From the Groningen Biomolecular Sciences and Biotechnology Institute and the Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Received for publication, November 28, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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Part of the dimer and B/C domain interface of the
Escherichia coli mannitol permease (EIImtl) has
been identified by the generation of disulfide bridges in a
single-cysteine EIImtl, with only the activity linked
Cys384 in the B domain, and in a double-cysteine
EIImtl with cysteines at positions 384 and 124 in the first
cytoplasmic loop of the C domain. The disulfide bridges were formed in
the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)3, and they
were visualized by SDS-polyacrylamide gel electrophoresis.
Discrimination between possible disulfide bridges in the dimeric
double-cysteine EIImtl was done by partial digestion of the
protein and the formation of heterodimers, in which the cysteines were
located either on different subunits or on one subunit. The disulfide
bridges that were identified are an intersubunit
Cys384-Cys384, an intersubunit
Cys124-Cys124, an intersubunit
Cys384-Cys124, and an intrasubunit
Cys384-Cys124. The disulfide bridges between
the B and C domain were observed with purified enzyme and confirmed by
matrix-assisted laser desorption ionization-time of flight mass
spectrometry. Mannitol did not influence the formation of the disulfide
between Cys384 and Cys124. The close proximity
of the two cysteines 124 was further confirmed with a separate C domain
by oxidation with Cu(II)-(1,10-phenanthroline)3 or by
reactions with dimaleimides of different length. The data in
combination with other work show that the first cytoplasmic loop around
residue 124 is located at the dimer interface and involved in the
interaction between the B and C domain.
The uptake and concomitant phosphorylation of a wide variety of
carbohydrates into bacterial cells is, in many cases, accomplished by
the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (1). In a cascade of phosphorylation reactions (Fig. 1), the phosphoryl group is transferred
from the energy donor phosphoenolpyruvate
(PEP)1 via the two general
components EI and HPr to the carbohydrate-specific components (Enzyme
II's), which transport and phosphorylate the carbohydrates (2). All
EII's2 have a similar
architecture and consist of the cytoplasmic A and B domains and a
membrane-embedded C domain. This article deals with the
mannitol-specific EII (EIImtl) of Escherichia
coli, in which the three domains are covalently linked. HPr
phosphorylates the A domain of EIImtl on
His554, which subsequently phosphorylates
Cys384 in the B domain. Mannitol in the periplasm is bound
by the C domain, transported into the cell via C and, while bound at
the cytoplasmic site of C, phosphorylated by the B domain.
EIImtl is most likely a dimeric protein and the subunit
interactions occur in the C domain (3-8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the
mannitol-specific phosphoenolpyruvate-dependent phosphotransferase
system of E. coli. Dotted arrows
indicate that the phosphoryl group transfer from HPr can proceed to
each of the EIImtl subunits and that inter- and intradomain
phosphoryl group transfer is possible as well.
Domain interactions and in particular the B/C domain interface play an important role in the catalytic cycle of EIImtl. The energy coupling mechanism involves conformational interaction between the B and C domain. The evidence for this notion is manifold. 1) Phosphorylation of the B domain increases the rate of transport 2-3 orders of magnitude (9, 10). 2) Modification or mutagenesis of the phosphorylation site in the B domain as well as removal of the cytoplasmic domains changes the mannitol binding kinetics of the C domain (11, 12). 3) Time- resolved fluorescence and phosphorescence spectroscopy showed that, upon phosphorylation of the B domain, Trp109 in the C domain becomes immobilized whereas Trp30 in the C domain becomes more flexible (13, 14). 4) Differential scanning calorimetry showed that the thermal stability of the C domain is higher in the presence of the B domain (15). 5) Isothermal titration calorimetry experiments indicated that a significant part of the structural changes upon the binding of mannitol to the C domain reside in the B domain. Approximately 50-60 residues are removed from the bulk water upon binding of mannitol, which was much less when the same measurements were done after removal of the B domain (16). 6). Close proximity of the B and C domain has been suggested for another PTS transporter, that is the BglF system of E. coli.3
To date, there is no structural information about the B/C domain or
dimer interface of EIImtl or any other EII. The topological
model of the C domain predicts 6 membrane-spanning -helices and two
large cytoplasmic loops (17), which is in accordance with the recently
resolved projection map of the C domain (7). The location of the loops,
however, is not known. A photocross-linking approach to identify the
B/C domain interface suggested that the end of the first cytoplasmic C
domain loop is located in the vicinity of Cys384 in the B
domain.4 Here we describe the
generation of disulfide bridges of cysteines within and between B and C
domains. This work demonstrates that the first cytoplasmic loop of the
C domain is in close proximity to the B domain active site and near the
dimer interface.
