From the Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland
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ABSTRACT |
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The mannose transporter of bacterial
phosphoenolpyruvate:sugar phosphotransferase system (PTS) mediates
uptake of mannose, glucose, and related hexoses by a mechanism that
couples translocation with phosphorylation of the substrate. It
consists of the transmembrane IICMan·IIDMan complex and the
cytoplasmic IIABMan subunit. IIABMan
has two domains (IIA and IIB) that are linked by a 60-Å long alanine-proline-rich linker. IIABMan transfers phosphoryl
groups from the phospho-histidine-containing phospho-carrier protein of
the PTS to His-10 on IIA, hence to His-175 on IIB, and finally to the
6'-OH of the transported hexose. IIABMan occurs as a stable
homodimer. The subunit contact is mediated by a swap of The carbohydrate transporters of the bacterial phosphotransferase
system (enzymes II of the
PTS)1 mediate uptake
concomitant with phosphorylation of hexoses and hexitols. They consist
of four functional units termed IIA, IIB, IIC, and IID that occur
either as individual subunits in a protein complex or as independently
folding domains of a multidomain protein. IIA and IIB sequentially
transfer a phosphoryl group from the phosphoryl carrier protein HPr to
the transported substrate. IIC and IID span the membrane and mediate
substrate translocation. Substrate translocation is activated by the
phosphorylation/dephosphorylation cycle of IIB (1-4). IIA and IIB of
certain transporters have regulatory activity in addition to their
"energy-transducing" function. For instance, IIAGlc of
Escherichia coli, the gene product of crr,
modulates the activities of adenylate cyclase (5, 6), glycerol kinase
(7), and of the membrane permeases for lactose and maltose (8-12). The IIB domains of some PTS transporters regulate the activity of antiterminator and transcription activator proteins (13). In the
absence of the cognate substrate, the IIB domain of the The tertiary and quarternary structures of IIA units from different
families of PTS transporters are completely unrelated. IIA occur as
monomers (IIAGlc) (7), stable dimers (IIAMan)
(19), or trimers (IIALac) (20) of identical subunits.
Similarly, the IIB units have different 3D structures but are monomeric
(21-23). The membrane-spanning IIC and IID subunits occur as
oligomers, mostly dimers (24-28). The multidomain composition of the
PTS transporters and their dimeric structure allows for various forms
of interallelic and intergenic complementation. For instance, the
coexpression of two mutated IICBGlc subunits of the glucose
transporter with inactive B and C domains, respectively, resulted in
complementation of transport activity (29). Complementation has also
been observed between inactive mutants of IICBAMtl (30, 31)
and between two inactive mutants of the paralogous transporters for Glc
and for GlcNAc (IICBGlc and IICBAGlcNAc) of
E. coli (32, 33). It is generally assumed that
complementation in vitro is because of the formation of
heterodimers between two different inactive subunits and not only to
transient association of different inactive homodimers.
The E. coli IIABMan subunit is a homodimer (see
Fig. 1A). Each monomer comprises two independently folding
domains, the A domain (residues 1-133) and the B domain (residues
156-323) connected by a 23-residue long alanine-proline-rich linker
(35, 36). The IIAMan domain contains a five-stranded
Only the A domain participates in the dimer interface. The
monomer-monomer interaction occurs through the interlocked -strands and
an extensive contact area between the IIA domains. The H10C and H175C
single and the H10C/H175C double mutants were used to characterize the
phosphoryl transfer between IIA to IIB. Subunits do not exchange
between dimers under physiological conditions, but slow phosphoryl
transfer can take place between subunits from different dimers.
Heterodimers of different subunits were produced in vitro
by GuHCl-induced unfolding and refolding of mixtures of two different
homodimers. With respect to wild-type homodimers, the heterodimers have
the following activities: wild-type·H10C, 50%; wild-type·H175C
45%; H10C·H175C, 37%; and wild-type·H10C/H175C (double mutant),
29%. Taken together, this indicates that both cis and
trans pathways contribute to the maximal phosphotransferase activity of IIABMan. A phosphoryl group on a IIA domain can
be transferred either to the IIB domain on the same or on the second
subunit in the dimer, and interruption of one of the two pathways
results in a reduction of the activity to 70-80% of the control.
INTRODUCTION
Top
Abstract
Introduction
References
-glucoside transporter (IIBCABgl) phosphorylates the antiterminator
protein BglG and thereby inactivates it. This way, IIBCABgl
feedback inhibits its own expression in the absence of a transportable substrate (inducer) (14, 15).
