From the Institute of Medical Microbiology and
Hygiene, University of Mainz, Obere Zahlbacher Strasse 67, D55101
Mainz, Germany and the ¶ Institute of Medical Biochemistry and
Genetics, Texas A & M University, College
Station, Texas 77843-1114
Received for publication, January 12, 2001, and in revised form, February 2, 2001
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ABSTRACT |
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Staphylococcal Staphylococcus aureus The conformation of the monomer is likely to be similar to that of the
homologous toxin leukocidin (6, 7), so that structural information is
available for both the initial and the final stages of pore formation
(Fig. 1A). In addition, two
intermediate stages can be distinguished: the membrane-bound monomer
and the heptameric pre-pore (8, 9). The pre-pore appears to be
irreversibly associated, but the -toxin forms heptameric pores
on eukaryotic cells. After binding to the cell membrane in its
monomeric form, the toxin first assembles into a heptameric pre-pore.
Subsequently, the pre-pore transforms into the final pore by membrane
insertion of an amphipathic
-barrel, which comprises the "central
loop" domains of all heptamer subunits. The process of membrane
insertion was analyzed here using a set of functionally altered toxin
mutants. The results show that insertion may be initiated within an
individual protomer when its NH2 terminus activates
its central loop. The activated state is then shared with the
central loops of the residual heptamer subunits, which results in
cooperative membrane penetration. This cooperation of the central loops
commences while these are still remote from the lipid bilayer.
Nevertheless, it is subject to modulation by the target membrane, which
therefore acts across a distance much like an allosteric effector.
However, while allosteric transitions usually are reversible, membrane
insertion of
-toxin is an irreversible event, and we show here that
it can proceed to completion in a domino-like fashion when triggered by
as little as a single foreign atom within the entire heptamer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-toxin, a protein of 293 amino
acid residues (Mr: 33,000), was the first
pore-forming bacterial cytolysin to be identified (1) and to date is
still one of the most studied members within this class of toxins. The
molecule is secreted as a water-soluble monomer that binds to
cytoplasmic membranes of eukaryotic target cells, where it subsequently
forms transmembrane pores of heptameric stoichiometry (2). The crystal
structure of the pore heptamer has been solved (3). The transmembrane part of the pore consists of an amphipathic
-barrel of 7-fold rotational symmetry, and it comprises the amino acid residues 118-140
of each subunit. Within this central loop, the odd-numbered residues
face the lumen of the pore, while the even-numbered ones are in contact
with the surrounding lipid bilayer (3-5).
-barrel is not yet inserted in
the membrane (Fig. 1B). During insertion, the central
loop requires assistance by NH2-terminal segments of the
polypeptide chain, which also undergoes a major conformational change
(10-12).
View larger version (48K):
[in a new window]
Fig. 1.
The structure of
-toxin and the four stages of pore assembly.
A, crystal structures of monomeric lukF and of the
-toxin
pore heptamer. Left, lukF is a component of staphylococcal
leukocidin that is homologous to
-toxin. This crystal structure
represents the monomer prior to membrane binding. The right
picture shows the
-toxin pore heptamer; one of the seven
subunits is shown in full. The stem of the
mushroom-shaped heptamer corresponds to the
membrane-inserted
-barrel that is traversed by the pore lumen. The
NH2 termini (residues 1-14) are shown in black
and the central loop domains (
-toxin, residues 115-144; lukF,
residues 114-141) in dark gray. The two domains are in
close contact in the monomer but are far apart after membrane
insertion. The figure was drawn with RasMol from 1pvl.pdb and 7ahl.pdb.
B, the four distinct stages of pore assembly: 1) the monomer
in solution, 2) the membrane-bound monomer, 3) the heptameric pre-pore,
and 4) the membrane-inserted pore.