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EXPERIMENTAL PROCEDURES |
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Chemicals-- Decyl-polyethylene glycol (dPEG) was synthesized by B. Kwant (Kwant High Vacuum Oil Recycling and Synthesis, Bedum, The Netherlands). Bovine pancreas L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was from Sigma and endoproteinase Glu-C of Staphylococcus aureus V8 was from Fluka. The bimaleimides o-PDM and p-PDM were from Aldrich and BMH was obtained from Pierce. EI and HPr were purified as described previously (18, 19). All chemicals used were analytical grade.
Construction of Plasmids for Expression of SSCS, SSCS-S124C, and IIChis-S124C-- Site-directed mutagenesis was performed with the Stratagene Quickchange mutagenesis kit. His-tagged EIImtl with cysteines at positions 110, 320, and 571 replaced by serine (SSCS) was constructed in pMaHisMtlAPr, which carries the gene for the N-terminal His-tagged EIImtl.5 Subsequently, SSCS-S124C was generated by replacing Ser124 with a cysteine. IIChis-CL was generated by replacing Cys110 and Cys320 by serine in pMaMtlIICHis, which carries the gene for the C domain with a C-terminal His-tag (16). IIChis-S124C was then generated by replacing Ser124 by a cysteine as described above. Mutations were identified by introducing silent restriction sites and confirmed by DNA sequence analysis.
Generation of ISO Vesicles and Purification of SSCS, SSCS-S124C,
and IIChis-S124C--
Growth of E. coli LGS322
(F thi-1, hisG1, argG6, metB1, tonA2, supE44,
rpsL104, lacY1, galT6, gatR49, gatR50,
(mtlA'p),
mtlDc,
(gutR'MDBA-recA))
carrying the various plasmids, and procedures to overexpress the mutant
proteins were identical to those described for wild-type
EIImtl (20). Inside-out (ISO) membrane vesicles containing
the mutant proteins were obtained as described (21). SSCS and
SSCS-S124C were purified by Ni-NTA-agarose affinity chromatography as
described for 6HEIImtl (14), except that dPEG was used as
the detergent. IIChis-CL and IIChis-S124C were purified as described
(16).
EIImtl Concentration Determination and Mannitol Binding and Phosphorylation Activities-- The dissociation constant for mannitol binding and EII or IIC concentration in vesicles was determined by flow dialysis after solubilization with 0.25% dPEG (22) with some modifications as will be described elsewhere.6 The mannitol phosphorylation activity was determined at 1 mM mannitol (23). The concentration of purified SSCS and SSCS-S124C was determined with the pyruvate burst assay, which determines the amount of PTS phosphorylation sites (24). The activities of IIChis-CL and IIChis-S124 were determined after formation of a heterodimer with EIImtl-G196D, using 33 µM mannitol as described (25).
Disulfide Cross-linking-- A solution of ~6 µM enzyme in ISO vesicles or 1 µM purified protein was brought to a final concentration of 5 mM DTT and 20 mM EDTA from stocks of 0.1 and 0.5 M, respectively. If appropriate, 90 nM EI was added. After 15 min of incubation at 30 °C, DTT and EDTA were removed on a Bio Micro-spin 6 column (Bio-Rad), equilibrated with 50 mM NaPi, pH 7.5, 0.1 mM EDTA, with (purified enzyme) or without (ISO vesicles) 0.25% dPEG. The buffer was deaerated with helium before addition of the detergent. Subsequently, HPr, MgCl2, dPEG, and PEP were added to phosphorylate the purified enzyme, provided EI was present; these additions increased the final volume by 50%. The same mixture without PEP was used to represent conditions in which the enzyme was not phosphorylated. The final concentrations of the components were 2 µM HPr, 5 mM PEP, and 5 mM MgCl2. After 5 min at 30 °C, disulfide bridge formation was initiated by oxidation with 0.1 volume of 3 mM Cu(II)-(1,10-phenanthroline)3 (CuPhe), followed by incubation at 30 °C for 30 min. The reaction was quenched by the addition of 65 mM EDTA from a stock of 0.5 M NaEDTA, pH 8.
