Interactions of Benzodiazepine Derivatives with Annexins*

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 273, No. 5, Issue of January 30, pp. 2885–2894, 1998 Printed in U.S.A.

Interactions of Benzodiazepine Derivatives with Annexins* (Received for publication, October 15, 1997, and in revised form, November 13, 1997)

Andreas Hofmann‡§, Achim Escherich¶, Anita Lewit-Bentleyi, Jo¨rg Benz‡, Ce´line Raguenes-Nicol**, Francoise Russo-Marie**, Volker Gerke‡‡, Luis Moroder¶, and Robert Huber‡ From the ‡Max-Planck-Institut fu¨r Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, the ¶Max-Planck-Institut fu¨r Biochemie, Abt. Bioorganische Chemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany, iLURE, Baˆtiment 209D, Centre Universitaire Paris-Sud, F-91405 Orsay, France, **ICGM, U332, INSERM, 22 rue Mećhain, F-75014 Paris, France, and the ‡‡Institut fu¨r Medizinische Biochemie, Universita¨t Mu¨nster, D-48149-Mu¨nster, Germany

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).

nels 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), plateletactivating 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

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 GABAbinding 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 chan-

Porcine annexin I was purified from bacteria expressing the recombinant protein (16). Recombinant human annexin II containing a Nterminal 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 (D1– 6), AV-N3 (D1–13), and AV-N4 (D1–14) 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).

X-ray Structure Determination * 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. This 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 fu¨r Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany. Tel.: 49-89-8578-2830; Fax: 49-89-8578-3516. This paper is available on line at http://www.jbc.org

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 5 b 5 160.93 Å, c 5 36.90 Å and contain one molecule per asymmetric unit (21, 22). Data

2885

1

J. Benz, unpublished results.

2886

Annexins and Benzodiazepines TABLE I Rmerge 5 SuI(k) 2 Î&u/SI(k), where I(k) and Î& are the intensity values of individual measurements and of the corresponding mean values; the summation is over all measurements Annexin III-BDA452

Annexin V-BDA452

Space group Cell constants (Å)

P21 a 5 42.69, b 5 69.47, c 5 51.25, b 5 95.51° Maximal resolution 2.3 Å Reflections measured 54047 Independent reflections 8264 Completeness 99.0% (` 22.3 Å) Rmerge 11.2%

R3 a 5 160.93, b 5 160.93, c 5 36.90 2.9 Å 35093 7534 95.4% (` 22.9 Å) 12.7%

TABLE II R 5 SiFou2uFci/SuF0u, where Fo and Fc are the observed and the calculated structure factors, respectively

FIG. 1. Structures of substances mentioned. BDA452, 3-(R,S)-(Ltryptophanyl)-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine2-one. BDA753, 3-(R,S)-all-L-(NH-Trp-Gly-Tyr-Ala-H)-1,3-dihydro-1methyl-5-phenyl-2H-1,4-benzodiazepine-2-one. BDA250, 1,3-dihydro-1methyl-5-phenyl-2H-1,4-benzodiazepine-2-one. DZM, 7-chlor-1,3dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one (diazepam). TRP, (1)-N-acetyl-L-tryptophan-amide. K201, 4-(3-(1-(4-benzyl)piperidinyl)propionyl)-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (14). were measured on a MAR image plate system (MAR Research, Hamburg) mounted on a Rigaku rotating anode generator (l 5 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 2 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). 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 mmol, for all other experiments: 1 mmol). 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 over-

Refinement Resolution Number of reflections (Fo . 2sFo) Number of non-hydrogen atoms R-factor Temperature factors Average B factor Root mean square deviation of B factors for bonded atoms Geometry root mean square deviations Root mean square deviation of bond lengths Root mean square deviation from planarity Root mean square deviation of bond angles Ramachandran plot Residues in most favored regions Residues in additional allowed regions Solvent statistics Number of water molecules Number of calcium ions