A variety of both recombinant and chemical modification techniques have
been applied to the -toxin pore to explore its application as a
biosensor and to extend its utility in cell biological research (13-15). The mutation characterized in the present study was initially designed for a cell biological experiment, too: the four amino acids
(YTRF) that were inserted at the turn of the membrane-penetrating loop
(following residue 129) represent a peptide signal for transferrin receptor endocytosis (16, 17). Accordingly, our intention was to
observe endocytosis of 129YTRF heptamers on nucleated
cells. This goal was not attained. However, the unique phenotype of
129YTRF could be exploited to reveal surprising features
concerning the molecular mode of pore formation. In particular, when
129YTRF was co-oligomerized on fibroblast membranes with a
series of point mutants of
-toxin, hybrid heptamers were detected
that were either fully active or entirely inactive depending on only one amino acid within a single subunit. A single amino acid
residue may thus trigger membrane insertion of the entire
-toxin
heptamer, whereby the NH2 terminus and the glycine-rich
central loop cooperate both intra- and intermolecularly in an intricate fashion.
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MATERIALS AND METHODS |
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Construction, Expression, Purification, and Chemical Modification
of -Toxin Mutants--
The mutant 129YTRF was
constructed by PCR mutagenesis according to Ref. 18 using two mutagenic
primers (coding sequence, GGT GAT ACA TAC ACG AGG TTC GGA
AAA ATT GGC GGC C; complementary, TTT TCC GAA CCT CGT GTA
TGT ATC ATC ACC AGT AAC; the four italicized codons represent the
insertion) and verified by DNA sequencing. The mutant gene was cloned
into the shuttle plasmid pDU1212 for expression in Staphylococcus
aureus DU1090 (19) and purified from bacterial culture
supernatants as described previously (20). The various single
cysteine mutants used in this study have been described before (4, 5,
21). The double mutant I5C/129YTRF was constructed by
cleaving the respective pDU1212-derived, single mutant plasmids with
KpnI and recombining the appropriate restriction fragments.
Analogously, the cysteine mutant I5C/G130C was subcloned from the I5C
and G130C single mutants. To achieve the formation of intramolecular
disulfide bonds with the latter mutant, the protein was transferred
into phosphate-buffered saline (pH 7.5) and supplemented with an
equimolar amount of dithiobisnitrobenzoic acid. After incubation at
room temperature for 15 min, disulfide bond formation was judged by
SDS-PAGE1 performed in the
absence of reducing agents.
Gel Electrophoresis and Western Blotting--
SDS-PAGE was
carried out under standard conditions but with gels and running buffers
not containing any disulfide reducing agents. Nondenaturing PAGE was
carried out using sodium deoxycholate (10 mM) in a
continuous buffer system (50 mM Tris, 50 mM
glycine; no adjustment of pH). Oligomerization was analyzed on
liposomes consisting of egg yolk phosphatidylcholine with cholesterol
(molar fraction, 40%) and prepared by membrane extrusion according to Ref. 22. Oligomer formation on fibroblasts was examined by Western blotting, which was carried out as described using a polyclonal rabbit-anti--toxin IgG antibody (23).
Fluorescence Assay of Hybrid Oligomer Formation-- This experiment was done as described previously (11). Briefly, the lytically active mutant K46C, thiol-specifically derivatized with fluorescein maleimide, was admixed with wild type toxin or 129YTRF, respectively, at various ratios. The mixtures were incubated with liposomes to induce oligomerization, which was confirmed by SDS-PAGE, and the fluorescein fluorescence was then assayed in a Spex Fluorimax fluorimeter (excitation wavelength, 488 nm; emission wavelength, 515 nm).
Assay for Competition in Binding of 129YTRF and Other Hemolysin Variants-- 10 µg of the mutant D108C, thiol-specifically derivatized with fluorescein maleimide, were added to fibroblasts grown to confluence in one well of a six-well culture dish. After incubation for 1 h at 37 °C, the supernatant was withdrawn, the cells washed with HBSS, and the cellular material recovered by solubilization with 3 ml of 0.5% SDS in HBSS. The sample was boiled for 5 min to ensure dissociation of the oligomers and fluorescein fluorescence quantified as above. Parallel samples were prepared with 10 µg of labeled K46C supplemented with 20 µg of either wild type hemolysin or 129YTRF, respectively.
