An unpaired charge on Asp-133 would thus inactivate the
carrier protein, and neutralization of this residue restores activity.
Along similar lines of reasoning one could explain the S61F
suppressor mutation. The aromatic side chain of Phe-61, which is
located one helix turn above Arg-64, might lower the polarity of the
environment around the carboxylate Asp-133, and thereby increase its
pKA. The unpaired charge of the carboxylate of
Asp-133 in LacS(R64C, S61F) would thus be neutralized at the prevailing
pH.
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To obtain further evidence for the hypothesis of the ion pair between Arg-64 and Asp-133, the double mutant LacS(R64C, D133C) was constructed. As anticipated, the transport activity and phenotype on lactose-MacConkey agar was similar to that of LacS(R64C, D133N) (Fig. 4). When the residues 64 and 133 are in close proximity, replacement of Asp-133 for an arginine might well be tolerated in the R64C background. This is indeed the case, LacS(R64C, D133R) shows 40% downhill transport activity compared with wild type LacS (Fig. 4). The P72L and G75S suppressor mutations are less easily explained, but might relate to a different position of helix II in the membrane, e.g. a position that places the unpaired charge at Asp-133 in a less unfavorable environment.
Second-site Suppressors of LacS (D71C)--
In contrast to the
mutations suppressing the R64C substitution, those suppressing D71C
were found in different regions of the LacS molecule. Most active was
the triple mutant LacS(D71C, R377H, D378N) with two mutations in
inter-helix loop 10-11. This mutant catalyzed downhill lactose uptake
at more than 60% the rate catalyzed by the wild type protein. It did
not catalyze significant p-driven uptake as was the case for the
other D71C suppressors. To dissect which of the mutations, R377H or
D378N, suppressed the defect of D71C, the corresponding "single"
mutants LacS(D71C, R377H), LacS(D71C, D378N), LacS(D71C, R377C),
LacS(D71C, R377D), and LacS(D71C, D378C) were constructed by
site-directed mutagenesis. The transport data clearly indicate that the
Arg-377 substitution is responsible for the gain of function (Fig.
4).
The second most active D71C suppressors (60% of the wild type) were LacS(D71C, R230C) and LacS(D71C, R230C, G546K, G572S). The R230C substitution is responsible for the restored activity, because the G546K and G572S mutations in the regulatory (IIA) domain of LacS did not alter uptake rate. Site-directed substitution of Arg-230 for an alanine or an aspartate also restored activity in the D71C mutant. The second-site suppressors LacS(L357F, T411I) and LacS(F261L, L357F, T411I) catalyzed downhill uptake at a rate that is 30% of the wild type. They have the mutation L357F in common, which is located in helix X. Because the double mutant LacS(D71C, L357F) constructed by site-directed mutagenesis showed a similar transport activity, we conclude that the Leu to Phe substitution at position 357 restored the transport activity (Fig. 4).
The second-site mutations P72S, A161T, A149V, and (S61F, P72L, T81I) are all located in the intracellular half of helix II or the intracellular loops 2-3 and 4-5. This is in accordance with a close approximation of helices II and IV, which was already concluded from the R64C suppressors. Strikingly, some of the mutations suppressing the D71C mutation are the same or similar to the ones suppressing the R64C mutation, e.g. the suppressor mutations S61F and/or P72L are found in the following combinations (R64C, S61F, P72L), (D71C, S61F, P72L, T81I), and (D71C, P72S).
The suppressor analysis together with the measurements of the constructed site-directed mutants reveals that all relevant pairs of mutations, except one, are located in the intracellular halves of helices II, IV, and X or the intracellular loops 2-3, 4-5, 6-7, and 10-11. The strongest indications for close approximation are found for helices II and IV (R64C, D133N), helix II and loop 6-7 (D71C, R230C), helix II and loop 10-11 (D71C, R377H), because these involve pairs of amino acids of opposite charge.
Substrate Protection of Maleimide Labeling of Single Cysteine Mutants in Helices II and IV
The N-terminal region of LacS has been proposed to be part of the actual cation binding site on the basis of conservation of charged residues, amphipathicity of helices II and IV, mutant analysis, and analysis of MelB fusions (1). The mutational analysis described here confirms that this region is indeed important for proton binding and/or coupling. This region, however, has never been directly associated with sugar binding and/or translocation. The proximity relations found for helices II, IV, X, and loop 10-11 suggest that helices II and IV are also involved in sugar binding and/or translocation.
Substrate protection of chemical modification is a means of showing
that a specific region in the protein is directly involved in binding
or at least conformationally coupled to the binding of substrate. Upon
alkylation of the cysteines at position 67 in helix II and 133 in helix
IV with NEM, downhill uptake is inhibited (Fig.
5, B and C). The
presence of a saturating concentration of nonmetabolizable substrate,
thiomethylgalactose (used at a concentration of at least 10 times the
apparent Km of the low affinity site) (6), protects
Cys-67 and Cys-133 from labeling by NEM (Fig. 5, B and
C). The control experiment shows that downhill uptake of
LacShis(C320A) is not affected by the incubation with NEM or TMG (Fig.
