DOI: 10.1021/cg901046f
Crystallization of the Membrane-Associated Annexin B1: Roles of Additive Screen, Dynamic Light Scattering, and Bioactivity Assay )
2010, Vol. 10 2528–2532
Fei-Xiang Ding,†,# Ye-Fen Xu,‡,# Arezki Azzi,§, Dao-Wei Zhu,§ Peter Rehse,‡ Chang-Qin Chen,‡ Shu-Han Sun,*,† and Sheng-Xiang Lin*,‡,§ †
)
Department of Medical Genetics, the Second Military Medical University, Xiangyin Road 800 Shanghai 200433, P. R. China, ‡Laboratory of Structural Biology with Visiting Scientists, Institute of Biochemistry and Cell Biology (IBCB), Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Yueyang Road 320 Shanghai 200031, P. R. China, and §Laboratory of Molecular Endocrinology and Oncology, Centre Hospitalier de Universit e Laval Research Center (CHUL, CHUQ) and Laval University, Qu ebec, G1V 4G2, Canada. #These authors contribute equally to this work. F.X.D. and Y.F.X. carried out the protein purification and crystallization in ICBC, SIBS. Present address: Al-Imam Mohammed Ibn Saud Islamic University, College of Medicine, Riyadh, 11578, Saudi Arabia. Received August 27, 2009; Revised Manuscript Received March 31, 2010
ABSTRACT: Annexin B1 (AnxB1) is a calcium-dependent phospholipid binding protein from Taenia solium cysticercus and has been reported to possess anticoagulant activity, to inhibit phospholipase A2, and to regulate membrane transport. Native AnxB1 and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. The results of dynamic light scattering analysis showed that Hepes buffer combined with low concentration salts (NaCl or CaCl2) was beneficial for preventing aggregation and for AnxB1 stabilization in the storage. After the additive screen, crystals have been yielded in the presence of guanidine hydrochloride (Gn-HCl). We determined that a low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity. Analysis of the crystal showed that in the presence of Gn-HCl, AnxB1 crystallizes in orthorhombic space group, which is modified from the cubic space group for crystals grown in the absence of Gn-HCl. A high quality data set (at 1.9 A˚) has been collected successfully for crystals of L-selenomethionine labeled protein in the presence of Gn-HCl, to solve the structure with the single anomalous dispersion method (SAD). The unit cell parameters are a = 102.35 A˚, b = 103.59 A˚, c = 114.60 A˚, R = β = γ = 90.00°.
Introduction Annexins are widely distributed calcium-dependent phospholipid-binding proteins in higher and lower eukaryotes, involved with numerous cellular processes including membrane fusion, ion channel activity, and heterocomplex formation with other proteins.1 Annexin B1 (AnxB1), a member of the annexin B subfamily, was discovered in Taenia solium cysticercus which triggers a form of verminosis called Taenia solium cysticercosis. AnxB1 contains 347 amino acids and shares 35% amino acid identity with hydra annexin B12 and human annexin A5. Similar to other annexins, AnxB1 is composed of four homologous repeats of about 70 amino acids and possesses all the highly conserved residues which are involved in salt bridge interactions. Compared with annexins A5 and B12, annexin B1 includes the slightly longer N-terminal domain (30 amino acids), as well as a connector region of 30 amino acids between domains II and III.1 Similar to other annexins, AnxB1 can bind to acidic phospholipid membranes in the presence of Ca2þ, which is facilitated by Ca2þ coordination within the AB and DE loop areas.2,3 Experiments in vivo and in vitro have demonstrated that AnxB1 protein had a high anticoagulation activity and a better affinity to the platelets than annexin A5.4-7 To study the three-dimensional structure, function, evolution, and application of annexins, we attempted to crystallize *Corresponding authors: (S.-H.S.) Tel: þ86-21-81871053. Fax: þ86-2181871053. E-mail:
[email protected]. (S.-X.L.) Tel: þ86-21-54921277. Fax: þ86-21-54921084. E-mail:
[email protected]. pubs.acs.org/crystal
Published on Web 05/04/2010
the membrane-associated AnxB1 supported by different screen approaches that are reported below. Experimental Section Expression and Purification of Native AnxB1. Recombinant AnxB1 was expressed in BL21 (DE3) bacterial cells using gene cloned into the expression vector pJLA503. Cells were grown in 2 YT medium with 100 μg/mL ampicillin at 30 °C and induced at 42 °C for a further 5 h when the OD600 reached 0.4-0.6. Cells were harvested by centrifugation and lysed by sonification in buffer A [20 mM Tris-HCl pH 8.5, 1 mM 1, 4-dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride (PMSF)]. The lysate was centrifuged and the supernatant was loaded onto a Q-Sepharose fast flow column and eluted with a 100-500 mM linear NaCl gradient in buffer A. Peak fractions were pooled and loaded onto a PhenylSepharose fast flow column, the flow-through contained AnxB1 was pooled, loaded on an Superdex-200 size-exclusion column and eluted with buffer B (20 mM Hepes-NaOH pH 7.5, 1 mM DTT). Peak fractions were pooled, concentrated, and stored at -80 °C until needed. The purity of the protein was assessed by SDS-PAGE and native-PAGE. Production of L-Selenomethionine-Labeled Annexin B1 (Sel-Met AnxBl). For expression of the selenomethionine (Sel-Met) derivative of the protein (Sel-Met AnxB1), the recombinant plasmid was transformed into BL834 (DE3). A small scale overnight culture was grown in M9 minimal media [6.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 mM MgSO4, 1 g/L NH4Cl, 0.1 mM CaCl2, 0.3% (v/v) glucose, 10 mL/L GIBCO MEM vitamin solution (Fisher Scientific)] supplemented with 50 μg/mL of ampicillin and 50 μg/mL of L-methionine, and used to inoculate the full scale culture. The culture was grown to an OD600 of 0.8 at 30 °C, harvested by centrifugation, washed twice, and resuspended in M9 minimal media supplemented r 2010 American Chemical Society
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Figure 1. Purification of native and Sel-Met AnxB1. (A) The purity of native and Sel-Met AnxB1 was detected by SDS-PAGE (15%), which showed a single band with a molecular mass of 38 kDa. Lane 1, native AnxB1; lane 2, Sel-Met AnxB1. (B) The homogeneity of native and Sel-Met AnxB1 was analyzed by native PAGE. Lane 3, native AnxB1; lane 4, Sel-Met AnxB1. (C) Mass spectrometry of native AnxB1. (D) Mass spectrometry of Sel-Met AnxB1.