Partial Digestion and Reduction--
The protein was partially
digested with 20 µg of trypsin or 100 µg of endoproteinase Glu-C/ml
of reaction mixture for 1 h at room temperature. The digestion of
the vesicles with endoproteinase Glu-C was done in the presence or
absence of 0.4% dPEG. The digestion was stopped by the addition of
SDS-PAGE denaturation buffer without -mercaptoethanol. If
appropriate, reduction was accomplished by the addition of 10 mM DTT after digestion.
Heterodimer Formation-- Heterodimers between 3 µM SSCS and 1 µM IIChis-S124C or 0.2 µM SSCS-S124C and 3 µM IIChis-CL were formed by mixing purified proteins, followed by an incubation at 30 °C for 30 min. To promote heterodimer formation between SSCS-S124C and IIChis-CL, 170 mM Na3PO4 was added from a 1 M stock solution at pH 7.6. This lowers the cloudpoint of the detergent (dPEG), in which the protein is solubilized. This treatment results in dissociation of the initially homodimeric enzymes and thereby facilitates the mixing of the species (26). Subsequently, the heterodimers were treated as described above for the disulfide cross-linking procedure except that EI, HPr, MgCl2, and PEP were omitted from the mixture.
Cross-linking with Dimaleimides of Varying Length-- 1 µM Purified IIChis-S124C was reduced and demetalated as described under "Disulfide Cross-linking." Cross-linking was initiated by adding to the protein, in 50 mM NaPi, pH 7.5, 0.1 mM EDTA plus 0.25% dPEG, 5 µM o-PDM, p-PDM, or BMH from a 10 times concentrated stock solution in N,N-dimethylformamide. The reaction was stopped with 10 mM DTT after incubation at 30 °C for 30 min.
SDS-PAGE Analysis and Immunoblotting--
SDS-polyacrylamide gel
electrophoresis was done with 10% acrylamide gels as described (27). A
denaturation buffer without -mercaptoethanol was used. The samples
were not boiled in denaturation buffer, because this leads to
aggregation. The proteins were visualized either by silver staining
(28) or by immunodetection after the proteins were transferred to
polyvinylidene difluoride membranes by semi-dry electrophoretic
blotting. Detection, using the Western LightTM
chemiluminescence detection kit with CSPDTM as the
substrate, was performed as recommended by the manufacturer (Tropix
Inc.). The first antibody was an anti-His antibody from Amersham
Pharmacia Biotech or Roche Molecular Biochemicals, and the second
antibody was an anti-mouse IgG alkaline phosphatase conjugate (Sigma).
MALDI-TOF Mass Spectrometry--
A Coomassie-stained band
containing the C domain, generated by tryptic digestion, was excised
from an SDS-polyacrylamide gel and completely destained with 50 mM NH4HCO3 in 40% ethanol.
Subsequently, the gel piece was washed three times with 200 µl of 25 mM NH4HCO3 and cut into pieces of
~1 mm3. A 200-µl volume of 50 mM
-mercaptoethanol in 25 mM
NH4HCO3 was added and, after 2 h of mixing
at room temperature, the peptides were extracted with two times 200 µl of 60% acetonitrile, 0.1% trifluoroacetic acid by 5 min of
sonication in a bath sonicator. The
-mercaptoethanol solution and
the extracts were pooled and dried in a SpeedVac. The last traces of
ammonium bicarbonate were removed by adding 10 µl of 1%
trifluoroacetic acid and subsequent drying in the SpeedVac. The dried
samples were dissolved in 5 µl of 50% acetonitrile, 0.1%
trifluoroacetic acid and sonicated for 5 min. Aliquots of 0.75 µl
were applied onto the MALDI target and allowed to air dry.
Subsequently, 0.75 µl of 10 mg/ml
-cyano-4-hydroxysuccinnamic acid
in 50% acetonitrile, 0.1% (v/v) trifluoroacetic acid was applied to
the dried samples, which was then allowed to dry again. MALDI mass
spectra were recorded with a Micromass Tofspec E MALDI time-of-flight
mass spectrometer operated in reflectron mode. Spectra were calibrated externally.
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RESULTS |
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Generation and Characterization of SSCS, SSCS-S124C, IIChis-CL, and
IIChis-S124C--
Two His-tagged EIImtl mutants were
constructed. SSCS is a single cysteine enzyme with only
Cys384 in the B domain. SSCS-S124C contains
Cys124 in the C domain in addition to Cys384.
