Interactions of Benzodiazepine Derivatives with Annexins*
Andreas
Hofmann
§,
Achim
Escherich¶,
Anita
Lewit-Bentley
,
Jörg
Benz
,
Céline
Raguenes-Nicol**,
Francoise
Russo-Marie**,
Volker
Gerke
,
Luis
Moroder¶, and
Robert
Huber
From the
Max-Planck-Institut für Biochemie,
Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried,
the ¶ Max-Planck-Institut für Biochemie, Abt. Bioorganische
Chemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany,
LURE,
Bâtiment 209D, Centre Universitaire Paris-Sud, F-91405 Orsay,
France, ** ICGM, U332, INSERM, 22 rue Méchain, F-75014 Paris,
France, and the 
Institut für
Medizinische Biochemie, Universität Münster,
D-48149-Münster, Germany
 |
ABSTRACT |
Human annexins III and V, members of the annexin
family of calcium- and membrane-binding proteins, were complexed within
the crystals with BDA452, a new 1,4-benzodiazepine derivative by
soaking and co-crystallization methods. The crystal structures of the complexes were analyzed by x-ray crystallography and refined to 2.3- and 3.0-Å resolution. BDA452 binds to a cleft which is located close
to the N-terminus opposite to the membrane binding side of the
proteins.
Biophysical studies of the interactions of various benzodiazepine
derivatives with annexins were performed to analyze the binding of
benzodiazepines to annexins and their effects on the annexin-induced
calcium influx into phosphatidylserine/phosphatidylethanolamine liposomes. Different effects were observed with a variety of
benzodiazepines and different annexins depending on both the ligand and
the protein. Almost opposite effects on annexin function are elicited
by BDA250 and diazepam, its 7-chloro-derivative. We conclude that
benzodiazepines modulate the calcium influx activity of annexins
allosterically by stabilizing or destabilizing the conducting state of
peripherally bound annexins in agreement with suggestions by Kaneko
(Kaneko, N., Ago, H., Matsuda, R., Inagaki, E., and Miyano, M. (1997)
J. Mol. Biol., in press).
 |
INTRODUCTION |
Benzodiazepines are well known pharmaceuticals used in the short
time therapy of insomnia and stress induced anxiety (2, 3).
Psychopharmaceutical effects are also reported for these substances,
but the molecular mechanism of their action is not yet well understood.
Benzodiazepines have been found to bind with high affinity to a defined
receptor population in the brain, that has been identified as the
GABAA receptor. The affinity of different benzodiazepine
derivatives to this receptor correlates well with their pharmacological
potency and their binding site is apparently localized on the receptor
close to the GABA-binding site (4, 5). The models of mechanism of
action proposed so far suggest a cooperative effect of both GABA and
benzodiazepine on the opening of the chloride channel. Recently,
diazepam was shown to increase the conductance of
GABAA channels up to 7-fold in rat cultured hippocampal
neurons (6). Benzodiazepine-related compounds are one of the most
important classes of bioavailable therapeutic agents with widespread
biological activities including anxiolytic, anticonvulsant, and
antihypnotic activities (7), cholecystokinin receptor A and receptor B
antagonists (8), opioid receptor ligands (9), platelet-activating
factor antagonists (10), human immunodeficiency virus trans-activator
Tat antagonists (11), GPIIbIIIa inhibitors (12), reverse transcriptase
inhibitors (13), and Ras farnesyltransferase inhibitors (14).
To these multiple actions of benzodiazepine compounds was added
recently the finding that the cardiac protective agent K201, a
benzothiazepine derivative, inhibits annexin V binding to actin in vitro (15). Its effect on annexin-induced calcium influx has also been studied and its binding site defined (1).
Based on these observations we analyzed in detail a potential
interaction between annexins and benzodiazepines. We report here that
complex formation occurs between annexins and various benzodiazepines
among which are the newly synthesized cholecystokinin-A and
cholecystokinin-B receptor antagonists, as well as known
pharamaceuticals like diazepam. Since the physiological function of
annexins is still not yet fully understood, the interaction of these
proteins with benzodiazepines might open new lines of investigation of the role of annexins in vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials
Porcine annexin I was purified from bacteria expressing the
recombinant protein (16). Recombinant human annexin II containing a
N-terminal elongation of six residues (MRGSFK) was purified from the
appropriately transformed bacteria as described (17). Human annexin III
(18), human annexin V (19), and human annexin VI, VIa, and VIb (20)
were purified as described. The N-terminal deletion mutants AV-N1
(
1-6), AV-N3 (
1-13), and AV-N4 (
1-14) were made by
introducing mutations in the annexin V wild-type cDNA,1 expressed and
purified according to the wild-type protocol. The synthesis and
biological properties of the benzodiazepine derivatives BDA452, BDA250,
and BDA753 (see Fig. 1) will be discussed
elsewhere. Diazepam (DZM) and 4-bromo-A23187 were purchased from Sigma
(Deisenhofen, Germany), N-acetyltryptophan-amide (Trp) from
Bachem (Switzerland), and the pentasodium salt of FURA-2 was from
Calbiochem (San Diego, CA).

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Fig. 1.
Structures of substances mentioned.
