From the Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The glucose transporter of the bacterial
phosphotransferase system (PTS) consists of a hydrophilic
(IIAGlc) and a transmembrane subunit
(IICBGlc). IICBGlc has two domains (C and B),
which are linked by a highly invariant sequence. Transport of glucose
by IIC and phosphorylation by IIB are tightly coupled processes. Three
motifs that are strongly conserved in 12 homologous PTS transporters,
namely two invariant arginines (Arg-424 and Arg-426) adjacent to the
phosphorylation site (Cys-421), the invariant interdomain sequence
KTPGRED, and two conserved histidines (His-211 and His-212) in the IIC
domain were mutated and the mutant proteins characterized in
vivo and in vitro for transport and phosphorylation
activity. Replacement of the strongly -turn favoring residues Thr
and Gly of the linker by
-helix favoring Ala results in strong
reduction of activity, whereas the substitutions of the other residues
have only minor effects. The R424K and R426K mutants can be
phosphorylated by IIAGlc but can no longer donate the
phosphoryl group to glucose. The H211Q and H212Q mutants continue to
phosphorylate glucose at a reduced rate but H212Q can no longer
transport glucose. Mixtures of purified R424K/H212Q and R426K/H212Q
have 10% of wild-type phosphorylation activity and when coexpressed in
Escherichia coli support glucose transport.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Escherichia coli has two transporters for glucose (1, 2). They act by a mechanism that couples translocation with phosphorylation of the substrate. Both transporters are components of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS).1 The IIAGlc-IICBGlc complex is specific for glucose, the IIABMan-IICMan-IIDMan complex has a broad substrate specificity for Glc, Man, GlcNAc, and other derivatives of Glc altered at the C2 carbon. The PTS comprises two cytosolic proteins, enzyme I and HPr, which sequentially transfer phosphoryl groups from PEP to the different carbohydrate-specific transporters (enzymes II). Number and substrate specificity of the transporters varies for different bacterial species (for review see Refs. 3-5). They have been grouped into four families (6) based on amino acid sequence comparisons. IICBGlc belongs to the glucose family presently comprising 25 members (Protein Domain Data base; protein.toulouse.inra.fr/prodom/prodom.html), the IIABMan-IICMan-IIDMan belongs to the mannose family comprising 6 members (7-12).
All PTS transporters consist of three functional units (IIA, IIB, IIC), which occur either as protein subunits or domains of a multidomain polypeptide (13). The glucose transporter consists of two subunits IIAGlc and IICBGlc. IIAGlc is a 18-kDa hydrophilic protein that is phosphorylated at His-90 (14). The 51-kDa IICBGlc subunit consists of two domains (15). The hydrophobic domain (IIC, residues 1-~380) spans the membrane eight times and contains the glucose binding site (16). The hydrophilic domain (IIB, residues ~380-477) is phosphorylated at Cys-421 (17). Phosphoryl groups are transferred from HPr via His-90 of IIAGlc to Cys-421 and hence to glucose (18). The IIC and IIB domain are connected by an heptapeptide sequence that is highly conserved in those two- and three-domain transporters of the glucose family that have the domain order CB(A). A chimeric protein consisting of the IICGlc domain of the glucose transporter and the IIBAGlcNAc domain of the GlcNAc transporter is active and glucose-specific (19). The IIC and IIB domains of IICBGlc can be expressed as separate polypeptides. The purified subclonal domains retain 2% of wild-type phosphotransferase activity when they are combined in vitro (20).
The three-dimensional structure of the IIBGlc domain, its interaction with the IIAGlc subunit and the structural consequences of its phosphorylation at Cys-421 have been analyzed by heteronuclear NMR spectroscopy (21, 22). A model of the transmembrane topology of the IIC domain has been derived from protein fusion studies and further confirmed by linker insertion mutagenesis (16).2 Point mutants of IICBGlc have been selected that facilitate Glc transport uncoupled of phosphorylation (23) and that retain glucose phosphorylation activity but have a strongly reduced translocation activity (24). All of these mutations are located in the IIC domain, and none was found so far in the IIB domain.