The phenotype of E. coli LGS322 expressing these mutants was analyzed on MacConkey agar plates with 1% D-mannitol. Both
strains formed purple-red colonies, indicating that the mutants
transport and ferment mannitol. Inside-out (ISO) membrane vesicles from LGS322 cells expressing both mutants were solubilized with 0.25% dPEG
and analyzed for mannitol binding and phosphorylation activities. The
dissociation constants for mannitol were 25 and 45 nM and the turnover values for phosphorylation of mannitol of these mutants were 5200 and 4900 min1 for SSCS and SSCS-S124C,
respectively. The turnover values for mannitol phosphorylation of
purified SSCS and SSCS-S124C were 3200 and 2700 per min, respectively.
These activities are similar to that of wild-type EIImtl
(22, 29). ISO membrane vesicles bearing IIChis-CL or IIChis-S124C, solubilized with 0.25% dPEG, and analyzed for mannitol binding, displayed dissociation constants for mannitol of 32 and 70 nM, respectively. These dissociation constants are similar
to previously determined values of the wild-type C domain generated by
tryptic digestion of the complete protein (22) or of the separately expressed IIC (8). In addition, IIChis-CL and IIChis-S124C could both
complement the mannitol-binding defective EIImtl-G196D for
phosphorylation activity in an in vitro assay, a result similar to that described for IIC (8). Overall, these kinetic data
clearly indicate that SSCS and SSCS-S124C are fully functional enzymes
and that both mutant C domains can bind mannitol and form functional
heterodimers with EIImtl.
Disulfide Bridge Formation in ISO Vesicles and Purified
Enzyme--
Fig. 2A shows the
results of CuPhe-induced oxidation of SSCS and SSCS-S124C in ISO
membrane vesicles, as visualized by immunoblotting with an antibody
raised against the N-terminal His-tag. The untreated proteins migrated
as a major band at ~60 kDa and are indicated as EII. The band at 36 kDa is a degradation product, whereas the one at 116 kDa (band 1) is
most likely the dimer. This dimer has been observed previously upon
extraction of the enzyme from the membrane (3) and is most likely not
held together by a disulfide bond, since it is resistant to reduction
with DTT. The amount of this band 1 is the same in all lanes. The
oxidation by CuPhe of both mutants resulted in the appearance of a
higher molecular weight band (band 2). The molecular mass was
~200 kDa but varied depending on the concentration of acrylamide that
was used (not shown). The formation of this band was almost completely
reversed by reduction of the sample with DTT, suggesting that it is
stabilized by a disulfide bridge. The reduction with DTT is not
complete in the particular sample in lane 5. However, in
lane 11 of Fig. 2A, lane 1 of Fig. 2B,
and in duplicate experiments complete reduction was observed. Both high
molecular weight bands (1 and 2) have been observed previously and both
were denoted as dimeric species (4, 5, 30). Also in this paper we refer
to band 2 as a dimeric species, but we cannot fully exclude the
possibility that it represents another oligomeric state as will be
discussed below. Since Cys384 is the only candidate for
disulfide formation in SSCS, the enzyme is thus capable of forming a
disulfide between the two Cys384 residues. This is an
important observation, because the dimer contacts are between the C
domains (8).
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Partial digestion of EIImtl with endoproteinase Glu-C generated a band at 33 kDa, which corresponds to the C domain without the A and B domains. Endoproteinase Glu-C instead of trypsin was used, because it did not cleave off the N-terminal His-tag within the 1-h incubation period. The cleavage pattern of digested SSCS was the same irrespective of whether the disulfide was formed or not. In addition, the cleavage pattern was unchanged upon reduction. This shows that the disulfide bridge resides in the domains that were degraded, which is consistent with the location of Cys384 in the B domain. However, if the same oxidation and digestion procedure was followed with oxidized SSCS-S124C, a new band at 50 kDa appeared (Fig. 2A, indicated by the arrow), irrespective of whether the digestion was done in the presence or absence of the detergent dPEG. This band disappeared upon reduction of the sample with DTT. This excludes the possibility that the protein was only partially cleaved under the oxidizing conditions. Based on its size, this band is probably the result of a disulfide bridge between Cys124 of both monomers, indicating that residue 124 is at the dimer interface. Very vaguely, some other products, which could include a disulfide bridge between Cys384 and Cys124 (see below) might be visible as well.