BDA452,
3-(R,S)-(L-tryptophanyl)-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one. BDA753,
3-(R,S)-all-L-(NH-Trp-Gly-Tyr-Ala-H)-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one. BDA250,
1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one. DZM,
7-chlor-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one (diazepam). TRP,
(+)-N-acetyl-L-tryptophan-amide.
K201,
4-(3-(1-(4-benzyl)piperidinyl)propionyl)-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (14).
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X-ray Structure Determination
Annexin V--
Rhombohedral annexin V wild-type crystals were
grown by vapor diffusion at room temperature against 1 mM
CaCl2, 1.9 M
(NH4)2SO4, 0.1 M Tris,
pH 8.0. Crystals were then soaked with 5 mM benzodiazepine in the harvesting buffer for several days. Co-crystallization was also
attempted, but failed as no crystal growth was observed. The crystals
with space group R3 have cell constants a = b = 160.93 Å, c = 36.90 Å and contain
one molecule per asymmetric unit (21, 22). Data were measured on a MAR
image plate system (MAR Research, Hamburg) mounted on a Rigaku rotating
anode generator (
= 1.5418 Å). Data analysis was performed with the
MOSFLM program package (23) and data reduction with the CCP4 program
suite (24). Data statistics are summarized in Table
I, the soaked crystals were isomorphous
to wild-type annexin V. Starting from the annexin V-WT structure,
refinement was initiated with X-PLOR (25), using the conjugate gradient
minimization. A 2Fo
Fc map was
calculated and used for inspection and model building of the
benzodiazepine on a graphics terminal with FRODO (26). Further rounds
of refinement were done to obtain good geometry which was checked with
the program PROCHECK (27). Table II
summarizes the refinement results. The topology of BDA452 was
constructed using an AM1 calculation with MOPAC (28).
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Table I
Rmerge = |I(k) I |/ I(k), where I(k) and
I are the intensity values of individual measurements and of
the corresponding mean values; the summation is over all
measurements
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Annexin III--
Soaking of pre-formed crystals in BDA452
solutions proved impossible and co-crystallization was therefore
attempted. Best crystals were obtained from a solution of 15 mg/ml
protein in 50 mM Tris-HCl buffer, pH 7.5, 20 mM
CaCl2, 2 mM benzodiazepine, and 1 M
(NH4)2SO4 in the drop, in vapor
diffusion against a well containing a double concentration of the
precipitating agent. Data were collected on the DW32 station of the DCI
storage ring at LURE, Orsay, which is equipped with a MAR image plate
system (MAR Research), using a wavelength of 0.97 Å. Data analysis was performed with the program DENZO and SCALEPACK (29, 55) and the data
reduction with the CCP4 program suite (24). The data statistics are
summarized in Table I. Since the data were nonisomorphous with both
wild-type annexin III data, as well as data of annexin III
co-crystallized with inositol phosphate (30), the structure analysis
had to be started using the rigid-body refinement option in X-PLOR
(25). Subsequent refinement was performed with REFMAC from the CCP4
program suite (24), and the solvent molecules were built and inspected
using O (31).
Vesicle Preparation
Phospholipid vesicles were prepared according to Reeves
and Dowben (32) by mixing phosphatidylserine and
phosphatidylethanolamine (Avanti Polar Lipids) at a molar ratio of
3:1 in chloroform (total lipid amount for centrifugation assay: 10 µmol, for all other experiments: 1 µmol). The solution was dried
under a stream of nitrogen for 30 min and then exposed to a stream of
water-saturated nitrogen for another 30 min. Lipids for the
centrifugation assay were covered with a 0.2 M saccharose
solution and the vesicles were allowed to swell overnight at 19 °C.
For the use in fluorescence titration experiments, the lipids were
covered with buffer (180 mM saccharose, 10 mM
HEPES, pH 7.4) and incubated at 37 °C for 2 h. The liposomes
for the calcium influx assay were covered with 2 ml of buffer F1 (100 µM FURA-2, 180 µM EDTA, 162 mM
saccharose, 5 mM HEPES, pH 7.4) and incubated at 37 °C
for 2 h. The vesicles were pelleted by centrifugation at
12,000 × g for 30 min. After resuspension in 200 µl
of buffer F2 (200 µM EDTA, 180 mM saccharose, 10 mM HEPES, pH 7.4) they were centrifugated again,
resuspended in buffer F2 and applied to a S200 spin column (Pharmacia).
After two additional centrifugation steps, the liposome pellet was
finally resuspended in 200 µl of F2. Aliquots of 20 µl were used
for the calcium influx assay. All final collection steps for the
different liposome preparations were done by centrifugation at
12,000 × g for 30 min.
Annexin and Benzodiazepine Binding to Phospholipid
Vesicles
Samples of 500 µl for binding assays contained the appropriate
concentration of the given benzodiazepine, a 20-µl aliquot of
phospholipid vesicles suspended in 5 mM TRIS, pH 7.4, 180 mM saccharose, and 1 mM CaCl2. The
components of the sample were mixed and after 10 min the phospholipid
vesicles were separated by centrifugation at 130,000 × g for 30 min (4 °C). Binding of benzodiazepines to the
vesicles was quantified by measuring the UV absorbance of the
supernatant at 280 nm with a Perkin-Elmer Lambda 17 UV/Vis
spectrophotometer. Control experiments were performed in the absence of
lipid vesicles. For calcium-dependent annexin binding 100 µg of annexin V (6 µM) from a highly concentrated stock
solution were added to the sample containing 1 µmol of lipid suspended in the above mentioned buffer, the appropriate amount of
CaCl2 and 100 µM BDA452. Centrifugation and
measurements followed the same protocol. As a control the annexin
binding assay was repeated in the absence of BDA452.
