(Received for publication, August 17, 1995)
From the
The interactions between the D-galactose-H symporter (GalP) from Escherichia coli and the inhibitory antibiotics, cytochalasin
B and forskolin, and the substrates, D-galactose and
H
, have been investigated for the wild-type protein
and the mutants Trp-371
Phe and Trp-395
Phe, so that the
roles of these residues in the structure-activity relationship could be
assessed. Neither mutation prevented photolabeling by either
[4-
H]cytochalasin B or by
3-[
I]iodo-4-azidophenethylamido-7-O-succinyldesacetylforskolin
([
I]APS-forskolin). However, measurements of
protein fluorescence show that both residues are in structural domains,
the conformations of which are perturbed by the binding of cytochalasin
B or forskolin. Moreover, both mutations cause a substantial decrease
in the affinity of the inward-facing site of the GalP protein for
cytochalasin B, 10- and 43-fold, respectively, but have little effect
upon the affinity of this site for forskolin, 0.8- and 2.6-fold
reductions, respectively. Both these mutations change the equilibrium
between the putative outward- (T
) and inward-facing
(T
) conformations, so that the inward-facing form is more
favored. They also stabilize a different conformational state,
``T
-antibiotic,'' in which the initial
interactions between the protein and antibiotics are tightened.
Overall, this has the effect of compensating for the reduction in
affinity for cytochalasin B, so that the respective overall K
values are 0.74- and 3.5-fold that of
the wild type, while causing a slight increase, 1.5- and 3.2-fold,
respectively, in affinity of the mutants for forskolin. The Trp-371
Phe mutation causes a 15-fold reduction in the affinity of the
inward-facing site for D-galactose, suggesting that this
residue forms part of the sugar binding site. In contrast, the Trp-395
Phe mutation has no effect upon the affinity of the
inward-facing site for D-galactose. These effects may be
related to the reduction in galactose-H
symport
activity only in the Trp-371
Phe mutant, although it still
effects active transport to the same extent as the Trp
Phe mutant. However, there is a 10-20-fold increase
in the K
values for energized transport
of D-galactose for both mutants.
The D-galactose-H symporter (GalP) (
)from Escherichia coli is homologous to a family
of mammalian glucose transporters (GLUT) (Maiden et al., 1987;
Baldwin and Henderson, 1989; Henderson and Maiden, 1990; Henderson,
1990; Henderson et al., 1992; Griffith et al., 1992).
The GalP and GLUT proteins are, therefore, predicted to have a similar
membrane topology, comprising 12 membrane-spanning
-helices, with
helices 6 and 7 connected by a cytoplasmic domain containing
60-70 amino acids (Mueckler et al., 1985; Griffith et al., 1992; Roberts, 1992). Moreover, GalP has many
properties in common with mammalian glucose transporters. The sugar
specificity of GalP is very similar to that of the human erythrocyte
(GLUT1) and the rat adipocyte (GLUT4) glucose transporters (Barnett et al., 1973; Rees and Holman, 1981; Cairns et al.,
1991; Walmsley et al., 1994b).
Another similarity is that GalP-mediated sugar transport is inhibited by the antibiotics cytochalasin B and forskolin (Henderson and Maiden, 1990; Cairns et al., 1991; Walmsley et al., 1993; Martin, 1993; Martin et al., 1994, 1995; Walmsley et al., 1994a, 1994b), which are potent inhibitors of glucose transporters (Baldwin et al., 1980; Baldwin and Lienhard, 1981; Shanahan, 1982; Gorga and Lienhard, 1981, 1982; Walmsley, 1988; Carruthers and Helgerson, 1991; Helgerson and Carruthers, 1987; Carruthers, 1990; King et al., 1991; Burant and Bell, 1992). The GLUT1 and GalP proteins can be photolabeled by these antibiotics, or derivatives of them, in a D-glucose-inhibitable manner (Cairns et al., 1984, 1987, and 1991; Holman et al., 1986; Holman and Rees, 1987; Karim et al., 1987; Wadzinski et al., 1990; Roberts, 1992; Martin 1993; Martin et al., 1994). The binding of either cytochalasin B or forskolin induces a quench in the protein fluorescence of both GLUT1 and GalP (Gorga and Lienhard, 1982; Carruthers, 1986; Pawagi and Deber, 1990; Chin et al., 1992; Walmsley et al., 1994; Martin, 1993; Martin et al., 1995). This phenomenon allowed the kinetics of the binding of cytochalasin B to GLUT1 and GalP to be monitored by stopped-flow fluorescence spectroscopy, showing that the mechanisms of binding of cytochalasin B to GLUT1 or GalP were similar (Walmsley et al., 1994a). Also, the mechanism for the binding of forskolin to GalP has been analyzed and is similar to that for cytochalasin B (Martin et al., 1995). All these data identified at least three conformational states of the transport proteins, changes in which were detected by measuring protein fluorescence.