-sheet (strand order 21345) covered by helices on either face
((
)4,
). Four strands are parallel, and the fifth
antiparallel strand which forms one edge of the sheet is swapped
between the subunits in the dimer. His-10, which is phosphorylated
during phosphoryl transfer from HPr to IIB, is located at the
topological switchpoint of the fold. Its imidazole ring is hydrogen
bonded to Asp-67, which acts as a general base increasing the
nucleophilicity of the imidazole ring (19). The B domain contains a
180° twisted seven-stranded
-sheet (strand order 3241567, 1-6 are
parallel and 7 is antiparallel) covered by helices on both faces, as
deduced from the IIBLev subunit which is 47% identical to
the IIBMan domain. His-175, which accepts the phosphoryl
group from His-10 and transfers it to the sugar, is located on an
exposed loop between the first
-strand and
-helix (23).
-strands and an extensive contact area of 1700 Å2 composed mainly
of hydrophobic residues. This confers high stability, and the
IIABMan dimer can be dissociated only concomitant with
complete denaturation (37). The B domain interacts with the
transmembrane IICMan·IIDMan complex of the
mannose transporter. The
IIABMan·IICMan·IIDMan complex,
which can be purified intact, has a stoichiometry closest to
2:1:2 (38-40). The IIABMan dimer can also be purified
as a soluble protein. Dissociated from the transmembrane
IICMan·IIDMan complex, IIABMan
has an elongated form. Ratios f/fo of
1.81 and 1.72 were calculated from the sedimentation coefficient
(s20,w = 3.7 S) determined by
analytical ultracentrifugation (37) and the diffusion coefficient (D = 4.73 10
7 cm2
sec
1) determined by dynamic light scattering,
respectively. The axial ratio of >10:1 derived from
f/fo (41) is compatible with a fully extended dimer (Fig. 1B)
composed of the central A dimer (50 Å along the major axis), the two
linkers (66 Å when fully elongated), and the two B domains (35 Å average diameter).
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Fig. 1.
Hypothetical model of the mannose transporter
complex. A, HPr and the two monomers of the
IIABMan complex are in different shades of gray.
The orientation of the IIB (PDB code 1BLE) and HPr (1POH, (55)) in the
complex with the IIA dimer (1PDO) are taken from the model proposed by
Schauder et al. (23). The active site histidines H10, H175,
and H15 are shown in ball and stick
representation. IICMan and IIDMan span the
membrane. The cartoon of IIABMan was produced using
MOLSCRIPT (56). B, backbone representation of
IIABMan with the alanine-proline-rich linkers in a fully
extended conformation. C, schematic representation of
cis and trans orientations of the IIA dimer
relative to the IIB domains with monomers. Active site contacts are
indicated in black.
It has been shown previously (34) that the active site mutants of
IIABMan, H10C, and H175C, are completely inactive when
assayed alone, but that approximately 3% of wild-type activity is
recovered when the purified proteins are mixed in a 1:1 ratio. Here we
show, that much higher activity is recovered when the purified mutants are mixed, completely unfolded with GuHCl, and then renatured. True
heterodimers form only under these drastic conditions. Phosphoryl transfer between subunits within the dimer is very efficient, whereas
transfer between different dimers is possible but inefficient.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains, Overproduction, and Purification of
Proteins--
E. coli WA2127HIC (manXYZ
ptsHIcrr (42)) was transformed with derivatives of pJFL encoding
wild-type and mutant IIABMan (34). IIABMan was
overexpressed and purified as described (34). Enzyme I and HPr were
purified, and membranes containing
IICMan·IIDMan were prepared as described (42,
43).
GuHCl Unfolding and Renaturation of IIABMan-- Stock solution of purified wild-type and mutant IIABMan were adjusted to a protein concentration of 5 mg/ml. Volumes from the different stocks were mixed to achieve the desired molar ratios or molar fractions. The mixtures were then split in two aliquots. One aliquot was diluted with 8 M GuHCl to a final concentration of 4 M GuHCl (37), and to the other aliquot, the same volume of buffer A (10 mM MOPS, pH 7.0, 50 mM NaCl, 0.5 mM dithiothreitol) was added. Both samples were incubated for 2 h at room temperature. Both samples were then diluted 20-60-fold with buffer A to the desired a IIABMan concentration (3-125 µg/ml) and incubated for another 2 h at 4 °C.