To further examine the nature of the Cys124-Cys124 disulfide bridge, the CuPhe-induced oxidation was repeated with purified SSCS-S124C. Fig. 2B shows the result of this experiment, visualized by immunoblotting. The same observations were made when the gel was silver-stained (not shown). The oxidation of purified SSCS-S124C also yields the reducible dimer band 2. Almost no band 1 was observed after purification, which probably indicates that the affinity between two monomers is decreased. Upon endoproteinase Glu-C digestion, the 50-kDa fragment was not observed. Instead, a His-tagged fragment of 42 kDa was visible in addition to the 33-kDa C domain band. This 42-kDa band disappeared upon reduction of the sample with DTT (compare lanes 3 and 4). Endoproteinase Glu-C digestion of the B domain will generate a 7.9-kDa fragment containing Cys384. The size of the 42-kDa band thus suggests that a disulfide bond is formed between the 33-kDa C domain harboring Cys124 and the 7.9-kDa B domain fragment with Cys384. In conclusion, the data in Fig. 2, A and B, point to the formation of two different disulfides, one intersubunit Cys124-Cys124 and one interdomain Cys124-Cys384. Further evidence for both disulfides will be supplied in the following sections.
Requirements for B/C Domain Disulfide Bridge Formation--
To
elucidate the composition of the disulfide bridges, the CuPhe-induced
oxidation was performed with purified SSCS-S124C and SSCS (Fig.
3). The two higher molecular weight bands
(1 and 2) were observed again with both proteins (lanes 2 and 14). These bands were not present upon reduction with
DTT and were much less intense or absent upon phosphorylation of the
protein at His554 and Cys384, confirming that
these bands, at least in SSCS, arise from a disulfide bridge between
the two Cys384 residues in the dimeric complex. Instead of
endoproteinase Glu-C, trypsin was used to define the cross-links.
Trypsin first cleaves in the linker between the C and B domain and,
subsequently, digests the A and B domain completely but leaves the C
domain intact except for the N-terminal His-tag (22, 31). The
proteolytically generated C domain can be observed on SDS-PAGE at 30 kDa, which is somewhat smaller than the endoproteinase Glu-C-generated
C domain. This is consistent with the removal of the His-tag and a
different cleavage site in the linker between the B and C domain. The
tryptic B domain fragment with Cys384 has a calculated mass
of ~1.9 kDa. Tryptic digestion of CuPhe-oxidized SSCS-S124C also
showed an ~30-kDa band, which was significantly broadened upwards
when the sample was not treated with DTT (compare lanes 6 and 7). This suggests that the broadened 30 kDa comprises the C domain plus the 1.9-kDa fragment of the B domain, analogous with
the 42-kDa band in Fig. 2B. Consistent with this conclusion are the following observations: (i) the broadening is not observed with
SSCS (Fig. 3: compare lanes 17 and 18); (ii)
phosphorylation prior to oxidation prevented the occurrence of the
broadening (compare lanes 6 and 8); and (iii) the
broadening was not observed in wild-type EIImtl or in
mutants of SSCS with cysteines at positions 158 or 199 (not shown). A
tryptic-generated C domain of SSCS-S124C, not treated with CuPhe, also
led to some broadening, which is probably due to spontaneous oxidation
(compare lanes 5 and 6). Another point to note is
that the addition of 100 µM mannitol did not have an effect on the occurrence of the broadening (lane 9).
Finally, dimeric C domain was not observed in the tryptic digest of
oxidized SSCS-S124C.
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Taken together, the broadening must be the result of a disulfide bridge between Cys384 and Cys124. To exclude the possibility that this disulfide bridge is an aspecific reaction between two accessible cysteines, another control experiment was performed (Fig. 3, lanes 11 and 12). With the sample in lane 11, oxidation was carried out after trypsin treatment, whereas in lane 12 it was carried out before trypsinolysis. Clearly, the broadening of the C domain band is no longer observed when the trypsin digestion preceded the oxidation. Thus, Cys124 is not capable of reacting with just any cysteine-containing peptide, present at the same concentration.