The efficiency of
photolabeling GLUT1 with antibiotics is maximal at 280 nm, suggesting
that a tryptophan residue may be activated prior to the covalent
attachment of the antibiotic. Moreover, the binding of antibiotics
induces a conformational change in GLUT1, where the environment around
one or more of the tryptophans must be perturbed. The sites of covalent
attachment for cytochalasin B and forskolin in GLUT1 have been
localized to putative -helices 10 and 11 by identifying the
photolabeled proteolytic fragments from GLUT1 (Cairns et al.,
1984, 1987; Holman et al., 1986; Holman and Rees, 1987; Karim et al., 1987; Wadzinski et al., 1990). The tryptic
digestion of the GalP protein, which has been photolabeled with
[4-
H]cytochalasin B, also produces a labeled
fragment (M
17,000-19,000) of almost
identical M
to that produced by digestion of
labeled GLUT1 (Cairns et al., 1991). There are two tryptophan
residues in GLUT1 that lie within this region, Trp-388 and Trp-412,
toward the cytoplasmic ends of putative helices 10 and 11, respectively
(Mueckler et al., 1985). Furthermore, cytochalasin B is
thought to bind to the inward or cytoplasmic facing site (Deves and
Krupka, 1978; Gorga and Lienhard, 1981), so that these tryptophan
residues seem likely candidates for forming a dynamic segment of the
transporter. The role of these residues has been investigated in
several studies in which mutant proteins, produced by site-directed
mutagenesis, were tested for their ability to bind antibiotics and
transport sugars (Katagiri et al., 1991, 1993; Garcia, et
al., 1992; Schurmann, et al., 1993). The eukaryotic
expression systems used did not provide sufficient amounts of the GLUT1
mutant proteins to allow them to be characterized in detail. The only
firm conclusion reached by these studies is that neither Trp-388 nor
Trp-412 is essential for photolabeling with either cytochalasin B or
forskolin but changing both residues does abolish photolabeling (Inukai et al., 1994).
The alignment of the amino acid sequence of GalP with that of GLUT1 indicates that these tryptophan residues are conserved in GalP and indeed within most members of this extensive family of sugar transport proteins (Griffith et al., 1992). It seems highly likely that these residues, Trp-371 and Trp-395, respectively, in the GalP sequence (Roberts, 1992), play similar roles in the function of the GalP and GLUT proteins. The present investigation has achieved a rigorous characterization of the role(s) of these residues in the binding of antibiotics, D-galactose, and protons, and in the subsequent translocation events. This investigation was only possible because the wild-type and mutant GalP proteins could be produced in high yield, from a strain of E. coli that overexpresses GalP (Roberts, 1992), which is amenable to detailed transient kinetic studies (Walmsley et al., 1994a, 1994b; Martin et al., 1995).
In this paper we have
determined the precise changes in the kinetic constants caused by
mutagenesis of the two highly conserved tryptophan residues in GalP on D-galactose transport. Changes in the inhibition by, and
binding of, cytochalasin B and forskolin were rigorously quantified.
Also, the effect of these mutations on photolabeling with
[4-H]cytochalasin B and
[
I]APS-forskolin, and on galactose-H
symport activities, were determined. Moreover, we have identified
these changes with different conformational states of the protein and
with the distribution between inward- and outward-facing forms.
The substitution of
Trp-371 by Phe in the D-galactose-H symporter
(GalP) seriously impaired its ability to transport D-galactose
under energized conditions, with more than a 10-fold increase in the K
and a 6-fold decrease in the V
, compared with the wild-type (Table 1).
We can assume that
p is unaffected at the low levels of
GalP expression applying in these experiments (see ``Materials and
Methods''). While the V
term is largely
governed by the rate constants for reorientation, the K
term is governed by both the rate constants for translocation and
the true affinity of the sugar binding site (Walmsley, 1988; Lowe and
Walmsley, 1986). The rate of sugar translocation will be governed by
the rate constants for reorientation of the unloaded and sugar-loaded
transporter. The increase in the K
for this mutant
may simply reflect a reduction in the rate constant for reorientation
of the loaded transporter, rather than any change in affinity. An
increase in the rate constant for reorientation of the unloaded
transporter would lead to a decrease in K
.
In
contrast, for the Trp-395 Phe mutant, there was about a 20-fold
increase in the K
, but the V
was only reduced to about half that of the wild type (Table 1). This suggests that this mutation has a more pronounced
effect on the affinity of the transporter for sugar, rather than on the
rate constants for translocation.
