(Received for publication, May 5, 1995; and in revised form, June 26, 1995)
From the
4-aminobutyrate metabolism in Escherichia coli begins
with transport across the cytoplasmic membrane via the GabP, which is
encoded by gabP. Although GabP is specific and exhibits poor
affinity for many cellular constituents such as the -amino acids,
the range of compounds recognized with high affinity has yet to be
investigated. In order to address this gap in knowledge, we developed a gabP-negative host strain, which permits evaluation of test
compounds for inhibitory effects on cloned GabP (expression inducible
by isopropyl-1-thio-
-D-galactopyranoside).
Using this inducible expression system, three structurally distinct categories of high affinity transport inhibitor were identified. The structural dissimilarity of these inhibitors significantly alters our view of ligand recognition by GabP. Any complete model must now account for the observation that inhibition of 4-aminobutyrate transport can be mediated either (i) by open chain analogs of 4-aminobutyrate, (ii) by cyclic amino acid analogs, or (iii) by planar heterocyclic compounds lacking a carboxyl group. Such results do not support a previously sustainable view of GabP that features a restrictive ligand recognition domain, unable to accommodate structures that differ very much from the native substrate, 4-aminobutyrate.
The metabolism of 4-aminobutyrate in Escherichia coli depends upon the gab gene cluster(1) , which
includes a regulatory gene, gabC, and three structural genes gabD (succinic semialdehyde dehydrogenase), gabT (glutamate:succinic semialdehyde transaminase), and gabP.
The latter encodes the 4-aminobutyrate transporter (GabP), a
hydrophobic, 466-residue polypeptide (2) that by hydropathy
analysis can be readily envisaged as a membrane protein with 12
transmembrane -helical domains. This putative topology is
characteristic of many carrier proteins from the plasma membrane of
bacterial and animal cells(3, 4, 5) . The
GabP is functional in whole cells and in right-side-out
vesicles(6) . Active accumulation of 4-aminobutyrate is
stimulated by membrane potential and inhibited by proton
ionophores(2) . Previous studies have indicated that GabP
exhibits considerable selectivity for 4-aminobutyrate and is able to reject the common
-amino
acids(2, 6, 7) .
The present study is focused on molecules that GabP can recognize rather than on those it can reject. An array of structurally diverse open chain amino acids and their heterocyclic analogs were shown to recognize the GabP with high affinity. These compounds caused a pattern of inhibition, which suggests that the ligand recognition domain of GabP should not be viewed as a restrictive envelope capable of accommodating only the native substrate, 4-aminobutyrate. Instead, there appears to be sufficient flexibility and/or space to allow larger amino acids, smaller amino acids, and conformationally constrained cyclic analogs to be recognized. Even highly planar heterocyclic structures lacking a carboxyl group can be potent transport inhibitors.
Figure 1:
Effect of gabP inactivation on
the time course of [H]GABA uptake by Escherichia coli K-12. E. coli DW1 (
,
)
and SK35 (
,
) were grown on minimal medium with glutamine as
the nitrogen source. Washed cells (in 100 mM potassium
phosphate (pH 7.0)) were exposed to 10 µM [
H]GABA (0.25 µCi/ml) with (opensymbols) or without (solidsymbols) 10
mM unlabeled GABA. At the times indicated, uptake was rapidly
quenched by the addition of a mercuric chloride stop solution. The
cells were harvested by vacuum filtration and radioactivity quantitated
as described under ``Experimental Procedures.'' Since some
data falling immediately along the base line were identical, certain
symbols have been offset slightly for
clarity.
LB medium (1% Bacto-tryptone, 0.5% Bacto yeast extract, 1% NaCl) supplemented with ampicillin (150 µg/ml) was used to grow the E. coli strains SK45 and SK55 for use in experiments with cloned gabP. Cells grown overnight (16 h) were diluted 100-fold into fresh medium (approximately Klett 10) containing 0.6 mM IPTG to induce high level gabP expression. Cells were grown for 3-4 doublings (Klett 130-150).
Transport reactions were initiated by adding 80 µl
of washed cells with rapid vortex mixing to 20 µl of solution
containing [H]GABA and other additions
(conditions indicated in figure legends). A metronome was used to
reproducibly time short uptake reactions (2-20 s). Uptake was
rapidly quenched by the addition of 1 ml of a ``stop
solution'' (buffer containing 20 mM HgCl
) to
the rapidly mixing reaction vessel. The ``quenched'' reaction
was vacuum filtered. The reaction vessel was then washed with 1 ml of
100 mM potassium phosphate (pH 7.0), 5 mM HgCl
, and this was applied to the same filter.
