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• W2092049962 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). 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). Benzodiazepines are well known pharmaceuticals used in the short time therapy of insomnia and stress induced anxiety (2Mutschler E. Mutschler E. Arzneimittelwirkungen-Lehrbuch der Pharmakologie und Toxikologie. 5th Ed. Wissenschaftliche Verlagsgesellschaft, Stuttgart1986: 145-148Google Scholar, 3Forth W. Forth W. Henschler D. Rummel W. Starke K. Allgemeine und spezielle Pharmakologie und Toxikologie. 7th Ed. Spektrum Akademischer Verlag, Heidelberg1996: 301-307Google Scholar). 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 (4Hommer D.W. Skolnick P. Paul S.M. Meltzer H.Y. Pharamcology: The Third Generation of Progress. Raven, New York1987: 977-983Google Scholar, 5Rang H.P. Dale M.M. Pharmacology 2nd Ed. Churchill Livingstone, Edinburgh1991: 634-635Google Scholar). 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 (6Eghball M. Curmi J.P. Birnir B. Gage P.W. Nature. 1997; 388: 71-74Crossref PubMed Scopus (136) Google Scholar). Benzodiazepine-related compounds are one of the most important classes of bioavailable therapeutic agents with widespread biological activities including anxiolytic, anticonvulsant, and antihypnotic activities (7Sternbach L.H. J. Med. Chem. 1979; 22: 1-7Crossref PubMed Scopus (397) Google Scholar), cholecystokinin receptor A and receptor B antagonists (8Bock M.G. Dipardo R.M. Evans B.E. Rittle K.E. Whitter W.L. Veber D.E. Anderson P.S. Freidinger R.M. J. Med. Chem. 1989; 32: 13-16Crossref PubMed Scopus (345) Google Scholar), opioid receptor ligands (9Romer D. Buscher H.H. Hill R.C. Maurer R. Petcher T.J. Zeugner H. Benson W. Finner E. Milkowski W. Thies P.W. Nature. 1982; 298: 759-760Crossref PubMed Scopus (182) Google Scholar), platelet-activating factor antagonists (10Kornecki E. Ehrlich Y.H. Lenox R.H. Science. 1984; 226: 1454Crossref PubMed Scopus (183) Google Scholar), human immunodeficiency virus trans-activator Tat antagonists (11Hsu M.C. Schutt A.D. Hooly M. Slice L.W. Sherman M.I. Richman D.D. Potash M.J. Volsky D.J. Science. 1991; 254: 1799-1802Crossref PubMed Scopus (225) Google Scholar), GPIIbIIIa inhibitors (12Bondinell, 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.Google Scholar), reverse transcriptase inhibitors (13Pauwels 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. Janssen P.A.J. Nature. 1990; 343: 470-474Crossref PubMed Scopus (737) Google Scholar), and Ras farnesyltransferase inhibitors (14James 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. Marsters Jr., J.C. Science. 1993; 260: 1937-1942Crossref PubMed Scopus (607) Google Scholar). 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 actinin vitro (15Kaneko N. Drug Dev. Res. 1994; 33: 429-438Crossref Scopus (95) Google Scholar). Its effect on annexin-induced calcium influx has also been studied and its binding site defined (1Kaneko N. Ago H. Matsuda R. Inagaki E. Miyano M. J. Mol. Biol. 1997; 274: 16-20Crossref PubMed Scopus (60) Google Scholar). 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. Porcine annexin I was purified from bacteria expressing the recombinant protein (16Seemann J. Weber K. Osborn M. Partan L.G. Gerke V. Mol. Biol. Cell. 1996; 7: 1359-1374Crossref PubMed Scopus (63) Google Scholar). Recombinant human annexin II containing a N-terminal elongation of six residues (MRGSFK) was purified from the appropriately transformed bacteria as described (17Thiel C. Weber K. Gerke V. J. Biol. Chem. 1991; 266: 14732-14739Abstract Full Text PDF PubMed Google Scholar). Human annexin III (18Favier-Perron B. Lewit-Bentley A. Russo-Marie F. Biochemistry. 1996; 35: 1740-1744Crossref PubMed Scopus (66) Google Scholar), human annexin V (19Burger A. Berendes R. Voges D. Huber R. Demange P. FEBS Lett. 1993; 329: 25-28Crossref PubMed Scopus (67) Google Scholar), and human annexin VI, VIa, and VIb (20Benz J. Bergner A. Hofmann A. Demange P. Göttig P. Liemann S. Huber R. Voges D. J. Mol. Biol. 1996; 260: 638-643Crossref PubMed Scopus (91) Google Scholar) were purified as described. The N-terminal deletion mutants AV-N1 (Δ1–6), AV-N3 (Δ1–13), and AV-N4 (Δ1–14) were made by introducing mutations in the annexin V wild-type cDNA, 1J. Benz, unpublished results. 