Calcium Influx Assay
The calcium influx into liposomes was monitored by using the
calcium-sensitive dye FURA-2 (33) and the FURA assay was performed following the protocol described by Berendes et al. (34). To increase the stability of the FURA liposomes, all solutions were saturated with Ar. A 20-µl aliquot of the FURA-loaded liposome suspension was mixed with 475 µl of buffer F2, and 5 µl of a 50 mM CaCl2 solution was added. The fluorescence
intensity was measured at 510 nm with the sample excited at 340 and 380 nm at time intervals of 1 min. After an equilibration time of 4 min the
protein was added from a concentrated stock solution and so was the
benzodiazepine derivative from a
Me2SO2-containing
stock solution. The Me2SO content of the sample did not
exceed 1% of the total sample volume in any experiment. Fluorescence measurements were carried out in 1-min intervals. At t = 36 min, 3 µl of a solution of Br-A23187 (0.1 mg/ml) was added to
determine the maximal possible calcium signal. Intensity measurements
were continued until t = 40 min. Data analysis was
performed by normalizing the fluorescence ratio
F(340 nm)/F(380 nm)
with respect to the maximal possible fluorescence ratio obtained from the values after addition of the ionophore Br-A23187 (36-40 min). The
normalized fluorescence ratio f is plotted versus
time, thereby yielding an influx curve. For further analysis the
slope
of the time interval 15-35 min was used as an activity
parameter ("steady state"). Alternatively, the initial slope
of
the influx curve, starting at t = 4 min, was analyzed.
The percentage of inhibition/activation was calculated using the steady
state slope and the initial slope, respectively, of the influx
experiment in the absence of benzodiazepine. Fluorescence
measurements were performed on a Perkin-Elmer 650-40 fluorescence spectrophotometer with a spectral bandwidth of 5 nm
(excitation slit) and 5 nm (emission slit). The shutter was closed
between the measurements to avoid photobleaching effects.
Fluorescence Titration
Binding of benzodiazepine derivatives to annexin was monitored
by quenching of the protein fluorescence, using a Perkin-Elmer 650-40 fluorescence spectrophotometer. Protein was added to 500 µl of buffer
(5 mM TRIS, 0.01% NaN3, pH 7.4) and the change
in fluorescence intensity was examined as a function of benzodiazepine concentration. The benzodiazepine derivatives were added in 1-µl aliquots from stock solutions (1 mM, 10 mM) in
Me2SO. The data were normalized with respect to protein
fluorescence intensity at an excitation wavelength of 280 nm. A control
experiment was performed recording the
concentration-dependent fluorescence of the
benzodiazepine derivative and, similarly, the protein was titrated with
Me2SO in 1-µl portions to yield the maximal possible fluorescence intensity in each titration step. These binding
experiments were also performed in the presence of PS/PE liposomes
(3:1) following an analogous protocol. The buffer used for the liposome
containing experiments (F3) consists of 180 mM saccharose,
10 mM HEPES, pH 7.4. Assuming a simple complex
formation,
|
(Eq. 1)
|
the fraction of complexed annexin is x([AL])
corresponding to the normalized fluorescence f([AL]) which can be
calculated by Equation 2,
|
(Eq. 2)
|
where F(annexin, Me2SO) is the
fluorescence intensity of annexin in the presence of Me2SO,
F(annexin, ligand) the measured intensity during the
titration and F(ligand) the fluorescence intensity of the
benzodiazepine. Division by F(annexin, 280 nm)
normalizes the experimental values with respect to protein fluorescence
without ligand and Me2SO.
The dissociation constant Kd was determined by
nonlinear least-squares fit of the data to a binding curve with a Hill
coefficient of n = 1.
The quenching of fluorescence intensity was also analyzed in terms of
the Stern-Volmer equation (35),
|
(Eq. 3)
|
where I represents the protein fluorescence intensity
in the presence of the ligand and I0 the
intensity in its absence.
Carboxyfluorescein Leakage Assay
Liposomes for the leakage assay were prepared as described (34),
except that 50 mM carboxyfluorescein (CF; obtained from Sigma, Deisenhofen, Germany) was included into the buffer to monitor leakage (36). Nonencapsulated CF was separated by gel filtration runs
on S200 microspin columns (Pharmacia). Leakage was investigated by
adding aliquots of the benzodiazepine derivatives to the vesicle suspension directly in the cuvette used for fluorescence determination. Excitation was set to 480 nm and the emission was detected at 540 nm.