Functional Assays of -Toxin Mutants on Rabbit Erythrocytes,
Human Fibroblasts, and THP-1 Cells--
Hemolytic titration was
carried out using rabbit erythrocytes (23) with visual reading after
120 min. For assays involving retention of marker molecules within
human fibroblasts, these were grown to confluence in 6 (K+)- or 96 (ATP)-well plates. The fibroblasts were washed
with HBSS and then treated with the various toxin variants for 1 h
at 37 °C; the wild type or mutant proteins used and their respective amounts are stated under "Results." Following incubation, the supernatant was withdrawn, and the cells were washed once with HBSS.
The cellular material was then recovered by lysis with Triton X-100
(Sigma; 1% in HBSS). Cellular ATP was assayed by luminometry, and
cellular K+ was determined by flame photometry as described
(24), whereby control cells incubated in parallel without toxins served
as standards. THP1 cells were treated analogously, except that they
were grown and treated with the toxin in suspension; washing and
recovery were performed by centrifugation.
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RESULTS |
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Construction and Pore-forming Properties of Mutant
129YTRF--
Four consecutive codons (YTRF: tyrosine,
threonine, arginine, and phenylalanine) were inserted between residues
Thr129 and Gly130 of -toxin. The
specific hemolytic activity of the ensuing mutant 129YTRF
(as assayed with rabbit erythrocytes) corresponded to that of wild type
toxin (data not shown). In contrast, when fibroblasts were treated with
129YTRF, no release of cellular K+ or ATP
occurred, whereas either marker was efficiently depleted by wild type
toxin (Fig. 2A). The
129YTRF mutant thus did not detectably permeabilize the
fibroblasts, although it readily formed oligomers on these cells (Fig.
2B). Similar results were also obtained with the lymphoid
cell line THP1 (data not shown). In sum, 129YTRF may form
either lytic or nonlytic oligomers, depending on the target
membrane.
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The 129YTRF Mutation Exerts a Dominant Negative Effect
upon Membrane Insertion of Wild Type Toxin into Fibroblast
Membranes--
To learn more about the functional properties of the
129YTRF mutant, we examined its interaction wild type
toxin. A constant amount of wild type toxin was admixed with
129YTRF at various ratios, and the mixtures were applied to
fibroblasts. Permeabilization of the cells was abolished when
129YTRF was employed in at least 2-fold excess over wild
type toxin (Fig. 3A). This
observed inhibition of wild type toxin by 129YTRF might
conceivably be due to a competition of the two species for a limiting
number of binding sites. The wild type toxin was therefore replaced
with a fluorescein-labeled mutant (D108C) to facilitate the
quantitation of the cell-bound active toxin. D108C indeed closely
resembled wild type toxin in permeabilizing the fibroblasts in the
absence but not in the presence of 129YTRF (data not
shown). Nevertheless, the amount of labeled D108C that became bound to
the fibroblasts was unaffected by 129YTRF (Fig.
3B), indicating that 129YTRF had not dislodged
D108C from its binding sites. We therefore considered the possibility
that the inhibitory effect of 129YTRF was due to the
formation of hybrid oligomers with the active toxin. The ability of
129YTRF to form hybrids with an active toxin species was
confirmed using an experimental approach outlined previously (11). The fluorescein derivative of another active cysteine mutant (K46C) exhibits pronounced self-quenching of fluorescence upon
oligomerization. When admixed to the labeled K46C, 129YTRF
was indistinguishable from wild type toxin in its ability to prevent
this self-quenching effect, indicating that both had formed hybrid
oligomers with K46C to the same extent. This strongly suggests that
oligomerization of all toxin species in question occurred entirely at
random. Then, the stoichiometry of the hybrid oligomers ensuing from a
binary mixture should be binomially distributed. With a 2-fold excess
of 129YTRF over wild type toxin, the fraction of
homogeneous wild type oligomers will then be virtually nil (0.05%),
whereas 6% of the oligomers will consist of 129YTRF only.
The majority of the oligomers will thus represent hybrids of the two
proteins of varying composition. The observed lack of permeabilization
therefore indicates that, within these hybrids, 129YTRF
suppresses the lytic activity of wild type toxin. This finding tallies
with the previous concept of cooperative membrane insertion of
the heptamer subunits (4, 11).