Assay for Phosphotransferase Activity-- In vitro phosphorylation of [14C]Glc was assayed by ion-exchange chromatography as described (34). 100 µl of incubation mixture contained 0.5 µg of enzyme I, 2.8 µg of HPr, and 0.5 µl of crude membranes (~4 µg of protein) containing the IICMan·IIDMan complex. The final concentration of renatured IIABMan varied between 3 ng and 130 ng/100 µl of incubation mixture. The exact values are indicated in the figure legends. The specific activity of [14C]Glc was 1000 cpm/nmol.
Assay for Protein Phosphorylation--
The rate and the extent
of protein phosphorylation was measured as described (45). The
incubation mixture (50 mM NaPi, pH 7.4, 5 mM MgCl2, 2.5 mM NaF, 2.5 mM dithiothreitol) contained, per 250 µl, 1.5 µg of
enzyme I, 2.5 µg of HPr, and 85 µg of IIABMan. The
phosphorylation reaction was started by adding to the incubation mixture at 24 °C [33P]PEP to a final concentration of
80 µM. Aliquots of 40 µl were withdrawn at the
indicated time points and diluted into 1 ml of 80% ammonium sulfate
solution at 4 °C. The protein precipitates were collected on glass
microfibre filters (GF/F, Whatman) under suction, washed, and counted
in a liquid scintillation counter. The background counts because of
enzyme I and HPr (less than 10%) were subtracted from the counts of
the complete system. Phosphorylated proteins were analyzed on 17.5%
polyacrylamide gels as described (21). 20-µl incubation mixtures
contained 134 µM [33P]PEP, 0.15 µg of
enzyme I, 0.46 µg of HPr, 10 µg of IIABMan, and 0.3 µl of IICMan·IIDMan-containing membranes.
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RESULTS |
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Functional Interaction of Subunits in IIABMan Dimers-- Wild-type IIABMan, H10C, H175C, and H10C/H175C double mutant were purified by phosphocellulose chromatography and gel filtration. A 1:1 mixture of purified H10C and H175C has about 5% of the specific activity of wild-type IIABMan. The activity increases nonlinearly at low concentration, and the concentration activity profile does not change after 24 h of preincubation (Fig. 2). These results suggest that the activity is because of transient association between two different inactive homodimers (second order reaction) and that monomers do not exchange to form heterodimers. However, when mixtures of H10C and H175C were denatured in GuHCl and then refolded by rapid dilution, a 20-fold higher specific phosphotransferase activity was obtained (Fig. 3A). When H10C and H175C were mixed in different proportions, the activity profile was bell-shaped with a maximum at a 1:1 molar ratio (Fig. 3B), as expected for a binomial distribution of active heterodimers and inactive homodimers. The activities of heterodimers between wild-type and mutated subunits was characterized in the following experiments. Constant amounts of wild-type IIABMan were mixed with increasing amounts of either H10C or H175C. One-half of the mixture was denatured with GuHCl and then renatured by dilution, the other was diluted only. The phosphotransferase activity remained approximately constant at all concentrations of H10C and H175C (Fig. 3C) independently of whether 100% of wild-type IIABMan occurs as homodimer (no GuHCl) or whether only 11% of IIABMan was in homodimers and the rest in heterodimers with an inactive subunit. The activity was linearly dependent upon the concentration of wild-type IIABMan when wild-type and H10C or H175C were mixed in different molar ratios, denatured, and then renatured (Fig. 3D). This suggests that the presence of a subunit with only one inactive domain in a heterodimer has no effect on the overall phosphotransferase activity of the wild-type subunit. Mixtures between wild-type IIABMan and an excess of the H10C/H175C double mutant were prepared to characterize the phosphoryl transfer between A and B domains on the same subunit. The concentration of wild-type IIABMan was kept constant, and the concentration of the double mutant increased to a maximum of 16:1 (Fig. 3, E and F). At a concentration ratio of 16:1, when only 6% of the wild-type protein is in homodimers and 94% in heterodimers with the double mutant, the activity is still 60% of the control and identical to the activity of the nondenatured mixture. The 40% decrease of activity is because of competition of the excess of inactive homodimers (8-fold over active homo- and heterodimers) for the IICMan·IIDMan complex. Competitive inhibition becomes more pronounced when the concentration of IICMan·IIDMan is rate-limiting. Under these conditions, the phosphotransferase activity is reduced to 50% when the concentration of wild-type homodimer plus heterodimer equals the concentration of the H10C/H175C homodimer (Fig. 3F).