MALDI-TOF Mass Analysis--
To demonstrate that the fragment,
which caused the broadening of the tryptic-generated C domain,
originated from the B domain, the protein was excised from the
SDS-polyacrylamide gel, reduced with -mercaptoethanol to cleave the
disulfide bond between the C domain and the B domain peptide, the
peptides were extracted with organic solvents, and the extract was
analyzed with MALDI-TOF MS. Fig. 4 shows
the mass spectrum of the extracted peptides. The spectrum only
contained 4 peaks, all of which could be assigned to tryptic B domain
fragments that contain Cys384. The peak at
m/z 1879.05 is the fully cleaved peptide with
residues 380-399 (expected m/z 1878.92), and the
peak at m/z 2007.05 represents the partially
cleaved fragments of residues 379-399 and/or 380-400 (expected
m/z 2007.02). This partial cleavage is due to the
presence of the RK and RKK sequences at the N and C terminus of these
peptides, respectively, that cannot be fully cleaved by trypsin. The
peaks at m/z 1954.79 and 2083.05 represent the
same peptides but with a
-mercaptoethanol adduct, which gives a mass
increase of 76 Da. If the same procedure was applied to the C domain
band that was reduced prior to SDS-PAGE and excised from the gel
following the same procedure no peptides were observed.
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The B/C Disulfide Bridge Can Cross the Dimer Interface--
To
this point, we have provided evidence for a B/C interdomain cross-link
between Cys384 and Cys124 in purified
SSCS-S124C, and a cross-link between the Cys124 of both
monomers in the enzyme in ISO membrane vesicles. In addition, the data
on SSCS provide unequivocal evidence for a disulfide across the dimer
interface between the Cys384 residues on each B domain. To
establish whether the disulfide between Cys384 in the B
domain and Cys124 in the C domain could be formed between
different subunits, heterodimers consisting of IIChis-S124C and SSCS
were subjected to CuPhe-induced oxidation. Each subunit in a
heterodimer provides only one of the cysteines that participate in the
disulfide bridge. Fig. 5A (lane 2) shows that when a 3:1 mixture of SSCS and
IIChis-S124C was oxidized, the same homodimer as observed with purified
SSCS alone (Fig. 5A, lane 4, and Figs. 2
and 3) was formed. In addition, a new band at ~90 kDa appeared,
whereas the IIChis-S124C band became less intense (lane 2).
This 90-kDa band can only be the heterodimer between SSCS and
IIChis-S124C. After reduction, both the SSCS dimer and the heterodimer
disappeared. This shows that the B/C domain disulfide bridge can cross
the dimer interface.
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The B/C Disulfide Bridge Can Be Formed Intramolecularly-- Next, we addressed the question whether or not the disulfide bridge between Cys384 and Cys124 can also be formed within one monomer. For this purpose, heterodimers of SSCS-S124C and a cysteine-less IIChis (IIChis-CL) were formed. It is crucial for this experiment that all the SSCS-S124C monomers are forming a heterodimer with a IIChis-CL monomer, such that only the intrasubunit disulfide bridge is possible. Therefore, an ~15-fold excess IIChis-CL was added and the heterodimer formation was facilitated by the addition of 170 mM Na3PO4, pH 7.6, which lowered the cloud point of the solution (see "Experimental Procedures"). Na3PO4 alone did not have an influence on the cross-linking behavior of SSCS-S124C (not shown). As can be seen in Fig. 5B, homodimers were no longer present upon oxidation with CuPhe, which is indicative of complete heterodimer formation (compare Fig. 2B, lane 2, and Fig. 5B, lane 2). Endoproteinase Glu-C digestion of the oxidized sample generated the 33-kDa C domain and the additional 42-kDa band (arrow in lane 4 of Fig. 5B); the latter is absent upon reduction of the sample with DTT. This experiment, therefore, proves that the B/C disulfide bridge can also be formed within one subunit.
Residue 124 Is Located Near the Dimer Interface--
The
CuPhe-induced cross-linking of SSCS-S124C in ISO membrane vesicles
corresponds most likely to a disulfide bridge between Cys124 of both monomers. The CuPhe-induced oxidation was
repeated with purified IIChis-S124C (Fig.
6). IIChis-S124C migrates as a monomer with an apparent molecular mass of 28 kDa in the presence of DTT. Upon
oxidation, a dimeric IIChis-S124C with an apparent molecular mass of 50 kDa was observed. To confirm the close proximity of both
Cys124 residues in one dimer, reduced IIChis-S124C was
subjected to cross-linking with dimaleimides of different lengths,
ranging from 7.7 to 15.1 Å. Fig. 6 shows that all three dimaleimides
also yielded the formation of the 50-kDa dimer. These data indicate that the residue at position 124 is located at the dimer interface in
purified IIChis-S124C.