When the amino acid sequences of the transporters belonging to the glucose family are compared, several regions of strong amino acid similarity can be discerned (Fig. 1). One includes the active site Cys-421 which is phosphorylated by IIAGlc and donates the phosphoryl group to the transported glucose. Besides Cys-421, Arg-424, and Arg-426 are also invariant. They are the only invariant arginines in the transporters of the glucose family. Arginines are frequently found in phosphate-binding and phosphate-catalytic sites where they can stabilize phosphate through hydrogen bonding and electrostatic interactions with their guanidino group (25). Here we characterize by Arg to Lys substitutions the functions of the two invariant arginines in the IIB domain. Surprisingly, the KTPGRED motif, the putative linker between the IIB and IIC domain, is also invariant. Although interdomain regions, linkers and hinges are enriched for residues such as alanines and prolines, glycines or glutamines (26-28) their amino acid sequence is usually not conserved. Here we address the question of how important the residues of this heptapeptide are for the function of IICBGlc. A third conserved region surrounds the invariant His-212, which has once been proposed to be a potential phosphorylation site (28). Although it has been shown convincingly that a cysteine of the B domain and not the C domain is phosphorylated (17, 29, 30), the possible function of the highly conserved His-212 in the enzyme II components of the glucose family has not yet been elucidated. Here we show that this residue is essential for transport but not for phosphorylation activity.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial Strains, Plasmids, and Growth Media--
E.
coli K12 ZSC112LG (glk manZ
ptsG:Cm) was used as
host for all experiments (20). Plasmid pTSGH11 encodes under the
control of Ptac a IICBGlc with a carboxyl-terminal
hexahistidine tag. pTSGH11 was constructed by ligation of the
SacII-HindIII vector fragment of pTSG11 (20) and
the SacII-HindIII insert fragment of pQEGH12
(31). pABG421 encodes the C421S mutant kanamycin resistance and the
P15A origin of replication (20). Cells were grown at 37 °C in LB
medium containing appropriate antibiotics. XL1-blue (Stratagene) was used for cloning and plasmid amplification.
Site-directed Mutagenesis-- The IICBGlc mutants H211Q, H212Q, K382A, T383A, G385A, R386A, E387A, D388A, R424K, and R426K were constructed using the gapped duplex procedure (20, 32). The mutant P384G was constructed by overlap extension mutagenesis (33). Mutant C421S was from (34). Mutant clones were identified by way of diagnostic restriction sites and sequencing. The restriction fragments carrying the mutants were inserted in to the ptsG gene on the expression plasmid pTSGH11.
In Vivo Complementation Assays--
ZSC112LG was doubly
transformed with pABG421 and pTSGH11 derivatives encoding the different
IICBGlc mutants and streaked on McConkey plates containing
ampicillin, kanamycin, and 0.4% Glc.
Overproduction and Purification of Proteins--
E.
coli ZSC112LG was transformed with derivatives of pTSGH11
encoding wild-type and mutant IICBGlc. IICBGlc
was overexpressed and purified by metal chelate affinity chromatography as described (20). Enzyme I, HPr, and IIAGlc containing
cell extracts were prepared, and the proteins were purified as
described (35).
In Vivo Transport and in Vitro Phosphotransferase
Assays--
Uptake of [14C]MG by bacteria was assayed
as described (24). Cells were grown to A600 = 0.5 in 250 ml of M9 mineral medium supplemented with 1% glycerol and
20 µM
isopropyl-1-thio-
-D-galactopyranoside, collected by
centrifugation, and resuspended in M9 medium without supplement. 0.33 ml of concentrated cell suspension were diluted with 0.77 ml of M9
medium and aerated for 10 min at 22 °C. Uptake was started by the
addition of 12 µl of 10 mM [14C]
MG (6000 cpm/nmol). Aliquots of 100 µl were withdrawn, diluted into 8 ml of
ice-cold M9 containing 0.4 mM
MG, filtered through GF
(Whatman) glass fiber filters under suction, and washed with 20 ml of
ice-cold 0.15 M NaCl. The filters were dried and counted. The dry weight was determined from 0.2 ml of the concentrated cell
suspension. In vitro phosphorylation of
[14C]Glc was assayed by ion-exchange chromatography as
described (1). 100 µl of incubation mixture contained 4 µl of cell
extract as source of enzyme I, HPr, and IIAGlc, 1 mM PEP, 1 mM [14C]Glc (1000 dpm/nmol), 0.1 mg of E. coli phospholipid suspension (Sigma). The phosphorylated proteins were analyzed on 17.5%
polyacrylamide gels as described (36). 20 µl of incubation mixtures
contained 75 µM [32P]PEP (1200 dpm/nmol),
0.7 µg of enzyme I, 1.4 µg of HPr, 0.3 µg of IIAGlc,
0.1 mg of E. coli phospholipids, and 5 µg of purified
IICBGlc and where indicated 3 mM Glc.