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Several "Breakthrough" Point Mutants Escape Inhibition by129YTRF on Fibroblast Membranes--
To characterize in
more detail the cooperation among the subunits of the heptamer, we then
tried to identify mutations that might complement the insertionally
deficient phenotype of 129YTRF. To this end, we screened an
existing collection of point mutants carrying unique cysteines
substituted for various residues within the cysteine-less wild type
-toxin molecule (4, 5, 21, 25). All these mutants were hemolytically
active, and although they varied to some extent in their permeabilizing
activity toward fibroblasts, they all effected a reduction of cellular ATP by at least 60% when employed at 10 µg/ml (data not shown). Accordingly, 10 µg/ml of each cysteine mutant in question was admixed
with a 3-fold excess of 129YTRF and applied to fibroblasts,
which were then assayed for cellular ATP. In most experiments, the
fibroblasts retained ATP, so that the mutant proteins resembled wild
type toxin in being inhibited by 129YTRF (Fig.
4). However, the figure also shows that
in several instances the cells had been permeabilized, indicating that
these mutants had escaped inhibition by 129YTRF. Mutations
of the latter type cluster in two regions: mutants S3C, I5C, I7C, and
D13C are located at the far NH2 terminus, while substitutions D127C, D128C, T129C, G130C, and K131C are in immediate proximity to the insertional mutation of 129YTRF. They will
collectively be referred to as breakthrough mutations in the
following.
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Breakthrough Mutants Effect Pore Formation when Co-assembled with
129YTRF into Hybrid Oligomers--
The lacking inhibition
of breakthrough mutants by 129YTRF might conceivably arise
in two different ways: first, the mutants might simply escape capture
by 129YTRF and oligomerize on their own; the lytic
homogeneous oligomers of a breakthrough mutant would then co-exist on
the cells with nonlytic 129YTRF oligomers. Second, the
breakthrough mutants might form hybrid heptamers with
129YTRF but then resist inhibition by the latter mutant, so
that the hybrids (or a fraction thereof) would insert into the membrane and form a pore. Fig. 5A shows
an experiment to distinguish between these possibilities that was done
with the breakthrough mutant G130C. Fibroblasts partially retain ATP
when treated with this toxin at 5 µg/ml; at 2.5 µg/ml, ATP is
nearly quantitatively retained. Strikingly, when the latter amounts of
G130C were supplemented with the nonlytic 129YTRF mutant in
excess, permeabilization was enhanced. This indicated that the two
mutant proteins had readily formed hybrid oligomers that were lytically
active. Similar results were also obtained with the mutants S3C, I5C,
or D13C in place of G130C (data not shown). Thus, both the
NH2-terminally and the centrally located breakthrough
mutations permit co-assembly with 129YTRF and prevail over
its functionally deficient phenotype within the hybrid heptamer.
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Nevertheless, Fig. 5A also shows that, with 2.5 and even with 5 µg/ml G130C, supplementation with 129YTRF does not achieve the same level of permeabilization as observed with 10 µg/ml G130C alone, indicating that the toxin mixture does not live up to the specific activity of G130C. This behavior might suggest that, within the hybrid heptamer, the two toxin species govern the insertion process in a co-dominant fashion, so that only above a certain threshold number of G130C subunits (e.g. two or four) would these elicit membrane insertion of the entire heptamer. In this case, the breakthrough activity should be titratable with a sufficient excess of 129YTRF. We therefore employed a constant amount of G130C that was admixed with increasing amounts of 129YTRF. Surprisingly, in this experiment, the permeabilizing efficacy of the toxin mixtures increased monotonously up to a 20-fold excess of 129YTRF (Fig. 5B). At the latter ratio, virtually all of the G130C monomers will be singly incorporated into hybrid oligomers that otherwise are comprised of 129YTRF subunits only. A single G130C subunit may therefore render a hybrid heptamer lytically active, and no threshold of subunit stoichiometry is apparent that would indicate a co-dominant control of membrane insertion of the hybrid heptamers.
