Interrelation between High and Low Affinity Tentoxin Binding
Sites in Chloroplast F1-ATPase Revealed by Synthetic
Analogues*
Jérôme
Santolini
,
Francis
Haraux
§,
Claude
Sigalat
§,
Laurence
Munier
, and
François
André
¶
From the
Section de Bioénergétique,
Département de Biologie Cellulaire et Moléculaire,
CEA-Saclay, and § Protéines Membranaires
Transductrices d'Energie (CNRS-URA 2096), DBCM-CEA Saclay,
bâtiment 532, F-91191 Gif-sur-Yvette Cedex, France
 |
ABSTRACT |
Eight synthetic analogues of tentoxin
(cyclo-(L-N-MeGlu1-L-Leu2-N-Me
ZPhe3-Gly4))
modified in residues 1, 2, and 3 were checked for their ability to
inhibit and reactivate the ATPase activity of the activated soluble
part of chloroplast ATP synthase. The data were consistent with a model
involving two binding sites of different affinities for the toxins. The
occupancy of the high affinity site (or tight site) gave rise to an
inactive complex, whereas filling both sites (tight + loose) gave rise
to a complex of variable activity, dependent on the toxin analogue.
Competition experiments between tentoxin and nonreactivating analogues
allowed discrimination between the absence of binding and a
nonproductive binding to the site of lower affinity (or loose site).
The affinity for the loose site was not affected significantly by the
modifications of the tentoxin molecule, whereas the affinity for the
tight site was found notably changed. Increasing the size of side chain
1 or 2 and introducing a net electrical charge both resulted in a
decrease of affinity for the tight site, but the second change
dominated the first one. The activity of different ternary complexes
enzyme-tentoxin-analogue depended on the nature of the toxin bound on
each site and not only on that bound on the loose site. This
demonstrates that the reactivation process results from an interaction,
direct or not, between these two binding sites. Possible molecular
mechanisms are discussed.
 |
INTRODUCTION |
F0F1 proton ATPases (or ATP synthases) are
bound to energy-transducing membranes and couple the phosphorylation of
ADP into ATP to the dissipation of a protonmotive force. They consist
of a transmembrane proton channel (F0) and an extrinsic
part (F1) bearing six nucleotide binding sites, catalytic
and noncatalytic. The F1 moiety is composed of five
different subunits named
,
,
,
, and
(stoichiometry
[3]
[3]
[1]
[1]
[1]). Subunits
and
bear the nucleotide binding sites and are disposed as a crown, the
subunit being located in the center of this structure (1-4). The
F0 moiety basically consists of three or four different subunits (Escherichia coli: a[1] b[2] c[9-12];
chloroplast: a[1] b[1] b
[1] c[9-12]), the mitochondrial
enzyme having additional subunits (5, 6). It is proposed that the
F0 moiety would work as a rotative proton-driven motor, the
rotor consisting of the c subunits (7), presumably arranged in a crown
(8). The rotation would be transmitted to the
subunit of the
F1 moiety (9), which should modify sequentially the three
catalytic sites located on
subunits (4) to induce ATP synthesis
(10). Experimental arguments have been presented against (11, 12) and
for (9, 13-15) the rotation of
. An essential feature of this model
is that the cooperative functioning among the three catalytic sites is
strictly related to the rotation of the
subunit and thus to the
proton pumping activity.
Tentoxin (TTX)1 is a natural
cyclic tetrapeptide
(cyclo-(L-MeAla1-L-Leu2-Me
ZPhe3-Gly4)),
produced by several phytopathogenic fungi of the Alternaria genus (16, 17). Under special conditions, this toxin induces a
chlorosis in some higher plants (18). It specifically inhibits ATP
synthesis in isolated chloroplasts (19). In vitro and at low
concentrations (10
8-10
7 M),
TTX inhibits the isolated chloroplast F1-ATPase (19-22),
but at higher concentrations (10
5-10
4
M), it strongly stimulates ATPase activity (21-23). At
these same concentrations, the effect observed on membrane-bound ATPase
(F0F1 complex) is restricted to a partial
release of inhibition, but the reactivated F0F1
complex recovers the ability to couple proton transport to ATP
synthesis (24). TTX dramatically disturbs the interactions among
different nucleotide sites of ATPase, whatever the toxin concentration
range (25, 26). Simultaneous perturbation of these interactions and
preservation of proton coupling in the TTX-reactivated form are
intriguing in the context of rotational catalysis. Understanding the
inhibitory and reactivating properties of TTX is therefore one of the
elements that may contribute to the elucidation of the mechanism of
energy coupling.
It has been demonstrated (27) that CF1 binds two molecules
of TTX on two sites of different affinities, which could be related to
the inhibitory and reactivating effects of this molecule. These binding
sites have not yet been identified, and the reasons for the specificity
of TTX for the CF1-ATP synthase of some higher plants
remain obscure (23, 28). TTX stabilizes and enhances the ATPase
activity of an
3
3 complex from spinach
CF1 (29), which proves that the
,
,
subunits are
not required for the stimulation effect of TTX but suggests that they
could be necessary for the inhibition.