Figure 2. Crystallization of AnxB1. (A) Needle-shaped crystals obtained from initial screening. (B) Clusters of needle-shaped crystals in Tris buffer. (C) The rod-shaped single crystals in Hepes buffer. (D) Plate-shaped crystals optimized by adding CaCl2. (E) Cube-shaped crystals optimized by varying pH and concentrations of NaCl, CaCl2, and PEG. (F) A high quality large single crystal obtained in the presence of Gn-HCl after additive screening. with 50 μg/mL of ampicillin. Cells were grown at 30 °C for 1 h to exhaust any remaining L-methionine in the media and then L-selenomethionine was added. Cells were incubated for 30 min before the culture was induced by heat shock at 42 °C for 5 h. Sel-Met AnxBl were purified as described above. The level of selenomethionine incorporation was regulated by the addition of unlabeled methionine to the growth media and monitored by mass spectrometry. Crystallization. Crystallization of native and Sel-Met AnxB1 was initially carried out with Crystal Screens I and II (Hampton Research, California, USA) at different temperatures. For crystallization, the protein was concentrated to 30 mg/mL. Drops containing equal volumes (1 μL) of protein sample and well solution were used to initiate hanging drop vapor-diffusion trials. Further screenings to find optimal crystallization conditions were performed by varying the temperature, protein concentrations, buffer, pH, salts, and additives.8,9 Dynamic Light Scattering Analysis. Dynamic light scattering analysis (DLS) measurements were performed at 277 K on a Wyatt Technologies DynaPro Titan dynamic light-scattering instrument
after filtration of the protein sample through a 0.02 μm filter, followed by injection into a 12 μL quartz cuvette, using a protein concentration of approximately 10 mg/mL. DLS data were collected and analyzed using DYNAMICS V6 software for the DynaPro Titan instrument (Wyatt Technology Corporation). Anticoagulant Bioactivity of AnxBl under Gn-HCl. Anticoagulant activity of AnxB1 was measured using modified kaolin partial thromboplastin time (KPTT) as previously described.10 Aliquots of 100 μL standard human plasma were mixed with 10 μL of protein sample containing different concentrations of Gn-HCl and 100 μL of kaolin active thrombofax. After incubation for 2 min at 37 °C, 100 mL of 25 mM CaCl2 was added. Fibrin formation was detected using a RackRotor Thrombolyzer (Behnk Elektronik, Norderstedt, Germany).
Results and Discussion Purification of Native and Sel-Met AnxB1. Typical yields were about 50 mg of native and Sel-Met AnxB1 purified by
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Figure 3. Intensity (line) and radius (circles) analyzed by DLS in different buffers. (A) Protein sample in 100 mM Tris-HCl pH 8.5, 1 mM DTT; (B) protein sample in 100 mM Hepes-NaOH pH 7.1, 150 mM NaCl, 1 mM DTT.