Another possibility that might be considered is that, within the hybrids, the breakthrough mutant molecules might insert on their own, while the 129-YTRF subunits would not; the partially inserted hybrid oligomer would then create a pore of reduced lytic capability. However, this assumption is at odds with the behavior of the same hybrid oligomers on a different cell type. The myeloid cell line THP-1, while readily susceptible to G130C or I5C alone, resists attack by mixtures of either mutant when admixed with 129YTRF (data not shown). Therefore, much like wild type toxin on fibroblasts, G130C or I5C are amenable to inhibition by 129YTRF, and they are thus not insertionally uncoupled.
In sum, neither co-dominant control nor uncoupling of membrane insertion may account for the experimental findings, so that the observed reduction in activity of G130C/129YTRF hybrid heptamers as opposed to the homogeneous G130C ones heptamers remains unexplained. This issue will be further considered below (see "Discussion").
The I5C Breakthrough Mutation Does Not Complement the129YTRF Insertion when Both Are Located on the Same
Molecule--
The existence of the NH2-terminal cluster of
breakthrough mutations indicated that, in one way or the other, the
amino terminus of a given subunit of the heptamer promotes membrane
insertion of the central loop of a neighboring subunit. This
stimulatory effect of the NH2 terminus might conceivably be
transmitted along different routes that are schematically depicted in
Fig. 6A. First, the two
molecular regions might be engaged in immediate interaction (as
represented by the full arrow). Alternatively, the
stimulatory effect might first be relayed to a corresponding element
in cis (long broken arrow) or in trans
(short broken arrow). To distinguish between these
possibilities, we combined the NH2-terminal breakthrough mutation I5C with 129YTRF to yield a double mutant, which
then again was functionally characterized within homogeneous and hybrid
heptamers. The results are summarized in Fig. 6, B-D. The
double mutant I5C/129YTRF alone did not exhibit any
permeabilization of fibroblasts, which is inconsistent with the
hypothesis of an immediate effect of the NH2 terminus upon
the central loop in trans (Fig. 6B). The double
mutant also failed to activate the 129YTRF single mutant
within hybrid heptamers, which is at odds with the assumption that the
NH2-terminal stimulus is first communicated in
trans to a neighboring NH2 terminus and from there to
the central loop in cis (Fig. 6C). However, much
like the 129YTRF single mutant, I5C/129YTRF was
readily complemented when co-oligomerized with I5C (Fig. 5D,
upper panel). Moreover, the double mutant also inhibited the activity of wild type toxin within hybrid oligomers (Fig.
5D, lower panel). These two findings support the
notion that the NH2 terminus primarily acts upon the
central loop in cis, which then shares its state of
activation with the other central loops within the heptamer. As
discussed below, this concept is also compatible with previous data
obtained with an NH2-terminal deletion mutant (Fig.
6E).
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The Amino Terminus and the Central Loop Region Are Close within the
-Toxin Monomer--
The amino terminus is located remotely from the
central loop within the heptamer (3), but the two regions are likely to be in close proximity within the monomeric toxin molecule
(cf. Fig. 1A). Their close cooperation during
membrane insertion suggests that this proximity be maintained in the
pre-pore stage. To address this question more directly, a double
cysteine mutant (I5C/G130C) was constructed. Fig.
7A shows that, in its
monomeric form, this protein was readily converted to an intramolecular
disulfide, the yield being around 50% in the experiment depicted.
Subsequently, the mutant protein was no longer hemolytically active,
and it failed to form SDS-resistant heptamers on membranes of model
liposomes (Fig. 7A). Interestingly, however, when the
liposomes were solubilized and electrophoresed with the nondenaturing
detergent deoxycholate, an oligomeric species was detected (Fig.