The results are expressed as,
|
(Eq. 4)
|
where Fi is the initial fluorescence
intensity before adding the protein, F is the fluorescence
reading at different times, and Fe is the final
fluorescence determined after adding Triton X-100 to the liposome
suspension (final concentration 0.1%).
 |
RESULTS |
Crystal Structure--
The crystal form of annexin V used, as well
as annexin III, have the Trp-187 containing loop of domain III exposed
on the surface of the protein. As described previously (37), five
-helices (A to E) form one domain, with the axes of helices A, B, D,
and E almost anti-parallel to each other, whereas the connecting helix C lies approximately perpendicular to them. The four domains (I to
IV) are arranged in a cyclic array with domains I/IV and II/III forming
two modules with pseudo 2-fold symmetry. In the center of the molecule
a prominent pore is created by helices IIA, IIB, IVA, and IVB, lined
with highly conserved charged or polar residues. The calcium-binding
sites are located on the convex side of the protein within a 17-amino
acid sequence called the endonexin-fold (38), which has been shown to
bind to the membrane (39). The N and C termini lie on the opposite,
concave side of the molecule.
The structure of annexin V in complex with the ligand BDA452 reveals
that the ligand is bound in a cavity (Fig.
2) which is located at the interface of
domains II, III, and IV at the concave side of the molecule. The
strongly bent BDA452 molecule (Fig. 3B) interacts with all domains
in that cleft in a hydrophobic manner, sharing a contact surface of
about 373 Å2 with the protein. The most prominent contact
residues are Thr-118 (loop IIB/IIC), Pro-119 (IIC), Glu-120 (IIC),
Arg-161 (loop IIE/IIIA), Asp-164 (loop IIE/IIIA), Val-203 (IIIC),
Arg-207 (IIIC), Ser-243 (IIIE), and Ser-247 (IVA) (Fig. 3A).
Despite a prevalent positive charge of the protein in the binding cleft
no notable polar interactions could be identified and the closest
distance of BDA452 to the protein atoms is above 3.1 Å. The
-imino
group of Arg-207 faces the 5-phenyl group of the ligand. No major
rearrangement of the protein structure is observed in the complex
structure when compared with the structure of ligand-free protein. A
contact of the ligand to the N terminus seems possible but could not be
identified in the structure since residues 1-4 are not defined in the
electron density. From the localization of the ligand on the concave
side we conclude that the membrane binding behavior of annexin V is not
affected directly by binding of BDA452.

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Fig. 2.
Surface representation of the annexin-BDA452
complexes. A, annexin III-BDA452. B, annexin
V-BDA452. The molecular surface of the protein is colored according to
the electrostatic surface potential (red, negative;
blue, positive). The BDA452 ligand is depicted as skeleton.
Figure was prepared with GRASP (51).
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Fig. 3.
A, structure of BDA452 bound to annexin
V. The bound ligand BDA452 is shown in yellow. Residues of
the binding cleft with the shortest distance to the ligand are
highlighted in bright blue. The protein backbone is colored
in dark blue. Figure was prepared with SETOR (52).
B, electron density of the bound ligand BDA452. The electron
density around the bound BDA452 is contoured at 1 cutoff. Whereas
the tryptophan moiety and the aromatic rings are well defined, the
seven-membered ring is only visible partially, thereby suggesting high
flexibility. The figure was prepared with FRODO (25).
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Several attempts were made to solve the crystal structure of annexin V
with other benzodiazepine derivatives analyzed in this study. Although
there is a high electron density peak in all of these structures no
reliable model building was possible for complexes AV-DZM, AV-BDA250,
and AV-BDA753, probably due to substantial disorder. This observation
is supportive of a remarkable flexibility of the benzodiazepine
derivatives even in the protein-bound state. Additionally, the binding
site might only be occupied partially.
The annexin III-BDA452 complex electron density shows a peak of
difference density in the analogous region as for annexin V. This peak
is, however, not sufficiently well defined to give detailed information
on the ligand structure. The best fit of the benzodiazepine obtained
indicates a nonspecific interaction, with the 5-phenyl group of the
ligand facing the side chain of Phe-206, with Arg-164 close by (annexin
III numbering). The indole moiety of BDA452 points toward the N
terminus, which is well defined up to Ser-2. The interaction with
BDA452 provokes slight displacements of the connecting segments between
domains II and III, and domains III and IV which line the binding
cavity.
Binding of Benzodiazepine Derivatives to Phospholipid
Membranes--
Lipid membranes and benzodiazepines may interact
directly as suggested by recent experiments, which show that different
benzodiazepine derivatives insert into lipid bilayers to different
extents (40). Binding of the benzodiazepine BDA452 to PS/PE liposomes
(3:1) was examined by the centrifugation assay. In this assay a
decrease of absorption in the supernatant is observed when compared
with the absorption curve of pure BDA452 (Fig.
4A). At an initial
concentration of 100 µM BDA452 approximately 80% of the
benzodiazepine is bound to the membrane. In the presence of BDA452 the
annexin V binding curve is not significantly affected if compared with
measurements without BDA452 (Fig. 4B).

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Fig. 4.
A, BDA452 is attached to liposomes. A
suspension containing PS/PE liposomes (3:1) (about 1 µmol total lipid
content), 1 mM CaCl2, and the appropriate
amount of BDA452 in 180 mM saccharose, 5 mM
TRIS, pH 7.4, is separated by centrifugation and the UV absorbance of
the supernatant is measured at 280 nm (filled squares).