Annexin III-BDA452

Annexin V-BDA452

20.0–2.3 Å All reflections 2742 0.200

10.0–3.0 Å 6451 2515 0.201

26.1 Å2 2.2 Å2

17.0 Å2 5.9 Å2

0.013 Å

0.013 Å

0.006 Å

0.009 Å

2.6 °

2.4 °

91.8% 8.2%

80.7% 18.6%

267 7

night 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 mM FURA-2, 180 mM 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 3 g for 30 min. After resuspension in 200 ml of buffer F2 (200 mM 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 ml of F2. Aliquots of 20 ml were used for the calcium influx assay. All final collection steps for the different liposome preparations were done by centrifugation at 12,000 3 g for 30 min.

Annexin and Benzodiazepine Binding to Phospholipid Vesicles Samples of 500 ml for binding assays contained the appropriate concentration of the given benzodiazepine, a 20-ml 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 3 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 mg of annexin V (6 mM) from a highly concentrated stock solution were added to the sample containing 1 mmol of lipid suspended in the above mentioned buffer, the appropriate amount of CaCl2 and 100 mM 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

Annexins and Benzodiazepines

2887

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-ml aliquot of the FURA-loaded liposome suspension was mixed with 475 ml of buffer F2, and 5 ml 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 Me2SO2containing 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 5 36 min, 3 ml 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 5 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 a of the time interval 15–35 min was used as an activity parameter (“steady state”). Alternatively, the initial slope b of the influx curve, starting at t 5 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 ml 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-ml 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-ml 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, Annexin 1 ligand º @annexin-ligand#

(Eq. 1)

the fraction of complexed annexin is x([AL]) corresponding to the normalized fluorescence f([AL]) which can be calculated by Equation 2, f~@AL#! 5 f0~A! 2 f~A! 5

F~annexin,Me2SO! F~annexin,280 nm! 2

F~annexin,ligand! 2 F~ligand! (Eq. 2) F~annexin,280 nm!

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 leastsquares fit of the data to a binding curve with a Hill coefficient of n 5 1. The quenching of fluorescence intensity was also analyzed in terms of the Stern-Volmer equation (35), I0 F~annexin,Me2SO! 5 5 1 1 Kqpc~ligand! I F~annexin,ligand! 2 F~ligand!

(Eq. 3)

where I represents the protein fluorescence intensity in the presence of the ligand and I0 the intensity in its absence.

2 The abbreviations used are: Me2SO, dimethyl sulfoxide; PE, phosphatidylethanolamine; PS, phosphatidylserine; CF, carboxyfluorescein; DZM, diazepam.

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).

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, F 2 Fi CF 2 leakage@%# 5 100p Fe 2 Fi

(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 a-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 calciumbinding sites are located on the convex side of the protein within a 17-amino acid sequence called the endonexin-

2888


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 1s 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).

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 e-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.

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


FIG. 4. A, BDA452 is attached to liposomes. A suspension containing PS/PE liposomes (3:1) (about 1 mmol 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 mM 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 mg of annexin V (6 mM) to PS/PE liposomes (3:1) in the presence (filled triangles) and absence (filled circles) of 100 mM BDA452 was measured by a centrifugation assay. Shown on the ordinate is 1 2 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 mM BDA452 in the presence of phospholipids and the appropriate amount of CaCl2 in the supernatant.

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 mM 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). Binding of Benzodiazepine Derivatives to Annexins—Fig. 5A shows the titration of 3 mM annexin V with BDA452 (0 –280 mM). 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 SternVolmer relations indicate a biphasic behavior. To ensure that the fluorescence quenching is due to a specific interaction of the

2889

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 mg of annexin V (3 mM) 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 5 26.1 mM. 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 mM). 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.

benzodiazepine derivative with annexin, a control titration experiment was done with N-acetyltryptophan-amide at concentrations from 0 to 32 mM. The addition of N-acetyltryptophanamide 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 mM 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

2890


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 SternVolmer 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. 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 mM) 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. 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 AVN1, 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). 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).