7B, lane 3). This oligomer migrated behind the
one observed when the mutant protein was reduced prior to membrane
binding (lane 4), but it could be reverted both to normal
electrophoretic mobility and to SDS-resistance by post-treatment with
dithiothreitol (Fig. 7, A and B, lanes
5). These results suggest that the NH2 terminus and
the central loop indeed remain in close contact at an early stage of
heptamer assembly. This contact probably is the structural basis of
their close cooperation in the subsequent process of membrane
insertion. The reduced electrophoretic mobility of the disulfide-bonded
pre-pore heptamer might reflect its lack of a protruding
-barrel,
which in case of the mature pore heptamer, confers additional electric
charge by the binding of deoxycholate to its hydrophobic outer
circumference (4). Our findings also provide another example of the
previous notion that the pre-pore stage may be further subdivided into
several forms that, among other features, are distinguished by their
susceptibility to dissociation by SDS (11).
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DISCUSSION |
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-Toxin is an important model system for the molecular study of
pore-forming proteins. Crystallization of the heptamer has provided the
first high resolution structure of a bacterial toxin pore (3), which
has recently been complemented by the crystal structure of the
homologous toxin leukocidin (lukF) in its monomeric state (6).
Comparison of the two structures highlights the crucial roles of both
the amino-terminal latch and the central glycine-rich loop in the
conformational transition associated with pore formation (Fig. 1). In
the monomer, the two segments are packed against the core of the
molecule, contacting each other at their tips. In the oligomer, both
have undergone a large move and now make long excursions from the core
of the protomer. At the same time, they are in close contact with their
respective counterparts of the neighboring subunits, the central loops
now forming the transmembrane
-barrel that is crucial to toxin
function. In addition to these structurally defined end points,
intermediate stages have been characterized by mutational analysis that
have outlined the way leading from the monomer to the final
membrane-inserted oligomer. It has thereby become clear that, in the
process of pore formation, oligomerization precedes membrane insertion
(8, 9). Insertion involves cooperation among neighboring protomers (4,
11).
Despite its various contacts within the monomeric and the oligomeric structures, respectively, the amino-terminal latch might conceivably contribute to membrane insertion through a variety of either intra- or intermolecular effects. The findings reported here now distinguish the essential role of the intramolecular effect the amino latch takes upon the central loop in cis. While this effect was observed here with individual (NH2-terminal breakthrough mutant) subunits within hybrid heptamers, it must be assumed to occur with all the subunits of an active (wild type) heptamer. This leads us to the notion that, in membrane insertion, the central loop receives dual activation both from the NH2 terminus in cis and from another central loop in trans. This model readily accommodates the second cluster of breakthrough mutations located within that loop, and it can also account for previous results obtained with an NH2-terminal deletion mutant (12). The latter protein exhibits impaired hemolytic activity, which it also imparts on wild type toxin within hybrid heptamers (Fig. 6E).
Another important prerequisite for membrane insertion to be initiated is that the oligomer receives activation from the target lipid bilayer, a fact that is strikingly illustrated by the finding that polymorphonuclear granulocytes resist toxin attack by preventing membrane insertion of the pre-pore altogether (5). The 129YTRF phenotype now shows that this activation may be available to a different extent with various naturally susceptible membranes. The 129YTRF mutant is active on its own on rabbit erythrocytes, it is inactive and also inhibitory toward all active toxin species tested on THP-1 cells, and it is either inhibitory or subject to activation on fibroblasts. The insertion of 129YTRF into fibroblast membranes triggered by breakthrough mutants exemplifies that, within an oligomer, individual protomers may transmit their activated state to neighboring subunits. On the other hand, the inhibitory action of 129YTRF shows that, despite available promotion by the membrane, a functional subunit may still fail to insert when in contact with drowsy neighbors. Therefore, in deciding on whether or not to proceed with membrane insertion, each subunit of the oligomer integrates signals received both from adjacent subunits and from the membrane. At what stage, then, does the membrane signal affect insertion of the central loop region? Consider a hybrid heptamer comprised of one wild type molecule and six molecules of 129YTRF. This heptamer will readily insert into the rabbit erythrocyte membrane, but not so into the fibroblast membrane. However, by substituting one molecule of, e.g. I5C for the wild type subunit, insertion into the fibroblast membrane will be restored. The NH2-terminal mutation therefore compensates for the insufficient membrane stimulus, and it does so by directly acting upon the central loop in cis. It thus turns out that the membrane signal takes effect, while the central loop is still in contact with the NH2 terminus and, hence, remote from the target membrane.