In both cases the data indicate a
significant reduction in the specificity (V/K
) of the mutant
transport proteins for D-galactose relative to the wild-type
transport protein. The specificity of the Trp-371 mutant was reduced
56-fold and that of the Trp-395 mutant 26-fold, relative to the wild
type (Table 1). However, it was demonstrated that the mutant
proteins catalyze energized transport by measuring the maximum
accumulation of 0.05 mMD-galactose in the presence
and absence of the uncoupler DNP (2 mM). The intracellular
concentration of D-galactose was measured at 4.2, 0.216, and
0.277 mM for the cells expressing wild type Trp-371
Phe
and Trp-395
Phe protein, respectively (using an intracellular
volume of 2.059 µl/mg dry weight of cells). The concentration of
intracellular D-galactose for cells treated with DNP was
measured at 0.036 mM, 0.028 mM and 0.036 mM for cells expressing the wild type, Trp-371
Phe and Trp-395
Phe proteins, respectively, indicating that the cells were
completely uncoupled. A comparison of the intracellular D-galactose concentrations of cells treated with and without
DNP showed that D-galactose was accumulated 116.67-, 7.71-,
and 7.69-fold by cells expressing the wild-type Trp-371
Phe and
Trp-395
Phe proteins, respectively (Fig. 1). The use of
the host strain JM1100, which has a GalK phenotype and is therefore
unable to metabolize D-galactose, ensured that any effects of
DNP on the metabolic enzymes would have no effect on the accumulation
of D-galactose
Figure 1:
The accumulation of D-galactose catalyzed by the Trp-371 Phe and Trp-395
Phe mutant proteins is active transport as demonstrated by their
sensitivity to uncouplers. The uptake of D-galactose (0.05
mM added concentration) catalyzed by the mutant proteins was
measured as described by Henderson and Macpherson(1986) after the cells
had been aerated in the presence and absence of 2 mM DNP for 3
min:
, Trp-395
Phe - DNP;
, Trp-395
Phe + DNP;
, Trp-371
Phe - DNP;
, Trp-371
Phe + DNP. Each point is the mean of triplicate
measurements; standard deviations are indicated by error bars.
A wild-type control was also carried out (data not shown) that
accumulated 8.65 ± 0.37 and 0.074 ± 0.0025 nmol D-galactose/mg cells dry weight in the absence and presence of
DNP, respectively. The intracellular concentrations of the untreated
cells were 116.67-, 7.71-, and 7.69-fold that of the DNP treated cells
for the wild-type Trp-371
Phe and Trp-395
Phe
cells.
We conclude that both mutants are still capable of energized sugar uptake against a concentration gradient, albeit at an efficiency 1% of wild type.
Figure 2:
The
Labeling of GalP with [4-H]cytochalasin B and
[3-
I]APS-forskolin. Inside-out membrane
vesicles were prepared from E. coli strain JM1100 expressing
the wild-type and mutant proteins using a French press as described
under ``Materials and Methods.'' These were photolabeled with
0.5 µM [4-
H]cytochalasin B or 50
nM [3-
I]APS-forskolin in the presence
of either 500 mM D or L-galactose. Samples (30
µg) of each membrane sample were separated on a 12.5%
SDS-polyacrylamide gel and stained with Coomassie Blue (A and C) and subjected to autoradiography (B) and
fluorography (D). The characteristic appearance of the GalP
proteins at 34-36 kDa is identified by the arrows. Fig. 1, A and B, show photolabeling by
[4-
H]cytochalasin B and Fig. 1, C and D, photolabeling by
[3-
I]APS-forskolin.
The labeled mutant GalP proteins were found to migrate to identical positions to the wild-type GalP protein on the gels (Fig. 2), confirming their identity.
The Trp-371 mutant GalP protein was less readily photolabeled with
[4-H]cytochalasin B, but more readily
photolabeled with [
I]APS-forskolin than the
wild-type protein (Fig. 2); D-galactose was less
effective at protecting the mutant against photolabeling, by either
inhibitor, than the wild-type protein (Fig. 2).
In the case
of the Trp-395 Phe mutant the extent of photolabeling with
cytochalasin B was moderately less than wild-type, whereas
photolabeling with [
I]APS-forskolin was greater
for the mutant than the wild type. However, substrate protected the
Trp-395
Phe mutant against photolabeling by both compounds
better than its protection of the Trp-371
Phe mutant.
We
conclude that neither Trp-371 nor Trp-395 is essential for
photoreaction with [4-H]cytochalasin B and
[
I]APS-forskolin, although we cannot rule out
the possibility that the Phe residues are susceptible to photolabeling.
In the case of the wild type,
both cytochalasin B and forskolin inhibited the transport of D-galactose, but cytochalasin B was a more potent inhibitor (Fig. 3). The reason for this difference is not clear, since
titration of the protein fluorescence of GalP, with these antibiotics,
indicates that the GalP protein has a similar overall affinity (K) for both of them (Table 2).
Figure 3:
The susceptibility of the Trp-371
Phe and Trp-395
Phe mutants to inhibition of D-galactose transport by cytochalasin B and forskolin.
Permeabilized cells, from E. coli strain JM1100, were
equilibrated with either 80 µM cytochalasin B, 80
µM forskolin or ethanol (solvent control) for 3 min. The
uptake of 50 µM
[1-
H]D-galactose was determined by
taking a sample 120 s after the addition of the sugar. The graph shows
percent transport compared with the ethanol control. Wild-type samples
with the addition of ethanol are the mean of 12 uptakes in three
separate experiments; samples with the addition of cytochalasin B are
the mean of six uptakes in two separate experiments and samples with
the addition of forskolin are the mean of three samples in a single
experiment. The mean values ± S.D. are shown. The mean values
for the uninhibited rates of D-galactose uptake (ethanol
control experiments) were 2.8 (±1.02), 0.125 (±0.06), and
0.25 (±0.02) nmol/mg/min for the wild-type, Trp-371
Phe,
and Trp-395
Phe mutants,
respectively.