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 mM CaCl2 in buffer F3. Normalization was carried out against the sample without Me2SO.

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 mg of annexin V (3 mM) 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 5 35.6 3 103 M21.

TABLE III Excitation wavelength: 280 nm, emission was monitored at 310 nm (AI, AII, and AV-WT) and 340 nm (AIII), respectively BDA452 Kd in mM

AI AII AIII AV-WT a b

65.6 932 34.8 26.1

NA, not analyzable. ND, not determined.

BDA452 with liposomes

Kq in 103

51.8 21.5 18.3 35.6

M

21

Kd in mM

4.23 NDb 89.2 27.0

Kq in 103

434 ND 18.5 14.2

M

BDA753 21

Kd in mM a

NA ND 147 378

BDA753 with liposomes

Kq in 103

NA ND 10.1 16.6

M

21

Kd in mM

1.00 ND 43.0 53.0

Kq in 103

222 ND 40.5 26.0

21 M


2891

FIG. 8. Typical calcium influx curve observed with annexin III and 200 mM CaCl2. An aliquot of 20 ml of the suspension of FURAloaded liposomes in a total volume of 500 ml of buffer F2, including 500 mM 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 5 4 min, 6.4 mg of annexin III (0.4 mM) is added. Data acquisition continues until t 5 35 min, where 5 ml of a solution of BrA23187 (0.1 mg/ml) is used to yield the maximal possible fluorescence. 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 mM. 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 3

P. Demange, personal communication.

FIG. 9. A, DZM does not cause membrane permeabilization at concentrations below 200 mM. 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 mg of annexin V (0.7 mM). 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 mM; open symbols, 250 mM 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 mM; open circles, 250 mM; filled triangles, 500 mM.

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

2892


TABLE IV The symbols indicate the following: a, activating; s, inhibiting; f, indifferent a/a0 indicates the half-maximal concentration according to steady state slope. b/b0 indicates the half-maximal concentration obtained by analyzing the initial slope; concentrations in mM. BDA452

a/a0

AII AV-WT AV-Nl AVIa AVIb AI AIII AV-N3 AV-N4 AVI a b

f s s s s a a a a a

s 0.5 a NDa ND 6.2 ND b NAb ND ND 46

BDA250

b/b0

a/a0 b/b0

DZM

a/a0 b/b0

BDA753

a/a0 b/b0

a 3.8 f ND ND a 57 44 ND ND 3.8 ND s ND ND f ND ND 5.3 s ND ND a ND ND a ND ND ND a ND ND a ND ND ND 33

ND, not determined. NA, not analyzable.

FIG. 10. Activation of annexin VI-induced calcium influx by BDA452. The steady state slope a (closed circles, solid line) and the initial slope b (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.

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). 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

FIG. 11. A, contradictory effects of BDA452 on annexin II. Data analysis of BDA452-dependent calcium influx assays with 21 mg of annexin II (1 mM). 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 mM 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.

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 benzodia-


2893

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).

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).

zepine 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. Binding of Benzodiazepine Derivatives to Annexins—Generally, the Kd values of the annexin-benzodiazepine complexes

2894


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. Acknowledgments—We are grateful to Lissy Weyher for skillful advice on fluorescence spectroscopy and Dr. Stefan Steinbacher and Dr. Andreas Bergner for helpful discussions. REFERENCES 1. Kaneko, N., Ago, H., Matsuda, R., Inagaki, E., and Miyano, M. (1997) J. Mol. Biol. 274, 16 –20 2. Mutschler, E. (1986) in Arzneimittelwirkungen-Lehrbuch der Pharmakologie und Toxikologie (Mutschler, E., ed) 5th Ed., pp. 145–148, Wissenschaftliche Verlagsgesellschaft, Stuttgart 3. Forth, W. (1996) in Allgemeine und spezielle Pharmakologie und Toxikologie (Forth, W., Henschler, D., Rummel, W., and Starke, K., eds) 7th Ed., pp. 301–307, Spektrum Akademischer Verlag, Heidelberg 4. Hommer, D. W., Skolnick, P. and Paul, S. M. (1987) in Pharamcology: The

5. 6. 7. 8. 9.