Above a concentration of 5 µM BDA452, significant
attachment of the benzodiazepine to the liposomes is observed. The
filled circles represent the UV absorbance in the
supernatant in the absence of liposomes. The results shown are the mean
of three independent preparations. B, annexin V binding to
phospholipid membranes is not affected by BDA452. The calcium dependent
binding of 100 µg of annexin V (6 µM) to PS/PE
liposomes (3:1) in the presence (filled triangles) and
absence (filled circles) of 100 µM BDA452 was
measured by a centrifugation assay. Shown on the ordinate is
1 a(280 nm) which represents the ratio
of binding of annexin to liposomes. a(280 nm) is
the absorbance of the supernatant at 280 nm normalized to the pure
annexin V absorbance. The dot-and-dash line was obtained by
considering the UV absorbance of 100 µM BDA452 in the
presence of phospholipids and the appropriate amount of
CaCl2 in the supernatant.
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Binding of Benzodiazepine Derivatives to Annexins--
Fig.
5A shows the titration of 3 µM annexin V with BDA452 (0-280 µM).
Binding of the benzodiazepine to the protein results in substantial
quenching of fluorescence emission intensity of the protein excited at
280 nm. Data analysis according to Equation 2 yields the dissociation
constants Kd, which are summarized in Table I.
Although the data shown in Fig. 5A might suggest a biphasic
interaction of BDA452 with annexin V, a monophasic model was applied to
all binding experiments, since neither the crystallographic results nor
data analysis according to Stern-Volmer relations indicate a biphasic
behavior. To ensure that the fluorescence quenching is due to a
specific interaction of the benzodiazepine derivative with annexin, a
control titration experiment was done with
N-acetyltryptophan-amide at concentrations from 0 to 32 µM. The addition of N-acetyltryptophan-amide
to an annexin solution does not result in any specific fluorescence
quenching within the concentration range tested (Fig. 5B).
Considerable scattering of data is observed in the presence of higher
N-acetyltryptophan-amide concentrations presumably due to
the high intrinsic fluorescence of this derivative. It has to be noted
that reproducible quenching data were only obtained with benzodiazepine
derivatives carrying a fluorophore group. Measuring of binding
parameters was therefore limited to BDA452 and BDA753. As mentioned
above, control titration experiments were also performed with annexins
and Me2SO revealing that the protein fluorescence is
affected by the presence of Me2SO in the sample solution
(Fig. 6A). The quenching
effect by the organic solvent, however, is strongly decreased, if PS/PE
liposomes and 200 µM CaCl2 are present in the
buffer (Fig. 6B). This indicates that the protein is
accessible to the quencher to a much lower extent in the membrane-bound
state. As concluded from the Kd values, BDA452 is
bound by one order of magnitude better than BDA753 for each annexin
tested. This might be due to the steric interference of the
tetrapeptide. Binding of BDA452 to annexins was not affected by the
presence of phospholipid vesicles, whereas binding of BDA753 was much
tighter in the presence as compared with the absence of phospholipid
membranes. We also attempted data analysis according to Stern-Volmer
Equation 3 where the quenching constant Kq is
obtained by fitting the plot of I0/I
versus c(ligand) to a linear equation (Fig.
7). Since Kq
represents an association constant the values are much higher for
BDA452 than for BDA753 (Table III), which
is in agreement with the conclusions drawn from the
Kd determination. Likewise the tendencies in the
binding behavior with and without lipids, respectively, are the same as
indicated by the Kd values. The Stern-Volmer analysis in terms of an association constant works well in the case of
tight binding between annexin and the ligand since one can assume
predominantly static quenching. In the case of weak binding (high
dissociation constants), the Kq values show less
agreement with the Kd values. Presumably, other effects than the formation of a nonfluorescent complex might apply for
the observed quenching behavior.

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Fig. 5.
A, fluorescence quenching of annexin V
upon addition of BDA452. Aliquots of BDA452 (stock solution in
Me2SO) are added to a solution of 52 µg of annexin V (3 µM) in 5 mM TRIS, pH 7.4, 1% i-PrOH,
0.01% NaN3. The ordinate values of
f([AL]) are calculated as described under "Experimental
Procedures" from the fluorescence intensities F at 310 nm
(excitation 280 nm). The solid line was obtained by fitting
a binding equation to the experimental data, yielding
Kd = 26.1 µM. B, the
addition of N-acetyltryptophan-amide does not affect annexin
V fluorescence significantly. N-Acetyltryptophan-amide is
added stepwise to an annexin V solution (3 µM). Buffering
and calculation of the f([AL]) values as in A.
Emission was measured at 310 nm (excitation 280 nm). In the
concentration range tested no significant fluorescence quenching of
annexin V occurs. Measurements at higher
N-acetyltryptophan-amide concentrations are not reliable due
to its high intrinsic fluorescence.
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Fig. 6.
Annexins are accessible to quenchers in
solution but much less in the membrane bound state. Fluorescence
intensities at 340 nm for annexin III (circles) and 310 nm
for annexin V (squares) are measured at an excitation
wavelength of 280 nm in the presence of increasing amounts of
Me2SO. Closed symbols, annexins in solution; measuring buffer, 5 mM TRIS, pH 7.4, 1% i-PrOH,
0.01% NaN3. Open symbols, annexins in the
presence of PS/PE liposomes (3:1) and 200 µM
CaCl2 in buffer F3. Normalization was carried out against the sample without Me2SO.