We have shown recently (30) that a very limited change in the molecule
of TTX (replacement of L-MeAla1 by
L-MeSer1) resulted in a dramatic loss of the
reactivating effect at high concentrations, although the inhibitory
effect at lower concentrations was unaffected. This led to the idea
that it was possible to discriminate inhibitory and activating effects
by an appropriate set of molecules derived from TTX. Because high
concentrations of MeSer1-TTX were able to prevent
CF1-ATPase reactivation by high concentrations of TTX, we
proposed that MeSer1-TTX could bind the reactivating site
competitively with TTX, giving rise to a poorly active form of the
enzyme. However, it cannot be excluded that MeSer1-TTX
prevents reactivation simply by chasing TTX from the high affinity
site. The question was whether the stimulation by TTX only involves the
low affinity binding site or the two binding sites of CF1.
To get information about possible cooperation among TTX binding sites,
we have used a kinetic approach consisting of studying the catalytic
properties of ternary complexes formed by CF1-
and
different TTX analogues. This approach involves various combinations of
analogues of different affinities for the inhibitory site and able or
not to reactivate the enzyme at high concentrations. The results
suggested that both binding sites participate in the formation of the
reactivated state. At the same time, we were able to characterize the
binding and effector properties of the set of TTX analogues modified in
various positions. This allowed us to make hypotheses about the domains
of the TTX molecule which are important for binding, inhibition, and
stimulation.
 |
MATERIALS AND METHODS |
Preparation and Assay of Solubilized, Activated
CF1-
--
The soluble chloroplast ATPase
(CF1) was extracted and purified from spinach
(Spinacia oleracea L.) leaves in the active form, devoid of
the inhibitory
(31). Storage conditions and determination of the
concentration of CF1-
were described previously (31). The assays were modified slightly with respect to previous conditions (30). The enzyme (80 µg ml
1) was activated by
incubation for 3 h, at room temperature, in a medium containing 20 mM Tricine and 3 mM dithiothreitol, pH 8. Its
activity, measured as described below, was constant for the entire
experiment (5-8 h), ranging between 4 and 6 µmol of hydrolyzed
ATP/mg of protein/min. For assays of ATP hydrolysis, the activated
enzyme was diluted 40-fold in the reaction medium containing 50 mM Tris-SO4, 0.18 mM
MgSO4, 40 mM KHCO3, pH 8.0. This
medium was supplemented with toxins at the indicated concentrations. After 5 min of incubation at 37 °C, the reaction was triggered by
adding 1 mM ATP (final concentration). Aliquots were taken up at different time intervals and analyzed for nucleotide contents by
high performance liquid chromatography, as described (30). The ADP
concentration increased linearly with time, which allowed measurement
of the rate of ATP hydrolysis. All of the rates displayed in the
figures were normalized to that of the control (not toxin-treated). A
rates versus concentration plot was fitted by a nonlinear
iterative algorithm using Microcal Origin 3.54 (Microcal Software).
With respect to our previous experimental conditions (30), the main change consisted in replacing the 1-h preincubation stage with toxin at
80 µg ml
1, at room temperature, by a direct 5-min
incubation in the reaction medium, at 37 °C. This time was
sufficient to get the maximal effect of toxins. Suppression of the
preincubation stage with TTX or derivatives had the advantage of
controlling the toxin/enzyme concentration ratio strictly.
Synthesis of TTX and Derivatives--
All toxins, including TTX,
were synthesized by Drs. Florine Cavelier and Jean Verducci,
Laboratoire des Amino acides, Peptides et Protéines,
Université Montpellier II, Montpellier, France. The synthesis and
structural properties of TTX and MeSer1-TTX have been
reported already (30, 32). Synthesis and structural features of the
other derivatives will be published elsewhere.
 |
RESULTS |
The Different Analogues of TTX--
Fig.
1 displays the different analogues of TTX
which were assayed for the inhibition and reactivation of
CF1-
ATPase activity. In four of them, the residue
methylalanine1 (MeAla1) was replaced,
respectively, by methylserine (MeSer1), by the benzyl ester
of methylserine (MeSer(Bn)1), by methylglutamate
(MeGlu1), and by the terbutyl ester of methylglutamate
(MeGlu(tBu)1). Two of them had their leucine replaced by
lysine (Lys2) or Z-protected lysine (Lys(Z)2),
respectively. Two others were modified on the
,
-dehydrophenylalanine residue, which was replaced either by an
,
-dehydrotyrosine (Tyr3) or by the methyl ester
derivative (Tyr(Me)3).

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Fig. 1.
Formula of the different TTX analogues.
The native molecule of TTX
[cyclo-(L-MeAla1-L-Leu2-MePhe[(Z) ]3-Gly4)]
is represented in the center. Each residue replacing
L-MeAla1, L-Leu2, or
MePhe[(Z) ]3 is labeled by the nomenclature of the
corresponding analogue.
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Inhibitory and Reactivating Effects of TTX and
L-MeSer1-TTX--
We have shown previously
that MeSer1-TTX inhibits CF1-ATPase with the
same efficiency as TTX at low concentrations, but it reactivates
CF1-ATPase poorly at high concentrations. This analogue also prevents the reactivation of ATPase by TTX (30). We have reinvestigated the effects of these two toxins to estimate
quantitatively the binding and catalytic parameters. Fig.
2 shows the effect of TTX and
MeSer1-TTX on the ATPase activity of CF1-
.
Although much less pronounced than with TTX, the reactivation by
MeSer1-TTX at high concentrations was effective. It was
possible to fit the data with a simple model involving two independent
binding sites for the toxin, the high affinity site being responsible for the inhibitory effect and the low affinity site being responsible for the reactivation (see Equation 12 under "Appendix"). TTX and MeSer1-TTX were found to have exactly the same affinity for
the first site (Kd1 = 0.038 µM) and also for the second site
(Kd2 = 39 µM for TTX,
Kd2 = 41 µM
for MeSer1-TTX). The only difference between the two toxins
was the ATPase activity of the complex having its two sites occupied:
220% of the control in the case of TTX but only 27% in the case of
MeSer1-TTX.