Finally, the filter was washed with 4 ml of buffer containing 5 mM HgCl
. The filter was dissolved in scintillation fluid
and the radioactivity (disintegrations/min) was calculated by a Beckman
LS3801 scintillation spectrometer using a stored quench curves and
automatic quench compensation (H number determination).
Since the [H]GABA concentration in all
experiments (10 µM) was set nearly equal to the published (6) K
(12 µM),
values for K
can be estimated to be about
1.8-fold lower than IC
. The concentration-response curves
used to estimate IC
spanned a range that was at least
100-fold (10-fold above and below the reported IC
).
As a prelude to the study of GabP inhibitors, a new
expression system consisting of a gabP-negative E. coli host strain and an IPTG-inducible gabP expression vector
was developed. The system exhibits a number of desirable properties,
including the ability to ascribe the vast majority of
[H]GABA transport (and the effect of test
compounds) to the cloned GabP.
The
utility of SK35 as a host strain for high level expression of gabP was shown by cloning the gene in pSCK380, a vector that includes a tac promoter and a lacIfor
effective suppression of the cloned gabP (SK35 host strain is
additionally lacI
). Placing the cloned gabP into SK35 resulted in a new strain, SK55, in which the
initial rate of [
H]GABA transport could be
induced 10-fold by IPTG (Fig. 2). A control experiment performed
with strain SK45 (containing the plasmid without the gabP insert) shows that induction of [
H]GABA
transport activity by IPTG requires cloned gabP. Comparison of
initial [
H]GABA (10 µM) transport
rates by strains DW1 (0.9 nmol/min/mg of protein) and SK55 (6.0
nmol/min/mg of protein) suggests that gabP is overexpressed by
about 6.5-fold following brief exposure to IPTG, a sugar that induces
expression from the tac promoter.
Figure 2:
lac control of gabP-dependent [H]GABA transport. E.
coli SK45 (
) and SK55 (
,
) were grown in LB
medium with (solidsymbols) or without (opensymbols) 0.6 mM IPTG to induce gabP expression. Washed cells (in 100 mM potassium phosphate
(pH 7.0)) were exposed to 10 µM [
H]GABA (0.25 µCi/ml). At the times
indicated in the figure, uptake was rapidly quenched by the addition of
a mercuric chloride stop solution. The cells were harvested by vacuum
filtration and radioactivity was quantitated as described under
``Experimental Procedures.''
Others (Niegemann et
al.(2) ) have cloned gabP for expression under
control of a tac promoter but were unable to directly
demonstrate induction with IPTG. The gene was found to be highly toxic,
and the permease was thus studied without induction to high level
expression. Moreover, the E. coli strain W3110 used in those
studies was a prototrophic (nearly wild-type) derivative of K-12, and
presumably contained a wild-type gabP on its chromosome. In
contrast, the present study shows that host strain SK35, is highly
defective for [H]GABA transport compared with the
parent strain DW1 (Fig. 1). Thus, [
H]GABA
transport measured in E. coli SK55 can be assigned
unambiguously to expression from the plasmid-borne gabP, and this transport activity is quite inducible by IPTG (Fig. 2). The [
H]GABA transport mediated
by the cloned gabP was energy-dependent and exhibited a K
of 10-12 µM (data
not shown). Thus, the IPTG-inducible [
H]GABA
transport activity expressed by E. coli SK55 conforms to
previous descriptions in the literature(2, 6) . E.
coli SK55 was used to characterize ligand recognition by GabP.
Figure 3:
Relationship between length of open chain
amino acid and potency for GabP inhibition. The inhibitory
potency (IC) of the open chain amino acids,
3-aminopropanoic acid (C3), 4-aminobutyric acid (C4),
5-aminopentanoic acid (C5), and 6-aminohexanoic acid (C6) was measured using E. coli SK55 that had been
grown in LB medium containing 0.6 mM IPTG to induce gabP expression. In order to estimate IC
, the washed cells
(in 100 mM potassium phosphate (pH 7.0)) were exposed to test
solutions containing 10 µM [
H]GABA
(0.25 µCi/ml) plus a range of inhibitor concentrations. After 1
min, uptake was rapidly quenched by the addition of a mercuric chloride
stop solution. The cells were harvested by vacuum filtration and
radioactivity was quantitated as described under ``Experimental
Procedures.''