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Fig. 7.
Stern-Volmer analysis of annexin V
fluorescence quenching with BDA452. Data were obtained from a
fluorescence titration experiment as described under "Experimental
Procedures." Aliquots of BDA452 (stock solution in Me2SO)
are added to a solution of 52 µg of annexin V (3 µM) in
5 mM TRIS, pH 7.4, 1% i-PrOH, 0.01% NaN3. Fluorescence readings were done at 310 nm (excitation
280 nm), I0 is the fluorescence intensity of the
annexin solution without BDA452. The solid line was obtained
by fitting the experimental data to Equation 3, yielding
Kq = 35.6 × 103
M 1.
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Table III
Excitation wavelength: 280 nm, emission was monitored at 310 nm (AI,
AII, and AV-WT) and 340 nm (AIII), respectively
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Effects of Benzodiazepines on the Annexin-induced Calcium Influx
into Lipid Vesicles--
The calcium influx activity of different
annexins and the influence of benzodiazepines was examined using FURA-2
loaded lipid vesicles and recording time dependent excitation spectra
after the addition of annexin and/or benzodiazepine derivatives.
Calcium influx curves were obtained by plotting the normalized
fluorescence ratio f versus time (Fig.
8). A control experiment was performed with DZM to exclude a possible membrane damage by the benzodiazepine derivative itself. Within the concentration range of the
annexin/benzodiazepine experiments no significant membrane
permeabilization was detected. At much higher concentrations (above 300 µM) the benzodiazepine leads to a considerable membrane
damage (Fig. 9A). As shown by different runs of the CF-leakage assay, the benzodiazepine derivatives used in this work do not cause significant membrane damage (Fig. 9,
B and C). Only BDA452 at higher concentrations is
able to increase the CF fluorescence intensity.

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Fig. 8.
Typical calcium influx curve observed with
annexin III and 200 µM CaCl2. An aliquot
of 20 µl of the suspension of FURA-loaded liposomes in a total volume
of 500 µl of buffer F2, including 500 µM
CaCl2 is measured for 5 min. The ratio
F340/F380 is obtained by
reading the fluorescence intensity at 510 nm while exciting at 340 and
380 nm, respectively. Ordinate values are normalized to the highest F340/F380
ratio. At t = 4 min, 6.4 µg of annexin III (0.4 µM) is added. Data acquisition continues until
t = 35 min, where 5 µl of a solution of Br-A23187
(0.1 mg/ml) is used to yield the maximal possible fluorescence.
|
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Fig. 9.
A, DZM does not cause membrane
permeabilization at concentrations below 200 µM. Results
from a calcium influx assay were performed with increasing portions of
DZM. The ordinate values shown are steady state slopes from
each influx experiment. Normalization was carried out against the
steady state slope of 13 µg of annexin V (0.7 µM). Only
at concentrations well above the ones used in the experiments with
annexin DZM causes considerable calcium influx by membrane damage.
B, effects of DZM and BDA452 on PS/PE liposomes (3:1). The
time dependent CF leakage, calculated according to Equation 4, shows
that DZM (circles) does not cause significant membrane
damage, whereas BDA452 (triangles) is able to cause
liposomes leakage at higher concentrations. Filled symbols,
50 µM; open symbols, 250 µM
benzodiazepine. C, BDA250 does not cause membrane leakage of
PS/PE liposomes (3:1). The CF assay was performed as in B.
BDA250 at different concentrations does not effect CF-loaded liposomes significantly. Filled circles, 50 µM; open circles, 250 µM;
filled triangles, 500 µM.
|
|
The effect of different benzodiazepine derivatives on different
annexins is not uniform (Table IV).
BDA452 was found to inhibit calcium fluxes induced by annexins AV,
AV-N1, AVIa, and AVIb, whereas with annexins AI, AIII, and AVI rates of
calcium fluxes are increased upon addition of the benzodiazepine
(Fig. 10). A similar effect was
observed with the N terminally truncated mutants AV-N3 and AV-N4.
Consequently, the question arose, whether the contact of the ligand
with the N-terminal region of the protein is essential and sufficient
for the macroscopic effect in the calcium influx assay. However, no
general correlation between the length of the N-terminal domain of
annexin V and the mode of effect of the benzodiazepine is observed.
While the truncated mutants AV-N1, AV-N3, and AV-N4 are differently
affected in the calcium influx assay albeit being unable to contact the
ligand, AIII and AI both have a longer N terminus than AV but are
activated by addition of BDA452. Similarly, AVI, a two-monomer annexin, is activated upon BDA452 addition. Annexin II shows a more complex behavior. Analysis of the steady state slope revealed a clearly inhibitory effect of BDA452 on this annexin, whereas the initial slope
indicates an activation (Fig.
11A).
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Table IV
The symbols indicate the following: , activating; ,
inhibiting; , indifferent
/ 0 indicates the half-maximal concentration according to
steady state slope. / 0 indicates the half-maximal
concentration obtained by analyzing the initial slope; concentrations
in µM.
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Fig. 10.