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Fig. 2.
ATPase activity of CF1-
versus TTX and MeSer1-TTX concentrations.
Conditions are as described under "Materials and Methods." ,
TTX; , MeSer1-TTX. Continuous curves result
from fitting the data with the Equation 12 of the "Appendix"
(two-sites model). The parameters obtained from the fits are displayed
in Table I. For details, see "Results."
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Effect of L-MeSer1-TTX in the Presence of
TTX at Reactivating Concentrations--
To know whether the activity
of these ternary complexes is governed by the nature of the toxin bound
on the low affinity site, on the high affinity site, or on both sites,
we have carried out the following experiment. MeSer1-TTX at
various concentrations was first mixed with TTX at a constant concentration (30 µM) in the reaction medium, then
CF1-
was added. After incubation, MgATP was added and
the ATPase activity measured. Fig. 3
(closed squares) shows the continuous decrease of the rate of ATP hydrolysis caused by the addition of increasing concentrations of MeSer1-TTX. The reaction rate actually depends on the
proportions and on the catalytic activities of the following four
ternary complexes (see "Appendix"): that bearing two molecules of
TTX (ET1T2), that bearing two
molecules of MeSer1-TTX
(EX1X2), that bearing TTX on the
tight site and MeSer1-TTX on the loose site
(ET1X2), and that bearing
MeSer1-TTX on the tight site and TTX on the loose site
(EX1T2). The activities of
ET1T2 and
EX1X2 (respectively, 220 and 27% of
the control) were known from the data of Fig. 2 fitted with Equation 12
under "Appendix" as well as the dissociation constants
Kd1 (0.038 µM) and Kd2
(40 µM), which are identical for the two toxins. This allowed us to fit the activity versus concentration data
points of Fig. 3 with the model developed in Equation 27 under
"Appendix." Since the concentrations of
ET1X2 and
EX1T2 are always identical because
of the identical affinities of TTX and MeSer1-TTX for the
enzyme, only the average of the activities of these two ternary
complexes can be determined. It was experimentally found to be 85% of
the control. If the catalytic activity was governed only by the nature
of toxin bound on the loose site, ET1X2 should have the same activity
as EX1X2, and
EX1T2 should have the same activity
as ET1T2. Thus, their average
activity would equal 125% of the control rather than 85% (the same
result should be found if the activity of the ternary complex depended only on the nature of the toxin bound on the first site,
i.e. if EX1X2 and
ET1T2 had, respectively, the same
activity as EX1T2 and
ET1X2). This is depicted by the
dashed curve in Fig. 3, which represents the theoretical
relationship between the rate of ATP hydrolysis and the
MeSer1-TTX concentration with the average activity of
ET1X2 and
EX1T2 set at 125%: it is clearly
not superimposable onto the experimental data.

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Fig. 3.
Rate of ATP hydrolysis as a function of
MeSer1-TTX concentration in the presence of 30 µM TTX. Conditions are as described under
"Materials and Methods." Solid curve, fitting of the
data with Equation 27 of the "Appendix" (competition between two
toxins of identical Kd1
and Kd2). Dashed
curve, theoretical values of the rate V calculated from
the same equation but assuming that VTX + VXT = 2.6. For details, see "Results."
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Inhibitory and Reactivating Effects of Different Synthetic
Analogues of TTX--
To discriminate better the functional role of
the low and high affinity TTX binding sites, we have used a another set
of molecules differing from TTX by a single residue. The effect of
these analogues on ATPase activity is shown in Fig.
4. All of these compounds inhibited the
ATPase but only at higher concentrations compared with TTX. The
reactivation was not observed in the case of toxins modified in
position 1 (Fig. 4a). Its level was not significant in the
case of Lys2-TTX and was moderate in the case of
Lys(Z)2-TTX (40% of the control, Fig. 4b).
Reactivation was marked in the case of Tyr3-TTX (about
75%) and maximal in the case of Tyr(Me)3-TTX (about 290%,
Fig. 4c).

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Fig. 4.
Effect of TTX and analogues on the ATPase
activity of CF1- . Conditions are as described under
"Materials and Methods." Panel a, analogues modified on
position 1 (MeAla); , MeSer(Bn)1-TTX; ,
MeGlu1-TTX; , MeGlu(tBu)1-TTX. Panel
b, analogues modified on position 2 (Leu); ,
Lys2-TTX; , Lys(Z)2-TTX. Panel c,
analogues modified on position 3 ( Phe); , Tyr3-TTX;
, Tyr(Me)3-TTX. Solid curves, fitting with
Equation 12 of the "Appendix" (the same two-sites model as in Fig.
2). Dashed curves, fitting with Equation 5 of the
"Appendix" (single-site model). The parameters obtained from the
fits are displayed in Table I. The fitted curve obtained with TTX in
Fig. 2 was redrawn (dots) in panels a,
b, and c for comparison.