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). Rhombohedral annexin V wild-type crystals were grown by vapor diffusion at room temperature against 1 mmCaCl2, 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 (21Huber R. Römisch J. Paques E.P. EMBO J. 1990; 9: 3867-3874Crossref PubMed Scopus (355) Google Scholar, 22Huber R. Berendes R. Burger A. Schneider M. Karshikov A. Leucke H. Römisch J. Paques E. J. Mol. Biol. 1992; 223: 683-704Crossref PubMed Scopus (236) Google Scholar). Data were measured on a MAR image plate system (MAR Research, Hamburg) mounted on a Rigaku rotating anode generator (λ = 1.5418 Å). Data analysis was performed with the MOSFLM program package (23Leslie A.G.W. Mosflm, Version 5.20. MRC Laboratory of Molecular Biology, Cambridge, UK1994Google Scholar) and data reduction with the CCP4 program suite (24CCP4 Acta Cryst. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). 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 (25Brünger A.T. X-PLOR, A System for X-ray Crystallography and NMR, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar), 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 (26Jones T.A. J. Appl. Cryst. 1978; 15: 24-31Google Scholar). Further rounds of refinement were done to obtain good geometry which was checked with the program PROCHECK (27Laskowski, 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.Google Scholar). Table IIsummarizes the refinement results. The topology of BDA452 was constructed using an AM1 calculation with MOPAC (28Dewar M.J.S. Zoebisch E.G. Healy E.F. Stewart J.P. J. Am. Chem. Soc. 1985; 107: 3902Crossref Scopus (15086) Google Scholar).Table IRmerge = Σ‖I(k) − 〈I〉‖/ΣI(k), where I(k) and 〈I〉 are the intensity values of individual measurements and of the corresponding mean values; the summation is over all measurementsAnnexin III-BDA452Annexin V-BDA452Space groupP21R3Cell constants (Å)a = 42.69, b = 69.47, c = 51.25, β = 95.51°a = 160.93, b = 160.93, c = 36.90Maximal resolution2.3 Å2.9 ÅReflections measured5404735093Independent reflections82647534Completeness99.0% (∞ −2.3 Å)95.4% (∞ −2.9 Å)Rmerge11.2%12.7% Open table in a new tab Table IIR = Σ∥Fo‖−‖Fc∥/Σ‖F0‖, where Fo and Fc are the observed and the calculated structure factors, respectivelyAnnexin III-BDA452Annexin V-BDA452Refinement Resolution20.0–2.3 Å10.0–3.0 Å Number of reflections (Fo> 2ςFo)All reflections6451 Number of non-hydrogen atoms27422515 R-factor0.2000.201Temperature factors Average B factor26.1 Å217.0 Å2 Root mean square deviation of B factors for bonded atoms2.2 Å25.9 Å2Geometry root mean square deviations Root mean square deviation of bond lengths0.013 Å0.013 Å Root mean square deviation from planarity0.006 Å0.009 Å Root mean square deviation of bond angles2.6 °2.4 °Ramachandran plot Residues in most favored regions91.8%80.7% Residues in additional allowed regions8.2%18.6%Solvent statistics Number of water molecules267 Number of calcium ions7 Open table in a new tab 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 mmCaCl2, 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 (29Otwinovski, Z., Proceedings of the CCP4 Study Weekend “Data Collection and Processing”, Sawyer, L., Isaacs, N., Bailey, S., 1993, SERC Daresbury Laboratory, UK.Google Scholar, 55Minor W. XDISPLAYF Programme. Purdue University, 1993Google Scholar) and the data reduction with the CCP4 program suite (24CCP4 Acta Cryst. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). 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 (30Perron B. Lewit-Bentley A. Geny B. Russo-Marie F. J. Biol. Chem. 1997; 272: 11321-11326Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), the structure analysis had to be started using the rigid-body refinement option in X-PLOR (25Brünger A.T. X-PLOR, A System for X-ray Crystallography and NMR, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar). Subsequent refinement was performed with REFMAC from the CCP4 program suite (24CCP4 Acta Cryst. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar), and the solvent molecules were built and inspected using O (31Jones T.A. Zou J.Y. Cowan S.W. Kjelgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Phospholipid vesicles were prepared according to Reeves and Dowben (32Reeves J.P. Dowben R.M. J. Cell Physiol. 1968; 73: 49-60Crossref Scopus (504) Google Scholar) 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 mmHEPES, 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 mmsaccharose, 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. 