Figure 4:
Inhibition of GabP by
conformationally constrained amino acid analogs. E. coli SK55
were grown in LB medium containing 0.6 mM IPTG to induce gabP expression. The washed cells (in 100 mM potassium phosphate (pH 7.0)) were exposed to 10 µM
[H]GABA (0.25 µCi/ml) plus the indicated
concentrations of either compound 2 (
) or compound 5 (
).
After 1 min, uptake was rapidly quenched by the addition of a mercuric
chloride stop solution. The cells were harvested by vacuum filtration
and radioactivity was quantitated as described under
``Experimental Procedures.''
Figure 5:
Inhibition of GabP by planar
heterocyclic amino acid analogs. E. coli SK55 were grown in LB
medium containing 0.6 mM IPTG to induce gabP expression. The washed cells (in 100 mM potassium
phosphate (pH 7.0)) were exposed to 10 µM [H]GABA (0.25 µCi/ml) plus the indicated
concentrations of either compound 7 (
), compound 8 (
), or
compound 9 (
). After 1 min, uptake was rapidly quenched by
addition of a mercuric chloride stop solution. The cells were harvested
by vacuum filtration, and radioactivity was quantitated as described
under ``Experimental
Procedures.''
There is a paucity of basic information on the structural
selectivity of the substrate recognition domain(s) of GabP.
Published studies indicate (i) that 4-aminobutyrate is recognized (6) with relatively high affinity (12 µM) and (ii)
that of the 20 common -amino acids, only threonine (6) and
aspartate (2) are weakly competitive (K
> 1 mM) with 4-aminobutyrate. These studies were
fundamentally important, showing that GabP is specific and able to
reject common amino acids from the cellular milieu.
On the other hand, rejected molecules (since they fail to interact with GabP) have provided limited information about the molecular architecture of the ligand recognition domain(s). In contrast, well-recognized ligands (i.e. those having high affinity for GabP) are presumably a good fit to the recognition domain and as such provide information to constrain our view of the permease molecule. Models of GabP structure and function ultimately have to account for the observed spectrum of recognizable ligands. The present study indicates that three structurally distinct ligand categories appear to recognize GabP with high affinity. These results place significant constraints on modeling of GabP structure and function.
The
results (Fig. 3) indicate that both the 4-carbon and the
5-carbon open chain amino acids can be recognized with high affinity
(approximately 10 µM), whereas longer or shorter molecules
are recognized with lower apparent affinity (IC > 100
µM). The diminished capacity of GabP to recognize the
3-carbon analog is consistent with previous studies, showing broad
rejection of the
-amino acids(2, 6) in which the
amino and carboxyl groups are attached to the same carbon atom. Thus,
any complete model of GabP would have to feature (i) ligand recognition
domain(s) spacious enough to accommodate amino acids larger than GABA
as well as (ii) a mechanism to prevent amino acids smaller than GABA
(or with less charge separation than GABA) from being effectively
recognized.
The space available within the GabP molecule for
recognition of larger amino acids has not previously been
characterized. The present study has probed this space, employing
cyclic amino acid analogs that are potent inhibitors of
[H]GABA transport. The cyclic analogs have
structures (i) that contain considerable bulk compared with open chain
structures and (ii) that lack the rotational freedom inherent in open
chain molecules. Despite these important structural differences, our
observations (Fig. 4) indicate that both compound 2 and
compound 5 are recognized with apparent affinities (50
µM) comparable with that of the native substrate,
4-aminobutyrate (compound 1).
It will be noted (Fig. 6) that both compound 1 and 6-aminohexanoate
(compound 3) are ``contained'' within compound 2. Thus, the cyclic molecule has the potential to be recognized
as an analog of either (or both) of these open chain compounds. If the
IC of cyclic compound 2 is described merely as the
weighted average of the IC
for either open chain compound
(no doubt a simplification of the actual situation) it follows
algebraically that the 6-aminohexanoate moiety contributes about 60% to
the overall affinity, while the 4-aminobutyrate moiety contributes the
remaining 40%. This highly simplistic analysis is in general agreement
with the notion that the bulky and relatively nonpotent
6-aminohexanoate moiety detracts considerably from, but does not
abolish, the inherent affinity (10 µM) of GABA (compound 1) for the permease. A similar analysis can be applied to
compound 5 as described below.