[32P]PEP was prepared as described (37).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alanine-scanning Mutagenesis of the Interdomain Linker--
The
amino acid sequence KTPGRED between the IIC and IIB domains is the most
highly conserved region in the transporters belonging to the glucose
family of the PTS (Fig. 1). Six of these
seven residues were replaced one at the time by alanine. Pro-384 was replaced with glycine because several attempts to replace it with Ala
by gapped duplex and polymerase chain reaction-based mutagenesis methods failed. The reason for this failure is not known. Mutant proteins were expressed in strain ZSC112G, which carries a
chromosomal deletion of ptsG. Six transformants produced red
colonies on McConkey glucose indicator plates, and only the G385A
mutant produced yellow colonies with a small red center. All the
IICBGlc mutants could be overepressed in the same amount as
wild-type protein and purified by Ni2+ chelate affinity
chromatography (results not shown). The purified G385A protein
displayed only 0.8% of wild-type activity, T383A had 10% of wild-type
phosphotransferase activity, whereas the other mutants had between 40 and 120% of wild-type activity (Fig. 2).
The very low activity of the G385A mutation was not due to protein
degradation, since the protein could be purified in the same amount as
the other IICBGlc mutants.
|
|
Function of the Invariant Arginines and Histidines--
The
IICBGlc mutants H211Q, H212Q, R424K, and R426K were
expressed in strain ZSC112G. The H212Q, R424K, and R426K mutants
produced yellow colonies on McConkey glucose indicator plates
indicating that they were unable to transport and phosphorylate glucose
in amounts sufficient to support fermentation of glucose (results not
shown). The H211Q mutant produced yellow colonies with a red center.
Uptake of
MG by whole cells expressing H211Q was reduced to less
than 10% of the wild-type control, whereas the H212Q, R424K, and R426K
mutants had no transport activity (Fig.
3).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Point mutations in three strongly conserved regions of the
IICBGlc subunit of the glucose transporter were
characterized with respect to their effect on protein phosphorylation,
nonvectorial glucose phosphorylation, and transport activity. The first
region of interest is the KTPGRED linker between the IIC and the IIB
domains, which is predicted to assume a loop structure (38, 39). The
T383A and G385A substitutions reduced the activity to less than 10% of
the control. Thr and Gly favor -turn structures, whereas Ala favors
-helices. Substitution of these
-turn residues at the center of
the hinge by Ala could lead to a stiffening of the linker with
concomitant reduced domain mobility. The G385A mutation is less active
than the two domains expressed as completely separated subunits (20) or
the transposition of the B domain from the carboxyl-terminal to the
amino-terminal end of the IIC domain. IIBCGlc with
circularly permuted domains has 40-70% activity provided the two
domains are linked via a flexible Ala Pro rich hinge
peptide.3 Pro is another
residue that strongly favors
-turn structure. The P384G substitution
reduced activity to 40%. Pro-384 could not be substituted by Ala for
unknown reasons. It is likely that the KTPGRED linker acts as a hinge
of precise but restricted mobility. In this respect it differs from the
Ala Pro rich linkers, which serve as universal joints in many unrelated
multidomain proteins including PTS proteins (26, 28, 40). The KTPGRED
sequence is highly conserved only in the two domain proteins with the
domain order CB but is missing in the two-subunit complexes of the
glucose family and in the transporters that have the circularly
permuted domain order BC.