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. The calcium influx into liposomes was monitored by using the calcium-sensitive dye FURA-2 (33Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) and the FURA assay was performed following the protocol described by Berendes et al. (34Berendes R. Burger A. Voges D. Demange P. Huber R. FEBS Lett. 1993; 317: 131-134Crossref PubMed Scopus (51) Google Scholar). 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 Me2SO 2The abbreviations used are: Me2SO, dimethyl sulfoxide; PE, phosphatidylethanolamine; PS, phosphatidylserine; CF, carboxyfluorescein; DZM, diazepam. -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 ratioF(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 versustime, thereby yielding an influx curve. For further analysis the slope α of the time interval 15–35 min was used as an activity parameter (“steady state”). Alternatively, the initial slope β 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. 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, Annexin+ligand⇌[annexin­ligand]Equation 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])=f0(A)−f(A)=F(annexin,Me2SO)F(annexin,280nm)−F(annexin,ligand)−F(ligand)F(annexin,280nm)Equation 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 (35Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 257-301Crossref Google Scholar), I0I=F(annexin,Me2SO)F(annexin,ligand)−F(ligand)=1+Kq∗c(ligand)Equation 3 where I represents the protein fluorescence intensity in the presence of the ligand and I0 the intensity in its absence. Liposomes for the leakage assay were prepared as described (34Berendes R. Burger A. Voges D. Demange P. Huber R. FEBS Lett. 1993; 317: 131-134Crossref PubMed Scopus (51) Google Scholar), except that 50 mm carboxyfluorescein (CF; obtained from Sigma, Deisenhofen, Germany) was included into the buffer to monitor leakage (36Wilschut J. Düzgünes N. Fraley R. Papahadjopoulos D. Biochemistry. 1980; 19: 6011-6021Crossref PubMed Scopus (434) Google Scholar). 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, CF−leakage[%]=100∗F−FiFe−FiEquation 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%). 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 (37Berendes R. Voges D. Demange P. Huber R. Burger A. Science. 1993; 262: 427-430Crossref PubMed Scopus (99) Google Scholar), five α-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 (38Geisow M.J. Fritsche U. Hexham J.M. Dash B. Johnson T. Nature. 1986; 320: 636-638Crossref PubMed Scopus (229) Google Scholar), which has been shown to bind to the membrane (39Voges D. Berendes R. Burger A. Demange P. Baumeister W. Huber R. J. Mol. Biol. 1994; 238: 199-213Crossref PubMed Scopus (162) Google Scholar). 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. 3 B) 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. 3 A). 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 ε-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.Figure 3A, 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 (52Evans S.V. J. Mol. Graphics. 1993; 11: 134-138Crossref PubMed Scopus (1249) Google Scholar). B, electron density of the bound ligand BDA452. The electron density around the bound BDA452 is contoured at 1ς 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 (25Brünger A.T. X-PLOR, A System for X-ray Crystallography and NMR, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar).View Large Image Figure ViewerDownload (PPT) 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. 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 (40Garcia D.A. Perillo M.A. Biochim. Biophys. Acta. 1997; 1324: 76-84Crossref PubMed Scopus (29) Google Scholar). 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. 4 A). 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. 4 B). Fig. 5 A 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. 5 A 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 withN-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. 5 B). Considerable scattering of data is observed in the presence of higherN-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 prese" @default.
• W2092049962 created "2016-06-24" @default.
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• W2092049962 date "1998-01-01" @default.
• W2092049962 modified "2023-09-26" @default.
• W2092049962 title "Interactions of Benzodiazepine Derivatives with Annexins" @default.
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