10. 11. 12.

13.

14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. 29.

30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.

Third Generation of Progress (Meltzer, H. Y., ed) pp. 977–983, Raven, New York Rang, H. P., and Dale, M. M. (1991) Pharmacology 2nd Ed., pp. 634 – 635, Churchill Livingstone, Edinburgh Eghball, M., Curmi, J. P., Birnir, B., and Gage, P. W. (1997) Nature 388, 71–74 Sternbach, L. H. (1979) J. Med. Chem. 22, 1–7 Bock, M. G., Dipardo, R. M., Evans, B. E., Rittle, K. E., Whitter, W. L., Veber, D. E., Anderson, P. S., and Freidinger, R. M. (1989) J. Med. Chem. 32, 13–16 Romer, D., Buscher, H. H., Hill, R. C., Maurer, R., Petcher, T. J., Zeugner, H., Benson, W., Finner, E., Milkowski, W., and Thies, P. W. (1982) Nature 298, 759 –760 Kornecki, E., Ehrlich, Y. H., and Lenox, R. H. (1984) Science 226, 1454 Hsu, M. C., Schutt, A. D., Hooly, M., Slice, L. W., Sherman, M. I., Richman, D. D., Potash, M. J., and Volsky, D. J. (1991) Science 254, 1799 –1802 Bondinell, W. E., Callahan, J. F., Huffman, W. F., Keenan, R. M., Ku, T. W. F., and Newlander, K. A. (1993) International Patent Application, WO 93/00095 Pauwels, R., Andries, K., Desmyter, J., Schols, D., Kukla, M. J., Breslin, H. J., Raeymaekers, A., Van Gelder, J., Woestenborghs, R., Heykants, J., Schellekens, K., Janssen, M. A. C., Clercq, E. D., and Janssen, P. A. J. (1990) Nature 343, 470 – 474 James, G. L., Goldstein, J. L., Brown, M. S., Rawson, T. E., Somers, T. C., McDowell, R. S., Crowley, C. W., Lucas, B. K., Levinson, A. D., and Marsters, J. C., Jr. (1993) Science 260, 1937–1942 Kaneko, N. (1994) Drug Dev. Res. 33, 429 – 438 Seemann, J., Weber, K., Osborn, M., Partan, L. G., and Gerke, V. (1996) Mol. Biol. Cell 7, 1359 –1374 Thiel, C., Weber, K., and Gerke, V. (1991) J. Biol. Chem. 266, 14732–14739 Favier-Perron, B., Lewit-Bentley, A., and Russo-Marie, F. (1996) Biochemistry 35, 1740 –1744 Burger, A., Berendes, R., Voges, D., Huber, R., and Demange, P. (1993) FEBS Lett. 329, 25–28 Benz, J., Bergner, A., Hofmann, A., Demange, P., Go¨ttig, P., Liemann, S., Huber, R., and Voges, D. (1996) J. Mol. Biol. 260, 638 – 643 Huber, R., Ro¨misch, J., and Paques, E. P. (1990) EMBO J. 9, 3867–3874 Huber, R., Berendes, R., Burger, A., Schneider, M., Karshikov, A., Leucke, H., Ro¨misch, J., and Paques, E. (1992) J. Mol. Biol. 223, 683–704 Leslie, A. G. W. (1994) Mosflm, Version 5.20, MRC Laboratory of Molecular Biology, Cambridge, UK CCP4 (1994) Acta Cryst. Sect. D 50, 760 –763 Bru¨nger, A. T. (1992) X-PLOR, A System for X-ray Crystallography and NMR, Version 3.1, Yale University Press, New Haven, CT Jones, T. A. (1978) J. Appl. Cryst. 