Figure 6:
Structural comparison of open chain and
cyclic amino acid analogs. The native GabP substrate is 4-aminobutyrate
or GABA (compound 1). Both open chain analogs (compounds 3, 4, and 6)
and cyclic analogs (compounds 2 and 5) are effective inhibitors of
[H]GABA transport (see Fig. 3and Fig. 4). It will be noticed that each open chain compound is
contained within the adjacent cyclic structure. Thus, the cyclic
compounds have the potential to be recognized in the context of either
(or both) of the open chain inhibitors.
Both the potent (IC = 10 µM) transport inhibitor,
5-aminopentanoate (compound 4), and the less potent (IC
= 100 µM) 3-aminopropanoate (compound 6) are ``contained'' within the structure of compound 5. Thus, the cyclic molecule has the potential to be recognized
as an analog of either (or both) of these open chain compounds. If the
IC
(50 µM) of compound 5 is analyzed
as the weighted average of the IC
values obtained for the
respective open chain inhibitors, then compound 6 and compound 4 (having IC
values of 100 and 10 µM,
respectively (Fig. 3)) must receive algebraic weights of 45 and
55%. In other words, the data are consistent with the idea that the
inhibitory potency of compound 5 derives about equally from its
5-aminopentanoate and 3-aminopropanoate moieties.
Regardless of
whether these simple comparisons and calculations have mechanistic
validity, it is clear that GabP recognizes both open chain and cyclic
molecules that are significantly larger than GABA itself. These large
amino acid analogs (Fig. 6) display amino and carboxyl groups on
a nonplanar carbon skeleton. In contrast, compounds 7-9 (Fig. 7) are highly planar and do not feature a carboxylic
acid group. Compound 7, the most potent (10 µM)
inhibitor in the planar series, is also the smallest and the most
flexible molecule. Decreasing the flexibility and increasing the
molecular size via addition of a second ring (compound 8)
reduced the potency 20-fold (200 µM). The relative
position of polar groups in the polycyclic compounds 8 and 9 was also important since the IC of compound 9 (2000 µM) was increased 10-fold over that for compound 8 (200 µM).
Figure 7: Structural comparison of planar heterocyclic GabP inhibitors. The structures of compound 7, compound 8, and compound 9 share in common the planar isoxazole ring. These molecules also lack the carboxyl group that is common to all of the open chain and cyclic amino acid analogs (Fig. 6).
The results presented above suggest
a minimal model (Fig. 8) for GabP in which GABA (compound 1) is a transported substrate that can enter the core of the
transport channel where it can become occluded by gating mechanisms
that are necessary (12, 13) for energy-driven (1, 2) accumulation. The inhibitory actions of the
structurally diverse set of molecules described in this communication
suggests additionally that portions of the transport channel may be
able to accommodate ligands far bulkier than GABA itself. Although the
possibility exists that some of these inhibitors act at an allosteric
ligand binding domain physically removed from the transport channel,
preliminary results from this laboratory suggest that
compounds 5 and 7 are indeed transported substrates (i.e. they necessarily enter the transport channel). Thus, we
favor the idea that most, and probably all, of the inhibitory compounds
discussed in this study act within or at the mouth of theGabP transport
channel.
Figure 8:
Model for ligand recognition by GabP. GABA (4-aminobutyrate) is a known substrate (2, 6) that can enter the GabP core to occupy an
occluded or gated space. Some mechanism of bidirectional gating is
necessary for active substrate accumulation by a carrier mechanism (i.e. carriers cannot behave as water-filled channels in which
the substrate has contiguous access to the intracellular and
periplasmic spaces). The nature of amino acid side chains (R-groups)
lining the transport channel or the access channels is completely
unknown, but the zwitterionic nature of the substrate strongly suggests
that polar residues will be important. The present study suggests
additionally that the working model must reflect properties of GabP
that allow [H]GABA uptake to be inhibited either
(i) by open chain GABA analogs, (ii) by cyclic, conformationally
constrained GABA analogs, or (iii) by planar heterocyclic compounds
having structures that are less obviously related to GABA than the
other categories of inhibitor. Whether any of these inhibitors can act
at an allosteric inhibitory site remains to be elucidated, but at
present the favored view is that the inhibitors interact with some
aspect of the transport channel. Compounds 5 and 7, the novel
heterocyclic inhibitors for which allosteric actions might be
suspected, interact with the channel since they are bidirectionally
transported.
Accordingly, compounds 5 and 7 are depicted at
the mouth of the GabP transport channel for illustrative purposes and
without any implication as to whether or not the permease can bind
these molecules simultaneously with
4-aminobutyrate.