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With each experiment, a control with pure wild-type IIABMan
was carried along as a reference for 100% activity and as control of
refolding yield. The activity recovered after rapid dilution of
wild-type IIABMan was 80 ± 30% (Table
I, column IIABMan homodimer).
The specific activity of heterodimers was calculated as follows. The
activity contributed by IIABMan wild-type homodimers was
subtracted from the total phosphotransferase activity of a mixture of
all dimers. The resulting difference was then divided by the
concentration of heterodimers in the mixture. The concentrations of
homo- and heterodimers were calculated from the binomial distribution.
The specific activities of the different dimers are summarized in Table
I. The turn-over number of wild-type IIABMan from
experiment to experiment varies between 2500 min1 and
1200 min
1. The H10C·H175C heterodimer has a turnover of
370 min
1. This is 37% of the activity of wild-type
IIABMan measured under the same conditions. The turn-over
numbers of heterodimers between a wild-type subunit and either H10C or
H175C are 50 and 45% of wild-type homodimer, and the turnover-number of a heterodimer between a wild-type subunit and a H10C/H175C double
mutant is 30%.
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Protein Phosphorylation-- IIABMan is phosphorylated with [33P]PEP in the presence of enzyme I and HPr and is dephosphorylated in the presence of IICMan·IIDMan and glucose (Fig. 4A). The H175C mutant is stably phosphorylated at His-10 but cannot be dephosphorylated because His-175 is missing. The H10C mutant is weakly phosphorylated although His-10 is missing. It is dephosphorylated in the presence of IICMan·IIDMan and glucose, indicating that phosphorylation occurred at His-175. Phosphorylation of H10C is HPr-dependent but much slower than phosphorylation of wild-type IIABMan (Fig. 5). His-175 must be phosphorylated by HPr directly. Contamination of H10C by IIABMan, which could complement the IIA function, can be excluded because H10C was isolated from an E. coli strain with a chromosomal deletion of the manXYZ operon. It is likely, that phosphorylation of IIB is a consequence of high local concentration of HPr which binds to mutated IIA and then nonspecifically delivers the phosphoryl group to a nearby His-175. Phosphorylation at His-10, whether in wild-type IIABMan or in H175C results in an increased stabilization of the IIABMan dimer against dissociation by sodium dodecyl sulfate, and this effect is not reversed as a consequence of dephosphorylation by IICMan·IIDMan and mannose (Fig. 4B).
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DISCUSSION |
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IIABMan consists of two domains, IIA and IIB, that
sequentially transfer a phosphoryl group from the phosphoryl carrier
protein HPr to the transported sugar. IIABMan is a
homodimer. The subunits are tightly linked through mutual exchange of
-strands between the
-sheets of IIA (19). The B domains, in
contrast, neither interact with each other nor strongly interact with
the IIA domains to which they are, however, covalently linked via 60-Å
long alanine-proline-rich linker (Fig. 1, A and B). Phosphoryl groups can be transferred from IIA to IIB on
the same subunit (cis), on different subunits
(trans), or both. Our results indicate that cis
and trans pathways are of comparable efficiency. Wild-type
IIABMan with four sites and four pathways (two
cis and two trans) per dimer has the highest
specific activity. The heterodimer between wild-type and H10C or H175C
with three active sites and only two pathways (one cis and
one trans) has 50% specific activity. The active monomer in
this heterodimer retains its full activity. Heterodimers with only one
functional A and one functional B domain and only one pathway
(cis or trans) retain between 30 and 40% activity. Taken together, this indicates that both cis and
trans pathways contribute to the maximal
phosphotransferase activity of IIABMan. A phosphoryl group
on a IIA domain can be transferred either to the IIB domain on the same
or on the second subunit in the dimer, and interruption of one of the
two pathways results in a reduction of the activity by 20% to 30% of
the control.
The results confirm our previous observation of interallelic complementation (34) and similar observations by others (26, 31, 46, 47). But in the case of IIABMan, the interpretation has changed. The weak complementation was because of phosphoryl transfer between randomly colliding homodimers. IIABMan monomers do not exchange, as evident from the structure of the IIA dimer (19). However, the long linker (Fig. 1B) allows sterically unconstrained interaction between IIA and IIB domains on different dimers. The linker allows the IIA dimer to dock on the IIBMan·IICMan·IIDMan complex in either of two orientations (Fig. 1C). The cis orientation is presented in Fig. 1A.