Interestingly, cytochalasin B and forskolin were more potent
inhibitors of the Trp-371 Phe mutant mediated D-galactose transport, relative to the wild type. This is most
likely due to the greatly impaired D-galactose binding
capability of the Trp-371
Phe mutant protein (higher K
) together with the higher affinity (lower K
) that the mutant has for both cytochalasin B and
forskolin compared with the wild-type protein (Table 2).
Furthermore, forskolin was more potent than cytochalasin B with the
Trp-371
Phe mutant (Fig. 3), consistent with the higher
affinity (lower K
) of the mutant for forskolin
compared with cytochalasin B (Table 2).
In contrast, both
cytochalasin B and forskolin were less effective inhibitors of the
transport of D-galactose mediated by the Trp-395 Phe
mutant, than by the wild-type, and were much less potent against the
Trp-395
Phe than against the Trp-371
Phe mutant (Fig. 3). The antibiotic forskolin had a significantly greater
inhibitor potency than cytochalasin B, with the Trp-395
Phe
mutant. This probably reflects the substantial difference in the
overall affinity (K
) of the Trp-395 mutant GalP
for forskolin and cytochalasin B (Table 2).
In order to assess
the importance of Trp-371 and Trp-395 in the binding of cytochalasin B
and forskolin, the intrinsic fluorescence levels of the wild-type and
mutant proteins were titrated with these antibiotics in the
stopped-flow apparatus. Although the changes in tryptophan fluorescence
of the mutant proteins were less than for the wild-type protein, they
were sufficient to allow detection of the binding of both cytochalasin
B (Fig. 4B and 5B) and forskolin (Fig. 4D and 5D) to both mutants. In each
case, the stopped-flow records were best fitted to a double-exponential
function. The total amplitudes of the fluorescence changes were
determined from the sum of the amplitudes of the two phases and are
plotted as a function of each antibiotic concentration in Fig. 4, B and D, and 5, B and D. A fit of each set of data to a quadratic equation for a
second-order binding process (Walmsley et al., 1993) yielded
the K values, and maximal fluorescence changes,
for cytochalasin B and forskolin given in Table 2.
Figure 4:
The binding of antibiotics to the Trp-371
Phe mutant GalP protein. The concentration dependence of the
observed rate of binding of cytochalasin B (A) and forskolin (C) to the Trp-371
Phe mutant GalP protein. The
concentration dependence of the total quench in the protein
fluorescence of the Trp-371
Phe mutant GalP protein induced by
the binding of cytochalasin B (B) and forskolin (D).
The
wild-type protein has a similar overall affinity for cytochalasin B and
forskolin, with K values of about 1.8-1.9
µM (Table 2). The binding of cytochalasin B causes a
larger quench in the protein fluorescence, of 7.6%, than forskolin,
which only causes a 4.2% quench (Table 2).
There was a slight
increase in the overall affinity of the Trp-371 Phe mutant GalP
protein for both cytochalasin B and forskolin, which was concomitant
with a small decrease in the fluorescence quench, relative to the wild
type. However, the differences from the wild type are small.
The
Trp-395 Phe mutation had a much more pronounced effect, causing
a 3.5-fold reduction in the overall affinity for cytochalasin B and
3.2-fold increase in the affinity for forskolin (Table 2).
Indeed, while the wild-type protein has similar affinities for
cytochalasin B and forskolin, the Trp-395
Phe mutant has an
11-12-fold difference in affinities for these antibiotics. In
addition, the fluorescence quenches with these antibiotics were reduced
to about half those for the wild type (Table 2).
Comparisons of the maximal fluorescence changes, corrected for the relative levels of expression, indicated that both Trp-371 and Trp-395 contribute to the fluorescence changes associated with the binding of cytochalasin B and forskolin to the GalP protein. Trp-395 contributes much more to the protein fluorescence signal, with cytochalasin B and forskolin, than Trp-371. Thus, although it is likely that neither residue is photolabeled by these antibiotics, they are both reporters of antibiotic binding, indicating that they form part of a dynamic segment, or segments, of the transporter, which might be part of the antibiotic binding site. Although neither residue is solely responsible for the fluorescence changes associated with the binding of cytochalasin B and forskolin, together, they are probably responsible for most, if not all, of the signals.
in which T and T
are, respectively, the
outward- and inward-facing forms of the unloaded transporters. In
addition, for GLUT1 there was an additional state T
(CB)
(Walmsley et al., 1994a). T
(A) and
T
(A) are two different conformations of the
transporter-antibiotic complex. For such a model the apparent
dissociation constant of the transporter-antibiotic complex is given by
the following equation,
with K, K
, and K
defined as K
= k
/k
, K
= k
/k
,
and K
= k
/k
. The second-order
association rate constant (k
) and dissociation
rate constant (k
) for the
transporter-antibiotic complexes were determined from the slope and
intercept, respectively, of a plot of the antibiotic concentration
dependence of the rate of the fast phase, and the dissociation
constants for these complexes were calculated from the rate constants (K
= k
/k
) (Table 2).