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For reactivating toxins (TTX, MeSer1-TTX,
Lys(Z)2-TTX, Tyr3-TTX, and
Tyr(Me)3-TTX), the data were fitted satisfactorily using
the same two-sites model, with the assumption that the complex bearing
a single molecule of toxin was fully inactive
(V1 set to zero, see Equation 12 under "Appendix"). When no reactivation occurred
(MeSer(Bn)1-TTX, MeGlu1-TTX, and
(MeGlu(tBu)1-TTX), a simpler model, involving only one
binding site (Equation 5 under "Appendix"), could fit easily the
data. In this latter model, to account for possible incomplete
inhibition, the enzyme-toxin complex was allowed to have an activity
different from zero, which was derived from the fit. In the case of
Lys2-TTX, the two models (two sites and single site) have
been used to fit the data because this toxin did not reactivate the
ATPase significantly at high concentration but was nevertheless shown to bind to the reactivating site (see below). In all cases, the dissociation constant for the inhibitory site
(Kd1) could be
determined. The values are summarized in Table
I, first column.
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Table I
Binding and catalytic parameters of tentoxin and analogues
Conditions are as described under "Materials and Methods."
Parameters are drawn from the data plotted in Figs. 2 and 4 for all
toxins and also from the data of Fig. 5 for Lys2-TTX, second
line. Kd1; dissociation constant from the high
affinity site; Kd2, dissociation constant from the low affinity
site; VI, relative activity of the complex bearing a
single molecule of toxin (assumed to be zero in the case of a two-sites
model); VA, relative activity of the complex bearing two molecules of toxin. The equations used were: V = (1 + [T]/Kd1) 1 + VI(1 + Kd2/[T] 1
for the single-site model, and V = (1 + [T]/Kd1 + [T]2/Kd1
Kd2) 1 + VA (1 + Kd2/[T] + Kd1Kd2/[T]2) 1
for the two-sites model, where V is the activity
normalized to that of the nontreated enzyme. See "Appendix"
for details.
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Effect of L-Lys2-TTX, a Nonreactivating
Compound, in the Presence of TTX at Reactivating
Concentrations--
The question is now to know whether the absence of
reactivation by MeSer(Bn)1-TTX, MeGlu1-TTX,
MeGlu(tBu)1-TTX, and Lys2-TTX results from a
default of binding or a nonproductive binding on the low affinity site.
The experiments carried out to address this question were based on the
same principle as that used in Fig. 3 for the poorly reactivating
analogue MeSer1-TTX. The effect of high concentrations of
nonreactivating analogues on the ATPase activity was checked in the
presence of reactivating concentrations of TTX.
Fig. 5 shows the effect of
Lys2-TTX on the ATPase activity in the presence of three
different concentrations of TTX in the reactivating range. The data
show that the addition of Lys2-TTX decreases the activity,
by limiting the reactivation by TTX, and that the concentration of
Lys2-TTX needed to prevent the enzyme reactivation
increases with the concentration of TTX (compare the three curves of
inhibition). This effect is consistent with a competition on the loose
site. To determine the binding parameters of Lys2-TTX and
the activity of the ternary complexes, we have fitted the data of Fig.
5 with the two-sites model described above (see also Equation 26 under
"Appendix"). In this model, the enzyme can exist under the
following states: E (without toxin),
ET1 (with TTX bound at the high affinity site),
ET1T2 (with TTX bound at both
sites), ET1X2 (with TTX bound at the
high affinity site and Lys2-TTX bound at the low affinity
site), and EX1X2 (with
Lys2-TTX bound at both sites). These states are the only
ones present at significant levels, when one considers the values of
Kd1 for TTX and for
Lys2-TTX. Fitting of the competition data allowed a refined
determination of the binding constant of Lys2-TTX for the
high affinity site and the estimation of its binding constant for the
low affinity site. The corresponding values of Kd1 and
Kd2 have been found close
to 2 µM and 55 µM, respectively. We also
determined the ATPase activities of the ternary complexes EX1X2 and
ET1X2 (called, respectively,
VXX and VTX). There were found almost negligible (about 5% of the control).

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Fig. 5.
Rate of ATP hydrolysis as a function of
Lys2-TTX concentration in the presence of three different
concentrations of TTX. Conditions are as described under
"Materials and Methods." , [TTX] = 9 µM; ,
[TTX] = 18 µM; , [TTX] = 32 µM.
Fitted curves were obtained with Equation 26 of the "Appendix"
(competition between two toxins). The dissociation constants
Kd1 and
Kd2 and the activity of
the CF1- ·TTX·TTX complex were known from data of
Figs. 2 and 4b and fixed at their values:
Kd1 = 0.038 µM; Kd2 = 38 µM; VTT = 2.22. The parameters
of Lys2-TTX were obtained from the fit. The three fits
gave: K d1 = 2 µM, K d2 = 55 µM, VXT = 0.05, VXX = 0.05.
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Effect of L-Lys(Z)2-TTX in the Presence of
TTX at Inhibitory Concentrations--
Contrary to the enzyme bearing
two molecules of Lys2-TTX, the enzyme bearing two molecules
of Lys(Z)2-TTX (EX1X2
complex) exhibited a significant ATPase activity, about 30% (Table I,
last column). The affinity of Lys(Z)2-TTX for the high
affinity site is low compared with TTX
(Kd1 = 1 µM
instead of 0.04 µM), but its affinity for the low
affinity site is somewhat better than that of TTX
(Kd2 = 11 µM versus 39 µM). This means
that, starting from the complex where the high affinity site is
occupied by TTX, one can fill specifically the low affinity site with
Lys(Z)2-TTX, forming the
ET1X2 complex. This was achieved by
adding various concentrations of Lys(Z)2-TTX to an enzyme
sample already containing TTX at a micromolar concentration. Fig.