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DISCUSSION |
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In this article we describe the generation of several disulfide bridges, indicative for close proximity, between two cysteines in the dimeric EIImtl. In the enzyme in ISO membrane vesicles, intersubunit disulfide bridges between both Cys384 residues and between both Cys124 residues are formed. Upon purification in the detergent dPEG, the intersubunit disulfide between both Cys384 residues is still formed. In addition, an interdomain cross-link between Cys384 and Cys124 is observed, which either can be formed as an intrasubunit or an intersubunit disulfide. The intersubunit disulfide between both Cys124 residues is not formed in purified EII, but it is formed in purified C domain harboring Cys124. It is important to stress that both the detergent-solubilized EII and IIC retain full binding capacity, and the soluble EII retains full mannitol phosphorylation activity. The observations made with the detergent-solubilized enzymes thus reveal structural information of functionally relevant conformations of the protein.
To form a disulfide bond, the C atoms of the cysteines
have to come within 3.8-4.5 Å of each other (32). The two
Cys384 and two Cys124 residues are thus in very
close proximity both at the B/C domain interface and the dimer
interface of EIImtl. The multiplicity of possible
disulfides is easy to understand in the light of the interdomain
dynamics that are essential for the entire phosphorylation and
transport cycle. The architecture and functioning of EIImtl
necessitates an interdomain flexibility in which the B domain interacts
with various domains at different stages in the catalytic cycle. (i)
The active sites of the A and B domains, each of which are proteins of
~15 kDa, must approach one another to transfer the phosphoryl group
from His554 on the A domain to Cys384 on the B
domain, then (ii) this same region of the B domain, containing a
phosphorylated Cys384, must interact with the C domain to
effect the conformational energy coupling which enables the
translocation and subsequent phosphorylation of mannitol. (iii) The
phosphoryl group can cross the dimer interface from the A domain of one
monomer to the B domain of the other monomer (illustrated in Fig. 1)
(33, 34) or from the B domain of one monomer to the mannitol bound by
the C domain of another monomer (25, 35). It is, therefore, logical that the active site Cys384 in the B domain is in close
proximity with different regions of the enzyme at different steps in
the catalytic cycle and that, during the cross-linking process, these
various domain interactions transiently occur and lead to the
cross-links observed. Upon purification in detergent, a different
pattern of the disulfide bridges is observed, which is additional
evidence for this dynamic situation.
The observation of different higher aggregated forms of EIImtl, all denoted as dimeric forms, is not new. Band 1 was observed upon extraction of the enzyme from the membrane, whereas band 2 appeared upon cross-linking via disulfides, dimaleimides, or lysine-specific cross-linkers (3-5, 30). Band 1 is insensitive to reduction suggesting that it is stabilized by noncovalent interactions rather than disulfides. In this regard, it is noteworthy that most observations of band 1 stem from measurements on protein extracted from the membrane but not purified. It is likely that its aggregation state is stabilized by lipids complexed to the protein. Band 2's position and, thus, its estimated mass varies with the degree of cross-linking in the polyacrylamide gel, but it is close to that of a tetramer. In a single-cysteine EIImtl, the tetramer can be generated when a single disulfide bond cross-links two dimers whose subunits are held together by native-like interactions, as in the case of band 1. The tetramer could also arise from two disulfide-bonded dimers that come together to form a tetramer. The band in between 1 and 2 (lane 3, Fig. 2A) could represent the loss of a monomer from a tetramer, which is only possible in the interdimer cross-linked tetramer. A possible tetrameric state of the enzyme, however, does not lead to other conclusions for the close proximity of the cysteines at positions 384 and 124. In the future we will examine the possible tetrameric nature of EIImtl in more detail with analytical ultracentrifugation as has been done for the lactose carrier of Streptococcus thermophilus (36).