A IIABMan mutant with His-86 on the IIA domain replaced by Asn was described to have the same properties as H175C mutant with an inactive IIB domain (34, 36). However, the x-ray structure of IIA showed that His-86 is in a surface-exposed loop and far from the active site. In addition, His-86 is not conserved in any of the homologous proteins (see below). Both observations make His-86 an unlikely target for mutations with a strong phenotype. Resequencing showed the supposed H86N mutation to contain the H175C mutation. We conclude that the H86N mutant is neutral and that vectors must have been exchanged by mistake.
Bacillus subtilis, Klebsiella pneumoniae, Vibrio furnissii,and Lactobacillus casei express transporters homologous to the mannose transporter of E. coli except that IIA and IIB are expressed as separate proteins subunits and not as two domains connected by an alanine-proline-rich linker (48-51). Using the Basic Local Alignment Search Tool (BLAST) program, IIAB homologs with alanine-proline-rich or Q-linkers (52, 53) were found in bacterial genomes2 (complete and in progress) of: Yersinia pestis, Actinobacillus actinomycetemcomitans, Enterococcus faecalis, Clostridium acetobutylicum, Streptococcus pneumoniae, and Streptococcus pyogenes. Why two forms of IIAB units and what function if any does the linker have? All things being equal, binding of the IIABMan dimer to the IICMan·IIDMan complex must be much stronger than binding of a monomeric IIB subunit because the dimer forms two contacts per molecule, whereas a IIB monomer forms only one (54). Although not covalent in the chemical sense, binding might become very strong, and IIAB remain membrane-bound for most of the time. Untying of IIB from the IIC·IID complex is necessary whenever IIB has a regulatory function and must diffuse to other targets. For example, monomeric IIBLev of B. subtilis is not only a subunit of the fructose transport complex, but it also can phosphorylate and thereby inactivate the transcriptional activator LevR (16-18). An analogous situation is observed in E. coli. The transporter for Glc and GlcNAc (IICBGlc·IIAGlc and IICBAGlcNAc) are homologous, but whereas IICBAGlcNAc is a three-domain protein, IIAGlc and IICBGlc are independent subunits. IIAGlc plays a pivotal role in regulation of catabolite repression and inducer inclusion, and it has been shown to interact with glycerol kinase, the transporters for lactose and maltose, and adenylate cyclase (5-12). These interactions with soluble and membrane-bound target proteins require that IIAGlc can freely diffuse through the cell.
The structural stability of the IIAB dimers and their mechanism of
phosphoryl transfer might be unique among the different families of
dimeric PTS transporters. Nevertheless, it indicates that interactions
between different subunits within a dimer (first order reaction) as
well as interactions between different dimers (second order reaction)
have to be taken into consideration when weak interallelic
complementation is observed. The ease with which stable heterodimers
can be generated by reversible unfolding will facilitate the
characterization by fluorescence energy transfer of domain motions that
might occur during phosphorylation and transport of mannose.
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ACKNOWLEDGEMENTS |
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We thank S. Mukhija (ARPIDA AG, Münchenstein) for the gift of [33P]PEP, S. D. Snyder (Protein Solutions Inc., Charlottesville) for determining the diffusion coefficient by dynamic light scattering, and S. Schauder for the help with preparing Fig. 1.
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FOOTNOTES |
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* This study was supported by Grant 31-45838.95 from the Swiss National Science Foundation and by contributions from the Ciba-Geigy Jubiläumsstiftung, Basel, Switzerland.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.
The atomic coordinates and structure factors (codes 1BLE, 1POH, and 1PDO) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
To whom correspondence should be addressed. Tel.: ++41
(0)31/631 43 46; Fax: ++41 (0)31/631 48 87; E-mail:
erni{at}ibc.unibe.ch.
2 http://www.ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html.
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ABBREVIATIONS |
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The abbreviations used are: PTS, phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (EC 2.7.1.69); IIABMan, hydrophilic subunit of the mannose transporter; IICMan and IIDMan, transmembrane subunits of the mannose transporter; HPr, histidine-containing phospho carrier protein of the PTS; PEP, phosphoenolpyruvate; GuHCl, guanidinium hydrochloride; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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