6 shows the ATPase activity measured in
such conditions. Two different concentrations of TTX were used. The addition of Lys(Z)2-TTX to CF1-
inhibited by
TTX did not restore any significant activity. The theoretical curve
displayed on Fig. 6, drawn using the same activity for
ET1X2 and
EX1X2, i.e. 30% of the
control, does not fit the experimental data. The activity of the
ET1X2 complex can indeed be
estimated to be less than 10% of the control, then different from that
of EX1X2. However, this
interpretation is valid only if the affinity of Lys(Z)2-TTX
for the loose site (Kd2 = 11 µM) does not depend on the toxin (TTX or
Lys(Z)2-TTX) bound to the tight site (see
"Discussion").

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Fig. 6.
Rate of ATP hydrolysis as a function of
Lys(Z)2-TTX concentration in the presence of two different
concentrations of TTX. Conditions are as described under
"Materials and Methods." , [TTX] = 0.5 µM; ,
[TTX] = 1 µM. Solid curves, theoretical
activities calculated with the same equation as used in Fig. 5
(Equation 26 under "Appendix"). The values of the dissociation
constants and the activity of the CF1- ·TTX·TTX and
CF1- ·Lys(Z)2-TTX·Lys(Z)2-TTX
complexes were drawn from data of Figs. 2 and 4b. The
activity of the CF1- ·TTX·Lys(Z)2-TTX
complex was set as the same value as that of the
CF1- ·Lys(Z)2-TTX·Lys(Z)2-TTX
complex, i.e. 30% of the control. Dotted curves,
theoretical activities calculated with the same assumptions except that
the activity of the CF1- ·TTX·Lys(Z)2-TTX
was set at 0.
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Effect of Different Nonreactivating Toxins in the Presence of TTX
at Reactivating Concentrations--
At high concentrations,
MeSer(Bn)1-TTX, MeGlu1-TTX, and
MeGlu(tBu)1-TTX did not reactivate the ATPase activity of
CF1-
. By competition experiments similar to those
carried out with Lys2-TTX and depicted above, we have
obtained the following results (data not shown): (i)
MeGlu1-TTX and MeGlu(tBu)1-TTX did not compete
at all with TTX for binding at the low affinity site; (ii)
MeSer(Bn)1-TTX competed efficiently with TTX, with a
Kd2 comparable to that of
the natural toxin, and its binding to the low affinity site gave rise
to a fully inactive complex.
 |
DISCUSSION |
The Two TTX Binding Sites Are Related to Inhibition and
Reactivation of CF1-ATPase Activity--
In a preceding
study (27), we detected the presence of two TTX binding sites on
isolated CF1 and CF1-
, using synthetic 14C-labeled TTX, and we related these two sites to the TTX
properties of inhibition of the CF1-ATPase activity below
0.1 µM and reactivation above 5-10 µM. The
present study, using TTX synthetic analogues, fully confirms the
existence of these two independent TTX binding sites and their direct
connection to the inhibition and stimulation processes. More, the
binding parameters have been determined carefully using the two-sites
formalism. For TTX, the value of
Kd2 derived from kinetic
experiments (40 µM) is consistent with that derived from
equilibrium dialysis experiments (70 µM). The
Kd1 values (0.04 µM from kinetic experiments and 1 µM from
14C binding studies) are different, but this can be
explained by the low temperature (4 °C) at which the dialysis
experiments were carried out. It has to be noticed that, although the
two TTX properties can be discriminated with some analogues, the
reactivation process was never observed solely, i.e. without
a prior inhibition resulting from the active binding to the first
site.
TTX Does Not Aggregate at High Concentrations--
The two-sites
formalism used here is relevant only if the formation of some toxin
aggregates does not compete with the binding of toxin to the enzyme.
Earlier NMR experiments suggested a possible self-association of TTX in
aqueous solution, at 3.5 mM, at
5 °C, and in the
presence of salts (32). However, only a part of the data was consistent
with this view: the modification of the chemical shift of the leucyl
proton and the decrease of the transverse relaxation time
T2 of given backbone protons. The first change could as well be ascribed to an intramolecular Leu-
Phe stacking, especially since we failed to observe other effects expected from intermolecular dipole-dipole interactions (nuclear Overhauser effects).
The T2 decrease can hardly be attributed
univocally to self-association of TTX molecules. It could indeed be
biased by chemical exchange (maybe not fully abolished in the NMR
experimental conditions), or it may reflect more complex hydrodynamic
properties than those generally assumed for small molecules.