Activation of annexin VI-induced calcium
influx by BDA452. The steady state slope (closed
circles, solid line) and the initial slope (open
triangles, dashed line) are plotted against the concentration of
BDA452 used in the FURA assay with annexin VI. Both parameters indicate
the strong activation of annexin VI due to BDA452. The curves follow a
saturation equation.
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Fig. 11.
A, contradictory effects of BDA452 on
annexin II. Data analysis of BDA452-dependent calcium
influx assays with 21 µg of annexin II (1 µM). Whereas
the steady state slope (filled circles) indicates an
inactivation of annexin II with increasing amounts of BDA452, the
initial slopes (open squares) point to an activation. In
terms of the total amount of calcium crossing the phospholipid membrane annexin II is not influenced by BDA452. Normalization was done against
the influx activity of 1 µM annexin II without BDA452. B and C, BDA250 and DZM display almost opposite
effects. Whereas DZM enhances the annexin-induced calcium influx (dose
dependent), BDA250 has no effect on the annexin V-induced membrane
permeabilization. The membrane function of annexin III is even
inhibited. B, annexin III; C, annexin V. Filled circles, BDA250; open squares, DZM.
|
|
To complete the general view on annexin-induced calcium influx we used
the smaller, commercially available benzodiazepine derivative DZM
(diazepam). Annexins AIII, AV, and AV-N3 are activated upon addition of
this derivative, whereas AI is not affected. It is very surprising in
this respect that the addition of BDA250 instead of DZM has an almost
opposite effect on the annexin behavior in the FURA assay, most likely
due to the missing 7-chloro substituent in BDA250. Annexin V is not
influenced by BDA250, whereas AI and AIII are inhibited (Fig. 11,
B and C).
 |
DISCUSSION |
Annexin V is known to display an ion channel-type activity under
certain conditions (37, 41, 42). Other annexins were also found to
cause cation fluxes through artificial membranes. On the other hand,
high annexin concentration leads to formation of two-dimensional
crystals on the membrane surface (39), a state that is
conduction-incompetent. Many parameters may influence the surface
concentration of annexin, e.g. the calcium concentration (43, 44), transmembrane potential (45), membrane composition (46)
etc.
The molecular mechanisms of annexin function on membrane surfaces are
not completely understood. There are several data supporting a membrane
stabilization by annexins at high concentrations, like the
two-dimensional crystal formation (EM), membrane rigidification (NMR),
and increase of seal resistance (patch clamp) (40, 45, 47,
49).3 A particular
interesting effect on membranes is the permeabilization elicited by
annexins, since these proteins do not insert into the bilayer.
Influx Mechanism--
We need to distinguish between three
different effects: (i) the interaction of the benzodiazepines with the
lipid membrane, (ii) the interaction of annexins with the membrane, and
(iii) the interaction of the annexin-BDA complex with the membrane in the presence of excess benzodiazepine. It is known from previous work (40) that benzodiazepines insert into lipid membranes to varying extents depending on their particular structure. The binding assay conducted in the present work indicates that about 80% of the
total benzodiazepine amount is attached to lipid vesicles at an initial
concentration of 100 µM. Additionally, it has to be taken
into account that the total lipid amount in the binding assay is by a
factor of 10 higher than in the calcium influx assay.
Hence, an alteration of membrane properties upon binding of
benzodiazepines has to be considered as well. The control experiments (FURA assay with DZM and the CF-leakage experiments) performed in
this work revealed that DZM and BAD452 cause membrane destabilization only at very high concentrations. To explain the results of our calcium
influx assays we propose a conformational change of the protein upon
binding of the ligand to a hydrophobic cleft at the interface of
domains II, III, and IV on the concave side of the annexin molecule as
shown in the crystal structure. Such binding may affect the
inter-module angle and the flexibility in different ways depending on
the annexin and the benzodiazepine derivative leading to activation or
inhibition of calcium influx.
Inhibition Mechanism--
Among the benzodiazepine derivatives
analyzed in this study, BDA452 inhibits calcium influx activity of
annexins AV, AV-N1, and AVIb, and BDA250 annexins I and III. Taking
into account the N-terminal sequences of annexins, BDA452 might be able
to contact the N terminus of AV and AVIb both of which have an alanine
in neighboring positions at the N terminus. This argument, on the other
hand, is not true for annexin V-N1 where residues 1 to 6 are missing.
Additionally, the contact is only possible with BDA452 because of its
tryptophan moiety being exposed to the protein exterior. It is
therefore surprising that BDA250 also displays inhibitory effects on AI
and AIII. These annexins, however, contain a longer N-terminal region
than AV and might be able to contact the ligand at other sites.
Moreover, both of them have a conserved serine (Ser-27; Fig.
12).

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Fig. 12.
Sequence alignment of annexins mentioned in
this study. Shown are only those sections which are near to the
binding site of BDA452 in annexin V. Conserved residues are highlighted in different blue colors, residues within the hydrophobic
cleft are rendered in yellow. The alignment was done using
the program PileUp of the GCG software package (54). The figure was
prepared with ALSCRIPT (48).
|
|
Crystal Structures--
The BDA452 ligand bound to annexin III
appears to have a somewhat different conformation when compared with
the annexin V-BDA452 structure (Fig.