As summarized in the Introduction, there is a lot of kinetic and thermodynamic evidence for conformational coupling at the B/C domain interface. However, there was no direct structural information about the location of this interface. Here, we present for the first time the location of at least part of the B/C domain interface, which is formed by the first cytoplasmic loop in the C domain and the region around the active-site cysteine in the B domain. The first evidence for the importance of this loop in the interaction with the B domain came from time-resolved fluorescence spectroscopy studies on a series of single-tryptophan containing mutants (13). Notably, a change in the time-resolved anisotropy of tryptophan 109 was observed upon phosphorylation of the B domain. The location around residue 124 is in a region which is conserved among mannitol-specific EIIs of different origin (37). Close proximity of this region of the C domain and the B domain active site was already suggested by photocross-linking experiments.4 Recent kinetic data on a series of phenylalanine to tryptophan replacements in this first cytoplasmic loop suggest that residues 126 and 133 are critical for the phosphorylation of mannitol but not for mannitol binding. The data suggest that these residues are located at or near the dimer interface and involved in the B/C domain interaction,7 which is consistent with the data presented here.
The location of the interface in the first cytoplasmic loop is very interesting, because all previously described mutations that have an influence on the functioning of EIImtl are located in the predicted second cytoplasmic loop. This is also the location of the GIXE motif (residues 254-257), which is highly conserved in all EII's and speculated to be involved in substrate binding (38). Replacement of Glu257 in this motif led to enzymes with no or only low affinity mannitol binding, depending on the substituent, as well as defective transport (39). Another region in the same loop is also important for activity. For instance, replacement of Gly196 or His195 led to enzymes, which exhibited no or low affinity binding for mannitol (25, 34, 40). Interestingly, a heterodimer of the inactive mutants E257A and H195A was significantly active in transport and phosphorylation of mannitol, suggesting that these residues are in close proximity (35). Also data on the glucose transporter of E. coli point to a role for the second cytoplasmic loop in substrate binding (41).
In conclusion, the data presented here suggests that residues 124 and
384 of both subunits can come within 5 Å of each other and are located
at the B/C domain and dimer interface. An intriguing question is what
happens at the B/C domain interface upon phosphorylation. Phosphorescence data of single tryptophan mutants in the C domain show
that a conformational change takes place upon phosphorylation of
Cys384, different from that upon mannitol binding (14).
With the current pairs of cysteines, however, it is not possible to
figure out what is happening precisely, because phosphorylation
prevents Cys384 from forming a disulfide. In the future, we
will screen several cysteine mutants in these regions of the protein to
determine the exact borders of the B/C domain and dimer interface and
changes therein upon mannitol binding and/or B domain phosphorylation. Eventually this will lead to a more detailed understanding of the
energy coupling mechanism at the B/C domain interface in
EIImtl.
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ACKNOWLEDGEMENT |
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Jaap Broos is acknowledged for critical reading of the manuscript and useful discussions.
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FOOTNOTES |
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* This work was supported by the Ministry of Economic Affairs, the Ministry of Education, Culture and Science, and the Ministry of Agriculture, Nature Management and Fishery in the framework of an industrial relevant research program of the Netherlands Association of Biotechnology Centers in the Netherlands (ABON).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.: 31-50-3634321;
Fax: 31-50-3634429; E-mail G.T.Robillard@chem.rug.nl.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M010728200
2 The nomenclature of the enzymes is: EII, carbohydrate-specific transport protein of the PTS; EIImtl, wild-type mannitol-specific EII of E. coli containing all four cysteines; SSCS, N-terminal His-tagged EIImtl with Cys110, Cys320, and Cys571 replaced with a serine; SSCS-S124C, SSCS with Ser124 in the C domain replaced with a cysteine; IIChis-CL, C-terminal His-tagged cysteine-less C domain with Cys110 and Cys320 replaced with serine; IIChis-S124C, IIChis-CL in which Ser124 is replaced with a cysteine. Numbering of residues, even in the His-tagged mutants, is always according to the numbering in the original sequence of EIImtl.
3 O. Amster-Choder, personal communication.
4 B. A. van Montfort, R. H. Duurkens, B. Canas, J. Godorac-Zimmerman, and G. T. Robillard, unpublished results.
5 E. Vos, personal communication.
6 E. Vos, J. Broos, and B. Poolman, unpublished data.
7 E. Vos, R. Ter Horst, G. T. Robillard, B. Poolman, and J. Broos, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PEP, phosphoenolpyruvate; ISO, inside-out; dPEG, decyl-polyethylene glycol; CuPhe, Cu(II)-(1,10-phenanthroline)3; PTS, phosphotransferase system; mtl, mannitol; HPr, histidine-containing protein; EI, enzyme I; MALDI-TOF MS, matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
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