Time-resolved fluorescence data were also supposed to reveal the
formation of tentoxin aggregates, in the 10 µM range
(32), on the basis of a heterogeneous decay of fluorescence, but the
two lifetimes thus determined (1.8 and 0.7 ns) were actually too close
and the method not sensitive enough to distinguish effectively between
a heterogeneous and a homogeneous decay. To overcome the ambiguities of
past studies, we carried out further investigations using different
spectroscopic techniques expected to detect stable aggregates via
modifications of the conformation of peptide bonds (CD) or of the
stretching vibrational modes of carbonyl groups (FTIR). No change in CD
spectra of TTX in aqueous solution was observed in the 5-1,000
µM range at temperatures between 5 and 37 °C (not
shown). FTIR difference spectra of TTX were recorded at 12 °C in
deuterated water solutions. Likewise, there were no variations observed
in the absorption domain of the stretching vibrational modes of
carbonyl groups (1,656 and 1,636 cm
1) in the 100-4,000
µM TTX concentration range (not shown). The stretching
mode of C=O groups is indeed sensitive to hydrogen bonding, and if
intermolecular associations took place or were stabilized via the
formation of hydrogen bonds, they would produce a detectable shift of
the wave number. These data do not support the existence of a micellar
equilibrium involving TTX molecules in the concentration range where we
observed the reactivation (10-1,000 µM). Finally,
definitive evidence for the absence of aggregates at high
concentrations of TTX in our experimental conditions came from simple
dialysis experiments using 14C-labeled TTX. These
experiments were carried out in the same medium as that used for ATP
hydrolysis activity measurements at 35 °C. The use of
14C-labeled TTX allowed the detection of very low
concentrations of the toxin in the dialysis buffer. The initial
diffusion rate of 14C-labeled TTX through the dialysis
tube, chosen with a cut-off at 1 kDa (experimentally checked with
molecules of different sizes), was strictly proportional to its
internal concentration ranging from 2 to 500 µM. This
demonstrates that the molecular mass of the diffusing species was
constant and always lower than 1 kDa. The molecular mass of TTX being
0.414 kDa rules out the possibility of
concentration-dependent aggregate formation. The
concentration of available monomers of TTX is identical to the total
concentration, which validates the enzymologic approach used in this
work.
Effects of Modifications of the TTX Molecule on Its Inhibitory
Properties--
All the analogues of TTX studied in the present work
have retained their inhibitory properties. In all cases, the ATPase
activity of CF1-
was completely lost when a single
molecule of toxin was bound. The only effect of the substitutions was
to decrease to various extents the affinity of the molecule for the
tight site, moderately for certain modifications (MeAla1
MeSer1, Phe3
Tyr3,
Phe3
Tyr(Me)3) and more drastically (up to
200 times) for others (in the increasing Kd1 order:
MeAla1
MeSer(Bn)1, Leu2
Lys(Z)2, MeAla1
MeGlu(tBu)1,
Leu2
Lys2, MeAla1
MeGlu1). Different molecular factors can account for this
Kd1 increase, such as the
introduction of longer chains, giving rise to additional steric
hindrance (MeAla1
MeSer(Bn)1,
MeAla1
MeGlu(tBu)1, Leu2
Lys(Z)2), or the introduction of a net electrical charge on
the molecule (MeAla1
MeGlu1,
Leu2
Lys2). The introduction of charges
seems to be more determining, since the replacement of a charged
residue by a neutral residue, even larger (MeGlu1
MeGlu(tBu)1, Lys2
Lys(Z)2), led
to some recovery of the affinity.
A first conclusion of our work is that the nature of residues 1 and 2 (N-MeAla and Leu in the natural molecule) is not so critical
for the inhibitory power of the molecule once it is bound to its site.
This can be related to previous structural results obtained by NMR (30,
32). Indeed, TTX and MeSer1-TTX exhibited the same
conformation of the cyclic backbone (cis-trans-cis-trans configuration of the amide bond sequence) and the same interconversion among four conformers. These structural properties have also been observed for all of the analogues of the residue
1.2 The conservation of the
conformational features of the molecules can account for the ability of
the molecule to inhibit the enzyme (VI = 0)
totally, and the variations of side chain can account for the changes
in the affinity for the tight site. However, changes in residue 3 did
not result in an increase in
Kd1. In fact, these modifications were confined to a substitution of the para
proton of the benzyl group, without any consequence on the rigid
configuration of the double bond of the
,
-dehydroamino acid.
Despite the introduction of polar groups on this side chain, there was
no repercussion on the affinity of the molecule for the tight site.
This result is not so unexpected if the binding of the molecule inside
the hydrophobic site involves a stabilization by an aromatic ring stacking.
Effects of Modifications of the TTX Molecule on Its Activating
Properties--
Whereas the only change in the inhibitory properties
of toxin derivatives is a variation of their affinity for the tight
site, the situation is quite different with regard to reactivating
properties. Noteworthy is that with the exception of
MeGlu1-TTX and MeGlu(tBu)1-TTX, all the
analogues were found to bind to the low affinity site with a
Kd2 comparable to that of
natural TTX, whether they were reactivating or not. The differences lie
in the effect of the molecule once bound to this site. It is possible
that the loose site has a more open configuration than the tight one,
which makes it less sensitive to steric hindrance variations and also to electrical charges (that can be shielded by water molecules). The
exceptions of MeGlu1-TTX and MeGlu(tBu)1-TTX,
however, remain to be explained.
In the absence of a structural model of the chloroplast ATPase species,
which differs from the mitochondrial species in its sensitivity to TTX
and in various structural features (notably regarding the
subunit),
it is still difficult to explain the various levels of reactivation
obtained with the different analogues bound on the two sites. For the
complexes bearing two molecules of the same toxin (homogeneous
complexes), a change of hydrophobicity of the molecule (30) can result
in a significant change in the reactivation level. Thus, the
replacement of a proton by a hydroxyl group (Ala
Ser; Phe
Tyr)
dramatically decreases the activity of the ternary complex when made in
position 1 and significantly when made in position 3. In the latter
case, this activity is restored when the labile proton is replaced
again by a more hydrophobic group (Tyr
Tyr(Me)). The comparison of
the reactivating properties of MeSer1-TTX and
Tyr3-TTX, two molecules slightly modified with unchanged
affinities for both sites, suggests that the nature of residue 1 is
more important than that of residue 3 in conferring to TTX its
reactivating properties. But also, as discussed below, the level of
reactivation was found to be dependent on the combination of the two
toxins bound (hybrid ternary complexes).