As shown in Table 2, the wild-type GalP protein was
characterized by similar association rates for cytochalasin B and
forskolin, but the dissociation of cytochalasin B was significantly
slower than forskolin. Thus, the T, or putative
inward-facing transporter, has a higher affinity for cytochalasin B
than forskolin.
In the case of wild-type GalP, the measured overall K for cytochalasin B is 4.6-fold greater than K
(Table 2), indicating that at least 78.4%
of the transporters are in the T
(or putative
outward-facing) conformation prior to the binding of cytochalasin B (K
(K
/K
) - 1) (Walmsley et al., 1994a). As such, the kinetics of the binding of
cytochalasin B do not lend any support to the existence of the
postulated T
(CB) conformation for GalP. However, there is
kinetic evidence to support the existence of all four postulated states
of the human erythrocyte glucose transporter (GLUT1) (Appleman and
Lienhard, 1985, 1989; Lowe and Walmsley, 1986, 1987; Walmsley and Lowe,
1987; Walmsley, 1988; Walmsley et al., 1994a).
On the other
hand, forskolin is bound 3.5-fold less tightly, to the wild-type GalP
protein, in the initial complex (e.g.
T(forskolin)), due to its faster rate of dissociation (Table 2). Accordingly, for forskolin, the overall K
is only marginally greater than K
. However, the binding of forskolin is biphasic,
indicating that this process involves at least two steps. Since there
is an equilibrium between the T
and T
conformations in the absence of bound ligand, K
and 1/K
must have similar values when
forskolin is bound (Martin et al., 1995).
The Trp-371
Phe mutant was also characterized by a concentration-dependent
linear increase in the rate of the fast phase of the binding of
cytochalasin B (Fig. 4A) and forskolin (Fig. 4C), yielding the kinetic parameters given in Table 2. For cytochalasin B, this mutation decreases the
association rate constant, while the dissociation rate constant is
increased, so that the affinity of the T
conformation was
reduced by 10.2-fold, relative to the wild type. The overall affinity
of the mutant for cytochalasin B is similar, or slightly greater, to
that of the wild-type (K
> K
for mutant), which indicates that, following the formation of the
initial complex, there is a further tightening in the binding of
cytochalasin B, as the mutant GalP protein undergoes the
T
(CB)-T
(CB) transition. Thus, the
T
(antibiotic) state is now detectable in this mutant.
In
contrast, the binding of forskolin to the Trp-371 Phe mutant
GalP protein was characterized by only small changes in the association
and dissociation rate constants; these were marginally higher and
lower, respectively, than the wild-type values, so that the mutant
protein had a higher overall affinity (lower K
)
for forskolin (Fig. 5C, Table 2).
Figure 5:
The binding of antibiotics to the Trp-395
Phe mutant GalP protein. The concentration dependence of the
observed rate of binding of cytochalasin B (A) and forskolin (C) to the Trp-395
Phe mutant GalP protein. The
concentration dependence of the total quench in the protein
fluorescence of the Trp-371
Phe mutant GalP protein induced by
the binding of cytochalasin B (B) and forskolin (D).
More
significantly, the Trp-395 Phe mutant was characterized by a
substantial reduction in the cytochalasin B association rate constant
and a moderate increase in the dissociation rate constant, so that the
affinity of the T
conformation was dramatically reduced by
42.7-fold, relative to the wild-type (Fig. 5A, Table 2). Again, the reduction in the affinity of the T
conformation was not matched by a similar reduction in the
overall affinity for cytochalasin B, indicative of the formation of the
T
(CB) conformation.
Surprisingly, the Trp-395 Phe
mutant was characterized by an apparently hyperbolic increase in the
rate of the fast phase of binding of forskolin (Fig. 5C). These data provide clear evidence that the
transporter-forskolin complex undergoes a further conformational
change, the T
(forskolin) to T
(forskolin)
transition. Presumably, for the wild-type protein, this transition
occurs at a rate that is too fast to be measured by stopped-flow
fluorescence spectroscopy. The data in Fig. 5C were
fitted to a hyperbolic function yielding respective minimal and maximal
rates of 3.6 (± 7.0) s
and 28.4 (±
5.8) s
and an apparent K
of 3.7
(± 2.7) µM. These values will correspond to those
for k
, k
, and K
, respectively, in . Using these
values and the measured K
, K
can be calculated as 0.41 from .