15, 24 –31 Laskowski, R. A., MacArthur, M. W., Smith, D. K., Jones, D. T., Hutchison, E. G., Morris, A. L., Naylor, D., Moss, D. S., and Thornton, J. (1994) PROCHECK, Programs to Check the Stereochemical Quality of Protein Structures, Version 3.0 Dewar, M. J. S., Zoebisch, E. G., Healy, E. F., and Stewart, J. P. (1985) J. Am. Chem. Soc. 107, 3902 Otwinovski, Z. (1993) in Proceedings of the CCP4 Study Weekend “Data Collection and Processing” (Sawyer, L., Isaacs, N., and Bailey, S., eds) SERC Daresbury Laboratory, UK Perron, B., Lewit-Bentley, A., Geny, B., and Russo-Marie, F. (1997) J. Biol. Chem. 272, 11321–11326 Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjelgaard, M. (1991) Acta Crystallogr. Sec. A. 47, 110 –119 Reeves, J. P., and Dowben, R. M. (1968) J. Cell Physiol. 73, 49 – 60 Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440 –3450 Berendes, R., Burger, A., Voges, D., Demange, P., and Huber, R. (1993) FEBS Lett. 317, 131–134 Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, pp. 257–301, Plenum Press, New York Wilschut, J., Du¨zgu¨nes, N., Fraley, R., and Papahadjopoulos, D. (1980) Biochemistry 19, 6011– 6021 Berendes, R., Voges, D., Demange, P., Huber, R., and Burger, A. (1993) Science 262, 427– 430 Geisow, M. J., Fritsche, U., Hexham, J. M., Dash, B., and Johnson, T. (1986) Nature 320, 636 – 638 Voges, D., Berendes, R., Burger, A., Demange, P., Baumeister, W., and Huber, R. (1994) J. Mol. Biol. 238, 199 –213 Garcia, D. A., and Perillo, M. A. (1997) Biochim. Biophys. Acta 1324, 76 – 84 Arispe, N., Rojas, E., Genge, B. R., Wu, L. N. Y., and Wuthier, R. E. (1996) Biophys. J. 71, 1764 –1775 Rojas, E., Pollard, H. B., Haigler, H. T., Parra, C., and Burns, A. L. (1990) J. Biol. Chem. 265, 21207–21215 Meers, P., Daleke, D., Hong, K., and Papahadjopoulos, D. (1991) Biochemistry 30, 2903–2908 Plager, D. A., and Nelsestuen, G. L. (1994) Biochemistry 33, 13239 –13249 Hofmann, A. (1997) Biochim. Biophys. Acta 1330, 254 –264 Meers, P. (1996) in Annexins: Molecular Structure to Cellular Function (Seaton, B. A., ed) pp. 97–119, Springer Verlag, Heidelberg Brisson, A., Mosser, G., and Huber, R. (1991) J. Mol. Biol. 220, 199 –203 Barton, G. J. (1993) Protein Eng. 6, 37– 40 Swairjo, M. A., Roberts, M. F., Campos, M. B., Dedman, J. R., and Seaton, B. A. (1994) Biochemistry 33, 10944 –10950 Liu, L., Tao, J. Q., and Zimmerman, U. J. P. (1997) FASEB J. 11, 3227 Nicholls, A., Bharadwaj, R., and Honig, B. (1993) Biophys. J. 64, A166 Evans, S. V. (1993) J. Mol. Graphics 11, 134 –138 Kraulis, P. J. (1991) J. Appl. Cryst. 24, 946 –950 Genetics Computer Group (1996) Wisconsin Package, Version 9.0, Genetics Computer Group Inc., Madison, WI Minor, W. (1993) XDISPLAYF Programme, Purdue University