13), although it is impossible to make
detailed comparisons due to the poor density of the ligand bound to
annexin III. The orientation of the ligand is similar in both proteins, while its shape is more open in annexin III. In both structures the
tryptophan moiety is pointing toward the N-terminal region of the
protein, but the length and conformation of the N termini is different
in the different annexins studied. The N terminus seems to define the
space within which the ligand can bind: thus Val-4 (annexin V) prevents
the ligand from a closer contact with domain I, which is accomplished
in annexin III, and the ligand is slightly rotated. On the other hand,
the change in position of domains II and III in annexin III as compared
with annexin V seems to be designed to ensure their similar
contacts with the ligand as in annexin V. Thus, the side chain
of Arg-164 (annexin III) maintains a similar contact with the
7-position of the benzodiazepine ligand in both structures. This
side chain could be important as a possible sensor for substitutions in
the 7-position of these ligands, since it is conserved throughout
nearly all annexins (except annexin VII). Surprisingly, the binding
pocket in both proteins is not as hydrophobic as one would expect (Fig.
2) but is positively charged which might be attractive for the
polarizable seven-membered ring of the benzodiazepine
derivative.

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Fig. 13.
Superposition of annexin III-BDA452 and
annexin V-BDA452. The difference in the relative position of the
third domain and the N termini of annexins III and V with BDA452 bound
to them. The backbone of annexin III is in red, with a
"ball-and-stick" representation of BDA452 in green,
while annexin V backbone is in blue, and the BDA452 bound to
it in yellow. Figure prepared with MOLSCRIPT (53).
|
|
Binding of Benzodiazepine Derivatives to Annexins--
Generally,
the Kd values of the annexin-benzodiazepine
complexes are by 1 order of magnitude higher than the concentration needed for affecting the calcium influx assay. This aspect is very
important with respect to the results from the influx assay being based
on initial benzodiazepine concentrations. Since these agents attach to
the lipid membrane, the free concentration in solution is lower than
the initial concentration. However, we did not correct the assay
concentrations for this adsorption effect. This means that a
significantly lower amount of benzodiazepine is required for
half-maximal binding than one would conclude from the binding
parameters determined in the above mentioned assays.
For annexin II very weak binding of BDA452 is observed. This could
explain the varying effect of BDA452 on the calcium influx activity of
annexin II and leads to the conclusion that this annexin is only
affected weakly by addition of the benzodiazepine derivative. Generally, the concentrations of benzodiazepines needed for binding to
annexins are rather high. Hence, any therapeutic effect of these drugs
on annexins are questionable.
BDA250/DZM--
Although no binding parameters could be determined
for the small benzodiazepine derivatives, significant effects in the
calcium influx assay can be achieved by varying the substitution at the benzodiazepine core. Exchange of the hydrogen at position 7 against a
chloro-substituent changes the indifferent effect of BDA250 on annexin
V into a considerable activation of the annexin-induced calcium influx,
with DZM being by one order of magnitude more potent than BDA250. The
behavior of annexin I and III is also significantly changed when
comparing these two agents, indicating that the energetic balance
between the conformers responsible for activation and inhibition of
influx is delicately tuned.
The sequence alignment of several annexins reveals that among the
residues in the hydrophobic binding cleft positions 118, 161, and 243 (annexin V numbering) are especially well conserved (Fig. 12). There
is, however, variance at other amino acid positions nearby. A dominant
sensor function of one of the residues remains to be elucidated.
Conclusion--
In the present work we examined the in
vitro interaction of annexins with benzodiazepine derivatives.
These interesting pharmacological agents are well known as
tranquilizing substances and some of them are also used in the therapy
of anxiety. Their ability of agonizing/antagonizing cholecystokinin-A
and -B receptor emphasizes their increasing importance. The first
interaction of similar agents with annexins was described by Kaneko
et al. (15). Recently, Liu et al. (50) reported
various effects of phenothiazines on aggregation and fusion of
liposomes where the heterotetramer [AIIp11]2 appears to
be strongly involved. We now show that annexins are able to bind
1,4-benzodiazepines with considerable affinities, thereby representing
putative benzodiazepine receptors. Whereas rather high concentrations
are needed for binding to annexins, the modulation of
annexin-induced membrane permeabilization requires a much lower amount
of benzodiazepines. The physiological role of these interactions
remains to be further investigated, but undoubtly must be taken into
account when considering pharmacology of these extremely potent
drugs.
 |
ACKNOWLEDGEMENTS |
We are grateful to Lissy Weyher for
skillful advice on fluorescence spectroscopy and Dr. Stefan Steinbacher
and Dr. Andreas Bergner for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the Fonds der Chemischen
Industrie (to A. H.) and European Council Grant ERBBIO4CT960083
(Biotechnology) (to the groups of V. Gerke, R. Huber, A. Lewit-Bentley,
and F. Russo-Marie).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: Max-Planck-Institut
für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany. Tel.: 49-89-8578-2830; Fax:
49-89-8578-3516.
1
J. Benz, unpublished results.
2
The abbreviations used are: Me2SO,
dimethyl sulfoxide; PE, phosphatidylethanolamine; PS,
phosphatidylserine; CF, carboxyfluorescein; DZM, diazepam.
3
P. Demange, personal communication.
 |
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