Importance of the Two TTX Binding Sites in the
Reactivation--
It is tempting to speculate whether the two TTX
binding sites are homologous domains of two different 
pairs, put
into different states by the asymmetry of the ATPase complex. Such a
situation has already been stated in the case of the binding of two
molecules of aurovertin to the bovine heart MF1 complex
(33), with the noteworthy difference that aurovertin is never
reactivating. If the two binding sites of TTX were located on different

pairs, the TTX molecule bound to the first 
pair could
block the enzyme, and the TTX molecule bound to the second pair could
unlock it.
An original result of this work deals with hybrid ternary complexes,
bearing natural TTX on the tight site and a synthetic derivative on the
loose site. By applying a simple model, with two independent binding
sites, we could estimate the activities of some of these hybrid
complexes and compare them with the activities of ternary complexes
bearing the same molecule on both sites (Table II). It is not possible, at the present
time, to understand the molecular rules that would determine the
activity of all of these ternary complexes, but there are two main
points. First, the activity depends on the toxin bound on the loose
site; compare, for example, the CF1·TTX·TTX complex
(220% activity) with the
CF1·TTX·Lys(Z)2-TTX and
CF1·TTX·Lys2-TTX complexes (negligible
activities). Second, the activity also seems to depend on the toxin
bound on the tight site; compare the
CF1·TTX·Lys(Z)2-TTX complex (negligible
activity) with the
CF1·Lys(Z)2-TTX·Lys(Z)2-TTX
complex (30% activity), and see also the competition between TTX and
MeSer1-TTX. So, although the CF1-ATPase enzymes
complexed with a single molecule of TTX or with a single molecule of an
analogue cannot be discriminated on the basis of their activity, they
are potentially different. This difference is revealed only when a
second molecule of the analogue is bound on the loose site. All of our
data are consistent with an interaction between the two TTX binding
sites.
View this table:
[in this window]
[in a new window]
|
Table II
ATPase activity of ternary complexes CF1 · toxin toxin
with respect to the activity of nontreated CF1
Conditions are as described under "Materials and Methods." The
first two columns indicate which toxins are bound to the high and low
affinity sites. Activities of homologous complexes, bearing two
molecules of the same toxin, were calculated from the magnitude of the
maximal reactivation (VA in Table I, see also Figs.
2 and 4). Activities of heterologous complexes, containing one molecule of TTX and one molecule of an analogue, were estimated from competition experiments (Figs. 3, 5, and 6). In the case of MeSer1-TTX, the
two different heterologous complexes cannot be discriminated, and only
the average of their activities is given. For details, see
"Results."
|
|
Cooperative Binding, an Alternative Hypothesis--
To fit our
data, we have considered only a simple model with two preexisting and
absolute Kd values, which assumes that binding of a
first toxin molecule to the complex has no influence on the affinity of
the complex for a second molecule. The activities of ternary complexes
are then the only way to detect interactions between the two sites.
However, it would be also possible to fit them with a model of
cooperative binding, and in this case the Kd2 of a given toxin
would depend on the toxin bound on the tight site. For example, data of
Fig. 6 could be fitted satisfactorily with a model assuming that all of
the ternary complexes have a negligible activity, provided
Lys(Z)2-TTX binds to the loose site with a
Kd2 equal to 100 µM instead of 11 µM, the value drawn from
Fig. 4. This means that replacing Lys(Z)2-TTX by TTX at the
tight site would dramatically decrease the affinity of
Lys(Z)2-TTX for the loose site. Even though the mechanism
differs from that assumed in our first model, this would demonstrate
even more directly the interaction between the two binding sites of the toxin. Understanding this interaction should be an important element in
the knowledge of the catalytic mechanism of
CF0CF1-ATP synthase.
 |
ACKNOWLEDGEMENTS |
We thank Véronique Mary for
extraction of the spinach chloroplast F1-ATPase. We are
indebted to Drs. Florine Cavelier and Jean Verducci for the
chemical synthesis of tentoxin and all of its analogues. Dr. Catherine
Berthomieu performed the FTIR analysis of tentoxin solutions.
 |
FOOTNOTES |
*
This work was supported by the Ministère de
l'Enseignement Supérieur et de la Recherche Contract ACC-SV5
(interface Chimie-Physique-Biologie) 9505221.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.:
33-01-6908-4432; Fax: 33-01-6908-8717; E-mail:
andre{at}dsvidf.cea.fr.
1
The abbreviations used are: TTX, tentoxin or
(cyclo-(L-N-MeAla1-L-Leu2-N-Me
ZPhe3-Gly4));
CF1, chloroplast F1 H+-ATPase;
CF1-
, chloroplast F1 H+-ATPase
devoid of
subunit; FTIR, Fourier transformation infrared spectroscopy; Lys2-TTX,
(cyclo-(L-N-MeAla1-L-Lys2-N-Me
ZPhe3-Gly4));
Lys(Z)2-TTX,
(cyclo-(L-N-MeAla1-L-Lys(Z)2-N-Me
ZPhe3-Gly4));MeGlu1-TTX,
(cyclo-(L-N-MeGlu1-L-Leu2-N-Me
ZPhe3-Gly4));
MeGlu(tBu)1-TTX,
(cyclo-(L-N-MeGlu(tBu)1-L-Leu2-N-Me
ZPhe3-Gly4));
MeSer1-TTX,
(cyclo-(L-N-MeSer1-L-Leu2-N-Me
ZPhe3-Gly4));
MeSer(Bn)1-TTX,
(cyclo-(L-N-MeSer(Bn)1-L-Leu2-N-Me
ZPhe3-Gly4));
Me
zPhe or
Phe,
,
-dehydrophenylalanine
N-methylated in Z configuration; Tyr3-TTX,
(cyclo-(L-N-MeGlu1-L-Leu2-N-Me
ZTyr3-Gly4));
Tyr(Me)3-TTX,
(cyclo-(L-N-MeGlu1-L-Leu2-N-Me
ZTyr(Me)3-Gly4));
Tricine, N-[2-hydroxy-1,1-bis(hydroxy-
methyl)ethyl]glycine.