5A).
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For the interpretation of the second-site suppressor mutants, it is
important to establish the nature of the transport-negative phenotype
of the R64C and D71C mutants. Because a direct binding assay is not
available (KD of lactose binding is in the millimolar range (14)), biotin-maleimide modification of the cysteines
in these mutants was determined in the presence and absence of a
saturating concentration of lactose (at about 10 times the apparent
Km of the low affinity site). After labeling, LacS
was purified by Ni+-affinity chromatography, and the amount
of biotinylated LacS was determined with streptavidin-alkaline
phosphatase. The substrate protection of biotin-maleimide labeling of
LacS(R64C) and LacS(D71C) demonstrates that lactose is still bound by
the mutants (Fig. 6). Equivalent
concentrations of glucose, which is not a substrate of LacS, did not
inhibit labeling of the cysteine residues with biotin-maleimide (not
shown). Because there are at least two binding conformations in LacS,
one facing the extracellular side and one facing the cytoplasm, we
cannot exclude the possibility that only one of the binding conformers
is intact, and that the other one is restored in the suppressor
mutants. It is, however, more likely that the suppressor mutations
relieve a defect in the translocation step, rather than in the binding
of lactose. Finally, the substrate protection of the labeling of the
cysteines at positions 64, 67, 71 (helix II), and 133 (helix IV)
indicated that the hydrophilic faces of these transmembrane segments
participate in ligand binding.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The aim of this paper was to localize the regions and residues in the lactose transport protein of S. thermophilus that are important for sugar and proton binding and translocation. From the analysis of the site-directed mutants and second-site suppressors, and from the assessment of substrate protection of site-directed modification, we conclude that the helices I, II, IV, VII, X, and XI and the intracellular loops 6-7 and 10-11 are involved in proton and sugar transport and propose a model for the helix packing in the LacS protein.
Mutagenesis of the (conserved) charged residues in helices II and IV
showed that a basic residue at position 64 and an acidic one at 71 are
essential for transport. Importantly, the capacity to bind is retained
in LacS(R64C) and LacS(D71C) as was shown from labeling studies with
biotin-maleimide in the presence and absence of lactose. The fact that
none of the D71C or R64C suppressors has regained the capacity to
catalyze p-driven uptake indicates that the energy-coupling
mechanism is much more sensitive to (small) structural changes in the
protein than sugar binding and translocation. Consistent with this
notion are the observations that residues Glu-67 and Ile-70 in helix II
and Asp-133 in helix IV are important for the coupled transport of the
sugar together with a proton (p-driven uptake), but not essential
for translocation per se. Mutants carrying a single cysteine
substitution at these positions catalyze downhill uptake and exchange
at wild type rates. Very similar observations have been made for a
number of substitutions in the inter-helix loop 10-11 (5, 17).
Furthermore, in MelB aspartate 55 and asparagine 58, equivalent to
positions 67 and 70 in LacS, are required for coupling of TMG transport
to sodium as a cation. Binding of - and -galactosides, on the
other hand, still occurs but now independent of sodium ions (25,
26).
In the mutants E67C, D133C, and K139C, and to a lesser extent I70C, the
uncoupled phenotype coincides with an increase in the efflux rate. How
can this gain of efflux activity be explained? In a transport protein
catalyzing the coupled transport of a solute (S) together with a proton
(H), the fully loaded (ESH) and the empty carrier (E) reorient their
binding sites upon translocation of a solute plus proton from out to in
(uptake) or in to out (efflux). The coupling efficiency decreases when
also the binary states of the carrier, ES or EH, are able to reorient
their binding sites (for a full account, see Ref. 27). These so-called
ES and EH leaks frequently occur (or become manifest) when one or more
critical residues are substituted. The rate-determining step in efflux down the concentration gradient by the LacS protein is the
reorientation of the empty carrier (Eout Ein). The increased efflux rate together with wild
type exchange and facilitated influx rates can be explained when the
reorientation EHout EHin has become faster
than the reorientation of the empty carrier in the wild type (EH leak).
In principle, the increased efflux rate can also be explained by an ES
leak type, but then one not only needs to invoke an ES leak pathway but
also must assume that the reorientation of the empty carrier from out
to in is faster than in the wild type
(Eoutmutant Einmutant faster than
Eoutwt Einwt).