His-211 and His-212 are conserved residues in the IIC domain, and Arg-424 and Arg-426 are two invariant residues immediately adjacent to the phosphorylation site of the IIB domain (Fig. 6). His-211 is not essential for any of the known IICBGlc functions. In contrast His-212, Arg-424, and Arg-426 are essential for vectorial transport of glucose. Phosphoryl transfer from Cys-421 to glucose is completely blocked in the arginine mutants and slow in the H212Q mutant. A mutant with a similar phenotype as R424K and R426K was also found in the IIB domain of the IIABMan subunit of the structurally unrelated mannose transporter (41). IIABMan R172Q can be phosphorlyated at His-175 but cannot donate the phosphoryl group to glucose. It is not clear what function Arg-424 and Arg-426 serve. In view of their proximity to the active site cysteine (Fig. 6) they could stabilize the phosphate in the bonded ground state or in the transition state during transfer from Cys-421 to Glc. Phosphotyrosine phosphatases, the second group of proteins that form phosphocysteine intermediates, provide strong evidence for such an interaction (42). Their active sites contain a cysteine and an arginine, separated by five residues. X-ray structures of these proteins indicate that the invariant arginine stabilizes the transition state with three hydrogen bonds between the guanidino group and two phosphate oxygens (43). However, since neither the IIB domains of the mannitol transporter nor the IIB domain of the cellobiose transporter have invariant arginines near the active site cysteine (44), alternative functions like stabilization of the interaction between the IIB and IIC domains during transport and phosphorylation of glucose should also be considered. In this case it is likely that second site suppressor mutations in the IIC domain can be found. The tight phenotype of the two arginine mutations will facilitate the selection of these mutants.
|
Transport activity is partially restored when H212Q and C421S are
expressed together, and phosphotransferase activity is restored when
purified H212Q is mixed with either R424K or R426K. This indicates that
IICBGlc subunits cooperate in phosphoryltransfer from
Cys-421 of one subunit to glucose bound to an intact IIC domain of the
second subunit. Since IICBGlc is a dimer (18) this
phosphoryl transfer most likely occurs between domains on different
subunits within the dimer. However, interdimer phosphoryl transfer
cannot be excluded, because molecular subunit exchange between purified
dimers has not been demonstrated for IICBGlc. Indeed, as
determined by fluorescence energy
transfer,4 intersubunit
exchange is very slow in the IIABMan dimer of the mannose
transporter where the subunits are interwined through a swap of strands
between the -sheets of each subunit (45).
His-195 and Gly-196 are two invariant residues in the members of the mannitol family of PTS transporters. Like His-212 of IICBGlc, His-195 of IICBAMtl was once considered as a potential phosphorylation site (46). His-212 and His-195 are located in the putative large cytoplasmic loop of their IIC domains (16, 47). His-195 and Gly-196 of IICBAMtl have been mutagenized and characterized (48-50). Like H212Q of IICBGlc, H195A and H195R of IICBAMtl were inactive but surprisingly could not be complemented with a C384S mutant (equivalent of the C421S mutant of IICBGlc) (48). The H195N mutant on the other hand was active (48). This indicates that His-195 of IICBAMtl and His-212 of IICBGlc are functionally nonequivalent despite their apparently similar position in the primary structure. In contrast, the G196D mutant, which had less than 0.1% of wild-type activity, could be complemented with the C384S mutation, indicating that the IIB domain can transfer its phosphoryl group to mannitol bound to the IIC domain (49). Complementation between IIC and IIB as well as between IIB and IIA domains therefore appears to be a general property of the dimeric PTS transporters. Although complementation between sites on different domains is most probable (51), noncomplementation between sites on different domains (36, 48) as well as complementation between sites on the same domain have both also been observed (36).
Acknowledgment-- We thank Ruedi Beutler for helping with the preparation of the similarity plot.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant 31-45838.95 from the Swiss National Science Foundation.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.: 41-31-6314346;
Fax: 41-31-6314887; E-mail: erni{at}ibc.unibe.ch.
1
The abbreviations used are: PTS,
phosphoenolpyruvate-dependent
carbohydrate:phosphotransferase system (EC 2.7.1.69);
IIAGlc, hydrophilic subunit of the glucose transporter;
IICBGlc, transmembrane subunit of the glucose transporter;
IIABMan, IICMan, IIDMan, subunits
of the mannose transporter of the PTS; IICBAMtl, mannitol
transporter of E. coli; HPr, histidine-containing
phosphocarrier protein of the PTS; PEP, phosphoenolpyruvate; MG,
-methyl-D-glucopyranoside.
2 R. Beutler and B. Erni, unpublished results.
3 R. Gutknecht and B. Erni, unpublished results.
4 B. Erni, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|