The Trp-395
Phe mutation has caused a large shift in the equilibrium between
T
and T
, so that about 29% of the transporters
are in the T
(or outward-facing) conformation in the
absence of ligands, while stabilizing the T
(forskolin)
complex (K
=
([T
(forskolin)]/[T
(forskolin)]
= 7.7). After equilibration with forskolin, the GalP-forskolin
complex is present as 11.3% T
(forskolin) and 88.7%
T
(forskolin). Furthermore, the affinity of the T
conformation for forskolin was reduced 2.5-fold. The above data
also provide minimal values for k
(k
> (k
+ k
)/K
) and k
(k
> k
) of 8.6 s
and 28.4
s
, respectively, indicating that the reduction in
the affinity of the T
conformation is largely attributable
to an increase in the dissociation rate constant.
Accordingly, the
substitution of Trp-395 for Phe causes a destabilization of the T (or outward-facing) conformation (K
3.63 for the wild-type GalP protein (Walmsley et al., 1994a), K
= 0.41 for Trp-395
Phe mutant GalP
protein), while stabilizing the T
(antibiotic) conformation.
A value of 1.2 can be calculated for K
, in the
binding of cytochalasin B to Trp-395
Phe mutant protein, since K
is known. Thus, at equilibrium, the
GalP-cytochalasin B complex is present as 54.5% T
(CB) and
45.5% T
(CB). This value for K
for the
T
(cytochalasin B) complex compares with a value of 7.7 for
the T
(forskolin) complex, so that this state is more stable
with bound forskolin.
The rate of binding of cytochalasin B to the Trp-371 Phe
mutant GalP protein was determined as a function of both the D-galactose and cytochalasin B concentrations (Fig. 6a). The data were fitted to an equation for
competitive inhibition (Walmsley et al., 1993, 1994a),
yielding values for the cytochalasin B association and dissociation
rate constants of 2.1 s
and 12.0
s
, respectively, and an apparent K
for D-galactose of 88.7 mM. The association and
dissociation rate constants compare well with those determined
independently by monitoring only the binding of cytochalasin B (Table 2). The K
for D-galactose
can also be determined from a fit of the amplitude data (protein
fluorescence quench) to an equation for competitive inhibition
(Walmsley et al., 1993), yielding a value of 78.6 mM (Fig. 6b). In addition, the K
for D-galactose was also determined by cytochalasin B
displacement, yielding a value of 71.7 mM (Table 3).
When these K
values for the mutant are compared
with those for the wild-type, they indicate that the Trp-371
Phe
mutation causes a 15-fold decrease in affinity of the inward-facing
conformation of GalP for D-galactose. This is consistent with
the fact that D-galactose affords less protection against
photolabeling of the mutant than the wild-type.
Figure 6:
Inhibition by D-galactose of the
binding of cytochalasin B to the Trp-371 Phe mutant GalP
protein. a, the observed rate (k
) of
binding of cytochalasin B to GalP (Trp-371
Phe) is plotted as a
function of the cytochalasin B concentration for a series of inhibitory D-galactose concentrations. Superimposed upon the data are the
best-fit lines for the following equation for competitive inhibition: k
= k
+ k
(CB)/(1 + (D-galactose)/K
), where k
and k
are the
association and dissociation rate constants for cytochalasin B and K
is the dissociation constant for D-galactose. This analysis yielded values of 2.1
(±0.08) µM
s
,
12.0 (±1.3) s
and 88.7 (± 13.7) mM for k
, k
, and K
, respectively. b, the D-galactose inhibition of the binding of cytochalasin B to
GalP (Trp-371
Phe). The binding of cytochalasin B to GalP was
monitored as the net quench in protein fluorescence (
F) when
vesicles were mixed with varying cytochalasin B concentrations in the
presence of a series of inhibitory D-galactose concentrations.
Superimposed upon the data are the best-fit curves for the following
equation for competitive inhibition:
F =
(
F
(CB))/(K
(1
+ (D-galactose)/K
)
+ [CB]), where
F
is the
maximal change in protein fluorescence and K
is
the dissociation constant for cytochalasin B. This analysis yielded
values of 7.42% (±0.25%), 1.50 (±0.36) µM,
and 78.6 (± 24.56) mM for
F
, K
, and K
, respectively. The symbols
represent 0 mM (
), 10 mM (
), 30 mM (
), 62.5 mM (
), 125 mM (
), 250
mM (
), and 500 mM (
) D-galactose.
Unfortunately, the
small signal change associated with the binding of cytochalasin B to
the Trp-395 Phe mutant GalP precluded an analysis of the
inhibition, by D-galactose, of the cytochalasin B binding
rate, in order to provide a measure of the affinity of this mutant for D-galactose. However, it was possible to obtain a value of the K
for D-galactose by displacement of
bound cytochalasin B, which yielded a value of 6.26 mM (Table 3). This suggests that the Trp-395
Phe
mutation produces little, or no change, in the affinity for D-galactose under nonenergized conditions. On the other hand,
the K
for the transport of D-galactose by
the mutant, under energized conditions, is approximately 20-fold higher
than for the wild-type. However, the mutant must still bind protons
since its affinity for D-galactose is increased under
energized conditions relative to that under nonenergized conditions (K
(inhibition of the binding of cytochalasin B)
> K
(transport measurements)) by 5.6-fold. This
difference in the K
and K
values for the Trp-395
Phe mutant (5.6-fold) is much lower
than for the wild-type (98-fold) or the Trp-371
Phe mutant
(127-157-fold). The fact that D-galactose affords
similar levels of protection to photolabeling by cytochalasin B and
[
I]APS-forskolin, under nonenergizing
conditions, of both the Trp-395
Phe mutant and the wild-type
GalP proteins is consistent with the similar K
values for D-galactose of these proteins.