Substrate protection of inactivation by alkylation of Cys-64, Cys-67, Cys-71 (helix II), and Cys-133 (helix IV) shows that these regions are not only involved in proton coupling but also conformationally active upon sugar binding or even directly interacting with bound sugar. The observation that the helices II and IV are involved in both cation and sugar binding can be explained by conformational coupling of sugar and proton binding or even structural overlap between the binding sites for sugar and proton. Similarly, the loop between helices 10 and 11 has been implicated in sugar binding (14, 28) as well as proton binding (5, 17). The observation that the majority of the MelB mutants isolated on the basis of TMG resistance, Li+ resistance, or Li+ dependence, but also site-directed mutants in helices I, II, and IV, exhibit simultaneous alterations in cation and substrate recognition (12, 25, 26) may be interpreted as an indication for structural overlap of the proton and sugar binding sites. Evidence for conformational coupling in MelB comes from (i) proteolytic studies showing that cleavage in inter-helix loop 4-5 is dependent on both cation and sugar binding (29) and (ii) fluorescence studies showing that the polarity of the environment of the fluorescent substrate changes in a sodium-dependent fashion (9).
All second-site suppressor mutants that cure the transport-negative
phenotype of LacS(R64C) and LacS(D71C), except one, are located in the
intracellular halves of helices II, IV and X or the intracellular loops
2-3, 4-5 and 6-7 and 10-11. The strongest indications for
neighboring residues are found in helices II and IV (R64C, D133N),
helix II and loop 6-7 (D71C, R230C), helix II and loop 10-11 (D71C,
R377H), because these involve pairs of amino acids of opposite charge.
On the basis of the second-site suppressor analysis and published data,
we propose a model for the packing of the transmembrane-spanning
helices of LacS in the membrane (Fig. 7).
In this model the helices I, II, IV, VII, X, and XI are in close
proximity and form at least part of the binding site and
translocation pathway of the transport protein. Because there are no
data, neither for LacS nor any other member of the GPH family, that
assumes a role in catalysis for helices III, V, VI, VIII, IX, and XII,
they have been designated as a second ring outside the core of
the protein.
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The helices II and IV are located next to each other and form part of the translocation pathway for several reasons: (i) these helices have a highly conserved amphipathic and substitution pattern within the GPH family (1); (ii) several conserved charged residues located in helices II and IV are important for coupled transport and/or cation binding in LacS (this study); (iii) the helices II and IV either interact directly with the sugar-substrate, or at least their conformation changes upon sugar binding (this study); (iv) second-site suppressor analysis has indicated that Arg-64 in helix II and Asp-133 in helix IV possibly form an ion pair (this study).
The loop between helices X and XI has been given a location in the core of the protein, because (i) solid-state nuclear magnetic resonance data have indicated that the residue Lys-373 in this loop is located less than 15 Å from the C-1 of bound galactose (14), (ii) a possible ion pair between Arg-377 with Asp-71 in helix II was found in the second-site suppressor analysis (this study), and (iii) ESR experiments have shown that the loop has a flexibility that is more in accordance with a location in the core of the protein than with a cytoplasmic location (14).
The intracellular loop 6-7 was placed in the proximity of
helices II and IV, because an arginine to cysteine mutation in this loop can restore activity of the D71C mutant. Putative
-helices X and XI and the loop between X and XI are proposed to be
part of the translocation pathway on the basis of the second-site
suppressor found between helices II and X (this study) and because
helix XI has a conserved amphipathic character (1).
The model is in accordance with, and accommodates, all published data
obtained for other members of the GPH family. The most relevant
published data are summarized here. Second-site suppressor analysis in
MelB (30-32) suggests interactions between helix II and helices IV,
VII, and X, and inter-helix loop 10-11 (Fig. 7, dotted
lines). Additional suppressor pairs were found between helices IV
and XI, and helices II and I. Moreover, the mutations that alter the
sugar and/or cation recognition of MelB, as isolated by selection for
TMG resistance or Li+ resistance, all lie in the regions
depicted here as the core of the carrier protein (25, 26). In some
cases the same residues are found as second-site suppressors and as
mutations changing the sugar-specificity, e.g. I352V in loop
10-11 of MelB. The specificity mutations found in helix VII of
MelB reside on the same face of the helix as the suppressor mutations
(32). Evidence for relatively close distance of the N- and C-terminal
regions also comes from fluorescence resonance energy transfer
experiments, where the tryptophans in inter-helix loop 2-3 and helix
IX of MelB were estimated to be 20 and 14 Å away from the bound
substrate, respectively (9, 10).
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ACKNOWLEDGEMENTS |
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We thank Dirk Jan Slotboom for his advice on the labeling studies and Erik Hamminga for DNA sequencing.
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FOOTNOTES |
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* This work was supported by funding from the European Community (Grants B10-4-CT-960129 and -960439) and the Human Frontier Science Program Organization.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.
To whom correspondence should be addressed: Tel.: 31-50-3634209;
Fax: 31-50-3634165; E-mail: b.poolman@chem.rug.nl.
Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M001343200
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ABBREVIATIONS |
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The abbreviations used are:
GPH, galactosides-pentosides-hexuronides;
NEM, N-ethylmaleimide;
p, proton motive force;
PCR, polymerase chain reaction;
TMG, methyl--D-thiogalactoside;
DTT, dithiothreitol;
MelB, melibiose carrier protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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and 
) or KPM with 10 mM TMG (
and 
). After 10 min of equilibration at
37 °C, 3 mM NEM (