Taken together, the above considerations unveil a striking resemblance
between membrane insertion of the -toxin heptamer and the
cooperative structural transition of an allosteric enzyme, which also
senses signals from extraneous ligands, transmits them to the remotely
located active site and integrates them among the subunits themselves.
There remains, however, one important difference: while the
reversibility of allosteric transitions is essential to their
regulatory role in enzyme activity, membrane insertion of the
-toxin
pore is an irreversible process. This irreversibility is strikingly
highlighted by the fact that a single mutant residue within the entire
heptamer may trigger insertion. Amazingly, in case of the breakthrough
mutant S3C, such lytic hybrids differ from the corresponding nonlytic
wild type/129YTRF hybrids by as little as a single sulfur
atom replacing an oxygen. In a reversible system, such a small change
should not effect much more than a slight shift of equilibrium; but, as
we can see here, it may suffice to trigger an irreversible,
self-sustaining reaction that proceeds to completion much like a
toppling cascade of dominoes.
Although the cellular acceptor sites for -toxin still await
molecular characterization, the limited number of toxin molecules found
on the surface of target cells indicates that there is selectivity in
toxin binding, and it also suggests that the toxin remains associated
with the binding sites after oligomerization. From this, it would
follow that, with different cells, these binding sites deliver a
different degree of activation to the pre-pore heptamer that is on the
verge of inserting. The question then arises whether or not, on a given
cell, all binding sites are homogeneous. If they were not, their
heterogeneity could account for the yet unexplained finding that the
hybrid oligomers of G130C (as well as the other breakthrough mutants)
and 129YTRF are less efficient in permeabilizing
fibroblasts than those consisting of G130C only. Let us consider a
single hybrid heptamer that is sitting on a fibroblast; n is
the number of its G130C subunits. All subunits are randomly bound to
either activating membrane sites or to nonactivating ones. One would
then expect the membrane stimulus to take effect only at those
activating sites that happen to carry a G130C subunit, while those
sites carrying a 129YTRF subunit would be silenced. Let
A be the fraction of activating sites and N the
fraction of nonactivating ones; A + N = 1. Then, the probability of membrane insertion of the hybrid oligomer
would equal 1
Nn, i.e. there
would be a direct relationship between the fraction of G130C subunits
and the readiness of an oligomer to achieve insertion. This would
explain the reduced specific activity of G130C/129YTRF
hybrids as opposed to homogeneous G130C oligomers. On the other hand,
when n G130C subunits would be singly incorporated into
n hybrid oligomers with 129YTRF, the probability
that at least one of them was inserted would again be 1
Nn, but (with n > 1) there would be
the additional chance that further oligomers would be inserted.
This consideration would account for the enhancement of efficacy of
G130C by its "titration" with increasing amounts of the nonlytic
129YTRF species. In sum, the experimental findings are in
line with the assumption that single cells may afford binding sites
that support membrane insertion to different degrees. We speculate that
this heterogeneity might be related to the lateral segregation of
lipids in cell membranes.
In conclusion, our study shows that membrane insertion of the -toxin
heptamer occurs in an intricately concerted manner resembling the
allosteric transition of an oligomeric enzyme. It is restricted or
promoted to a varying extent by different natural target membranes, whereby the molecular nature of the incremental cell membrane constituents remains to be elucidated. At the same time, membrane insertion is exceedingly susceptible to minimal changes of protein structure, which reflects the irreversible nature of the event of
insertion. Ultimately, therefore, we are left with the question why a
pore-forming toxin that, after oligomerization on the target membrane,
is committed to an irreversible, one-hit mode of action is activated in
a fashion similar to allosteric enzymes that must reversibly adjust to
the prevailing metabolic situation.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft (SFB 490). This work contains part of the M.D. thesis of R. Schnabel.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.: 49-6131-393-2865; Fax: 49-6131-393-2359; E-mail: avaleva@mail.uni-mainz.de.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M100301200
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HBSS, Hanks' balanced salts solution.
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REFERENCES |
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