Figure 7:
A D-galactose-promoted alkaline
pH change is demonstrated by the Trp-395 Phe mutant of GalP but
not by the Trp-371
Phe mutant. E. coli strain JM1100
expressing either the wild-type, Trp-371
Phe, or Trp-395
Phe mutant GalP proteins were grown overnight on rich media containing
20 mM glycerol as described under ``Materials and
Methods.'' D-Galactose (3.3 mM) was added to an
anaerobic suspension of cells of the indicated strain (17.3 mg, dry
mass) in 3.7 ml of 150 mM KCl, 2 mM glycylglycine, pH
range 6.3-6.5. The pH was measured as described by Henderson and
Macpherson(1986). An upward deflection represents an alkaline pH
change.
In complete contrast, cells carrying the
Trp-371 Phe mutation did not show any sugar-H
symport activity ( Fig. 7and four additional measurements
in two separate cultures). This important difference from both the
wild-type and the Trp-395
Phe mutation is discussed further
below. It is possible that much higher concentrations of D-galactose need to be added to reveal sugar-H
symport, given the much reduced affinity of this mutant for sugar
(see above), but this could not be achieved in the experimental system
used for measuring the pH changes.
From the results in this paper we conclude that residues Trp-371 and Trp-395 are located in conformationally dynamic positions within the GalP symporter, with both contributing to the changes in tryptophan fluorescence induced by the binding of antibiotics. Although neither residue is photolabeled by cytochalasin B or forskolin, this does not preclude the possibility that these residues interact directly with the antibiotics, which would be consistent with the observations discussed below.
The mutation of Trp-371 and Trp-395 to phenylalanines reduces the affinities of the initial GalP-cytochalasin B complexes by at least an order of magnitude. However, this is compensated for by a further conformational change in which the binding of the cytochalasin B is tightened, so that the overall affinities of the two mutants for cytochalasin B are similar to that of the wild-type protein.
In contrast, the affinities of the initial GalP-forskolin complexes formed by the two mutant proteins are similar to that of the wild-type protein. But again, there is a further tightening in the interaction between the antibiotic and the transporter, so that forskolin is bound more tightly by the mutants than by the wild-type, indicating that these residues interact differentially with cytochalasin B and forskolin.
The differential interaction of
cytochalasin B and forskolin with GalP is best illustrated by the
Trp-395 Phe mutant. The replacement of Trp-395 by Phe causes
about a 43-fold reduction in the affinity of the initial complex for
cytochalasin B, but only a 3-fold reduction for forskolin. There is
about a 3.5-fold reduction in overall affinity for cytochalasin B,
while the overall affinity for forskolin is enhanced by about 3.2-fold.
The increase in the overall affinity relative to those of the initial
complexes is largely due to destabilization of the outward-facing
conformation (>78% of wild-type GalP molecules are outward facing in
the absence of ligands), so that the inward-facing conformation of the
Trp-395
Phe mutant, to which the antibiotics bind, is favored in
the absence of ligands (K
= 0.41 and 71%
are inward-facing). This is similar to the situation that prevails for
human erythrocyte glucose transporters (GLUT1), where the inward-facing
conformation progressively predominates at subphysiological
temperatures (K
= 0.25 and 80% of the
transporters face inwards at 20 °C; Lowe and Walmsley, 1987;
Walmsley, 1988). Furthermore, in both the case of the Trp-395
Phe mutant GalP and GLUT1, there is evidence that these proteins
undergo a further transition following the binding of cytochalasin B (e.g. in each case K
< K
): the T
(CB) to T
(CB)
transition that further stabilizes the protein-cytochalasin B
interaction.
The nature of the T(antibiotic) to
T
(antibiotic) transition is not known, but one possibility
is that it represents a partial reorientation of the transporter
(Martin et al., 1995). The binding of antibiotics may induce
the closure of the binding site, by acting as transition-state
analogues, which do not allow complete reorientation. In the case of
wild-type GalP, there is no kinetic evidence to support the existence
of the T
(CB) state (Walmsley et al., 1994a) but
there is for the T
(forskolin) state (Martin et
al., 1995). This suggests that the reorientation process may
proceed to a smaller extent with cytochalasin B than with forskolin
(Martin et al., 1995). This suggestion is consistent with the
present studies, which show that the Trp-395
Phe mutant binds
forskolin more tightly than cytochalasin B (K
= 1.2 and 7.7 for cytochalasin B and forskolin,
respectively). Moreover, the T
to T
transition
for the Trp-395
Phe mutant is at least an order of magnitude
faster when cytochalasin B, rather than forskolin, is bound. In the
case of wild-type GalP and the Trp-371
Phe mutant, this
transition is too fast to be measured by stopped-flow fluorescence
spectroscopy for both cytochalasin B and forskolin. Clearly, the
T
(CB) and T
(forskolin) states are not
equivalent and may be reached by different pathways.