2
F. André, unpublished results.
 |
APPENDIX |
The Single-site Model--
The binding equilibrium is governed
by Kd1, the dissociation
constant of the enzyme-toxin complex,
|
(Eq. 1)
|
where [E] is the concentration of the free form of
the enzyme, [ET] the concentration of the complex, and
[T] the concentration of free toxin. If [Et]
refers to the total concentration of the enzyme, the relative
concentrations of the two forms of the enzyme are
|
(Eq. 2)
|
|
(Eq. 3)
|
If v0 and vI are
the rates of the reaction catalyzed by the forms E and
ET, respectively, the total reaction rate is
|
(Eq. 4)
|
The rate V normalized to the control reaction rate
v0 is
|
(Eq. 5)
|
with VI = vI/v0.
In the experimental plots, the free concentration of toxin, [T], will
be identified to the total concentration of toxin
[T]+[ET], since the toxin is always in large excess
under our conditions. This approximation also applies to the two-sites
model.
The Two-sites Model--
Let us consider the binding equilibria
where two molecules of toxin are successively bound to sites called 1 and 2 (Scheme 1, where
ET1 refers to the complex with one molecule of
toxin bound to the high affinity site
(Kd1),
ET2 to the complex with one molecule of toxin
bound to the low affinity site
(Kd2), and ET1T2 to the complex with the toxin
bound to the two sites. One has
|
(Eq. 6)
|
|
(Eq. 7)
|
and
|
(Eq. 8)
|
Because experimentally
Kd1
Kd2, one can consider
that [ET2] is negligible. The relative
concentrations of the different states are therefore
|
(Eq. 9)
|
|
(Eq. 10)
|
and
|
(Eq. 11)
|
To simplify, one assumes that binding of a single toxin molecule
fully inhibits the enzyme, i.e. the activity of the state ET1 is 0. The reaction rate V (with
toxin) normalized to that of the control (without toxin) becomes
|
(Eq. 12)
|
where VA is the ratio between the
catalytic activities of the states
ET1T2 and E.
Competition between Two Different Toxins at the Two Binding
Sites--
Let us consider the same equilibria where two different
toxins (T and X) are bound. One has
|
(Eq. 13)
|
|
(Eq. 14)
|
Kd1
Kd2.
|
(Eq. 15)
|
K
d1
K
d2.
|
(Eq. 16)
|
By convention, T refers to TTX and X to an analogue. Indexes 1 and 2 still refer to the high and low affinity sites, respectively. K
d1 and
K
d2 are the dissociation constants corresponding to toxin X. The concentrations of all of the possible complexes are given by
|
(Eq. 17)
|
|
(Eq. 18)
|
|
(Eq. 19)
|
|
(Eq. 20)
|
|
(Eq. 21)
|
|
(Eq. 22)
|
|
(Eq. 23)
|
|
(Eq. 24)
|
and
|
(Eq. 25)
|
At this stage, some approximations can be made. Because
Kd1
Kd2 and
K
d1
K
d2, the states
ET2 and EX2 can be
neglected. This is true because the competition experiments were made
with the tight site almost fully occupied. In addition, one still
assumes that the states ET1 and
EX1 have no catalytic activity. The reaction
rate normalized to that of the control is then
|
(Eq. 26)
|
where VTT, VXX,
VTX, and VXT are the
activities of the ET1T2,
EX1X2,
ET1X2, and
ET2X1 complexes, respectively,
normalized to the activity of the E state.
Equation 26 was used to fit the data of Fig. 5, where
Lys2-TTX competes with TTX, and the data of Fig. 6, where
Lys(Z)2-TTX competes with TTX. The values of [T] and
[X] were experimentally known, and
Kd1,
Kd2,
K
d1, and
VTT were determined from the data of Figs. 2 and
Fig. 4. K
d2,
VTX, and VXX were derived from the fit. VXT was set at different values,
which did not affect the quality and the parameters of the fit, because
the contribution of the EX1T2 form
is negligible. So VXT could not be
determined.
Particular Case of Competition between Two Different Toxins of
Identical Affinities--
In Equation 26, if
Kd1 = K
d1 and if
Kd2 = K
d2 (case of the competition
between TTX and MeSer1-TTX), the rate equation becomes
|
(Eq. 27)
|
This equation was used to fit the data of Fig. 3.
Kd1,
Kd2,
VTT, and VXX were
determined previously from the data of Fig. 2.
VTX and VXT cannot be
discriminated in Equation 27; only their sum (or their average) can be
derived from the fit. If the activity of the ternary complex depended
only on the nature of the toxin bound on the second site, thus
VXT = VTT and
VTX = VXX, then
VXT + VTX = VXX + VTT. The same
equality should be found if the activity of the ternary complex
depended only on the nature of the toxin bound on the first site,
i.e. VXT = VXX and
VTX = VTT. This equality can be checked easily with the results derived from the fit.
 |
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