The much higher
value of the overall K for D-galactose
(0.67 mM, Walmsley et al. 1994b) compared with K
(0.06 mM, Table 1) implies that
energization increases the affinity of the sugar binding site (Cairns et al., 1991; Walmsley et al., 1994). The affinity of
the inward-facing site for D-galactose was determined to be
5.8 mM by the inhibition of the rate of binding of
cytochalasin B (Walmsley et al., 1994b). The affinity of this
site was also determined by D-galactose titration of the
fluorescence of 8-anilino-1-napthalenesulfonate bound to GalP, yielding
a K
of 7.2 mM, from which the affinity of
the outward-facing site was calculated to be 13.8 mM (Walmsley et al., 1994b). A comparison of the K
values obtained for D-galactose binding to the mutants
with that for the wild-type will be informative of any changes in the
sugar binding site. On the other hand, a comparison with the K
values for transport should be informative as to
whether these mutations have affected the communication between the
proton and sugar binding sites. The K
for the
binding of D-galactose to the inward-facing site of the
Trp-371
Phe mutant GalP was 11-15-fold higher than for the
wild-type, suggesting that this residue may be involved directly in the
interaction with the internal sugar. The reduction in the affinity of
the sugar binding site probably accounts for the increase in the K
for transport, which is about 10-fold higher
than that for the wild type. In contrast, the K
for the binding of D-galactose to the inward-facing site
of the Trp-395
Phe mutant GalP was similar to that of the
wild-type, suggesting that this residue does not interact directly with
the sugar. However, the transport K
was increased
about 20-fold relative to the wild type, so that it is only about
5.6-fold lower than the K
for sugar binding to the
inward-facing site. This might have indicated that the primary effect
of the Trp-395
Phe mutation was to decrease the coupling between
the putative proton and sugar binding sites, but its sugar-H
symport activity was hardly diminished, while that of the Trp-371
Phe mutant was apparently abolished. However, both mutants were
still able to accumulate galactose to about the same extent, in an
uncoupler-sensitive manner. This implies that the Trp-371
Phe
mutant is not fundamentally impaired in
H
-translocation, although its apparent affinity for
protons may be diminished by changes in its binding of sugars. These
intriguing observations are the subject of further investigations,
aimed at determining the site(s) of H
binding, the
participation of H
in the discrete kinetic events we
have identified, and its possible pathway during translocation through
the GalP protein.
Garcia et al.(1992) have mutated the
corresponding tryptophan residues in GLUT1 (Trp-388 and Trp-412) to
leucine and glycine residues. Essentially, they found that the GLUT1
Trp-388 mutants had a decreased sensitivity of transport to
cytochalasin B, while the sensitivity of the Trp-412 mutants was
unaltered. This contrasts with GalP, where the Trp-371 mutant-mediated D-galactose transport is more sensitive to cytochalasin B
inhibition than the wild-type, which is more sensitive than the Trp-395
mutant. For GLUT1, the Trp-412 mutant, but not the Trp-388 mutant, was
shown to have reduced transport activity. Both of the Trp mutants of
GalP have reduced transport activities, with the Trp-371 mutant having
the lowest specificity constant. On the other hand, Katagiri et al. (1991 and 1993) have reported that both the Trp-388 Leu and
Trp-412
Leu mutants of GLUT1 have specificities (V
/K
) that are reduced to
20 and 15% of the wild type. However, the Trp-388
Leu mutation
was reported to cause a decrease in the specificity constant (V
/K
) for zero-trans uptake
that was not compensated for by a corresponding change in the
specificity constant for zero-trans efflux (Katagiri et al.,
1993); this contradicts the Haldane relationship for a passive
transport system and is impossible (V
/K
(influx) = V
/K
(efflux); Lowe and
Walmsley, 1987). Both of the mutants had reduced levels of
photolabeling with antibiotics (Katagiri et al., 1991, 1993).
Schurmann et al.(1993) have reported that the Trp-388
Leu mutation decreases both the photoaffinity labeling of GLUT1, with
[
I]APS-forskolin, and the glucose transport
activity; while the Trp-412
Leu mutation does not affect the
photolabeling but abolishes transport activity. These later studies are
more consistent with those reported herein on GalP, in which the
transport activity (V
/K
)
for the Trp-371
Phe and Trp-395
Phe mutants is reduced to
1.7 and 3.8% of the wild-type level, respectively. However, in contrast
to GLUT1, the low specificity constant for the Trp-395
Phe
mutant was shown to be largely due to a substantial increase in the K
for transport under energizing conditions.