three chromatographic steps from 1 L culture, and the preparations were homogeneous as revealed by SDSPAGE. Analysis of AnxB1 by native gel and mass spectrometry confirmed that it consisted of a highly homogeneous polypeptide chain (Figure 1). AnxB1 contains seven methionine residues, and the mass spectrum of Sel-Met AnxB1 (Figure 1C,D) showed that this protein used for crystallization consisted of an 82% overall replacement of methionine sulfur by selenium. Crystallization. In the initial screening, the protein sample was in Tris buffer (20 mM Tris-HCl pH 8.5, 1 mM DTT). Small needle-shaped crystals (Figure 2A) were obtained after several days in Sparse Matrix Screening condition 1 (2 M NaCl, 10% (w/v) PEG 6000) at 277 K. No crystals were obtained in such screen at 295 K. Attempts to optimize condition No. 1 by buffer addition produced a cluster of needles (Figure 2B) in 2 M NaCl, 0.1 M Tris-HCl pH 8.5, 10% (w/v) PEG 6000 and single rod-shaped crystals (Figure 2C) in another condition [2 M NaCl, 0.1 M HepesNaOH pH 7.5, 10% (w/v) PEG 6000]. Streak-seeding with these needles did not improve the size of the crystals. Thus,
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the protein solution was exchanged into Hepes buffer and condition 1 was optimized further by adding CaCl2, and single plate crystals were yielded (Figure 2D) in 2 M NaCl, 0.1 M Hepes-NaOH pH 7.5, 10% (w/v) PEG 6000, 0.2 M CaCl2. When varying pH and concentration of NaCl, CaCl2, and PEG, cubic crystals (Figure 2E) were obtained in 0.1 M Hepes-NaOH pH 7.1, 1.6 M NaCl, 0.27 M CaCl2, and 8% (w/v) PEG 6000 and they reached 0.5 0.3 0.3 mm. However, the crystals (diffracted to 3.1 A˚) needed further improvement for data collection. Thus, we attempted to identify an additive that would improve the crystal quality. Large diffraction-quality crystals were obtained in the presence of 0.06 M Gn-HCl in the well. The crystals grew to maximum dimensions of 0.8 0.5 0.3 mm (Figure 2F), and they were transferred to a solution containing the reservoir solution and 40% (v/v) glycerol before being freshly frozen in liquid nitrogen for data collection, yielding 1.8 A˚ complete data set at NSLS (National Synchroteon Light Source, Brookhaven National Lab, NY, USA). Dynamic Light Scattering Analysis. The results of DLS experiments showed that the protein in different conditions was mainly present in a monomeric form, and the change of buffers or addition of salts could transform minor oligomers to monomers (Figure 4). The sample in buffer A (100 mM Tris-HCl pH 8.5, 1 mM DTT) showed more aggregation than that in buffer B, and as time went on, an increase of a hydrodynamic radius of particles was observed (Figures 3A and 4A). The sample in the buffer B (100 mM Hepes-NaOH pH 7.1, 1 mM DTT) showed little aggregation which did not increase over time (Figure 4B), suggesting the beneficial role of Hepes buffer to stabilize the protein sample and avoid the aggregation. The oligomeric proteins can be dissociated into monomers after addition of NaCl (Figures 3B and 4C,D,E), and these figures better illustrated how the size distribution changes from bimodal, broader peak, to nonomodal, narrower peak, during the increase in NaCl concentration. Therefore, NaCl was not only the main precipitant in the crystallization condition, but also showed an effect to reduce aggregation. Similar to NaCl, CaCl2 was efficient in preventing aggregation (Figure 4F). As we all know, AnxB1 is a Ca2þ-binding protein. CaCl2 not only provides screening of electrostatic interactions between the protein molecules but may also involve specific interaction with AnxB1. Since Gn-HCl is a chaotropic agent, the stability of protein was also analyzed by DLS in the presence and absence of Gn-HCl. No difference was observed between the two samples (Figure 4G,H). These results showed that Hepes buffer at pH 7.1 combined with low concentration salts (NaCl or CaCl2) was beneficial for preventing aggregation and for a good storage of AnxB1, coincident with the improved crystallization condition. Anticoagulant Bioactivity of AnxBl under Gn-HCl. GnHCl is widely used to study the folding and stability of proteins. It is reported that Gn-HCl (at low concentration) binding to specific sites of protein can influence the overall conformation or activity of proteins and metal ion-protein interaction.11 The above crystallization experiment of AnxBl showed that Gn-HCl at low concentrations could improve the crystal quality. In order to examine whether Gn-HCl had any effect on anticoagulant bioactivity of AnxBl, prolongation of clotting time activity induced by AnxBl in the presence of Gn-HCl was assayed using KPTT (Figure 5). Low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity which reached a
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Figure 4. DLS analyses of AnxB1 in different buffers. (A) Protein sample in 100 mM Tris-HCl pH 8.5, 1 mM DTT; (B) protein sample in 100 mM Hepes-NaOH pH 7.1, 1 mM DTT; (C) protein sample in 100 mM Hepes-NaOH pH 7.1, 10 mM NaCl; (D) protein sample in 100 mM Hepes-NaOH pH 7.1, 20 mM NaCl; (E) protein sample in 100 mM Hepes-NaOH pH 7.1, 150 mM NaCl; (F) protein sample in 100 mM HepesNaOH pH 7.1, 270 mM CaCl2; (G) protein sample in 100 mM Hepes-NaOH pH 7.1, 1.6 M NaCl, 270 mM CaCl2; (H) protein sample in 100 mM Hepes-NaOH pH 7.1, 1.6 M NaCl, 270 mM CaCl2, 60 mM Gn-HCl.
maximum value at 60 mM Gn-HCl. This activity, however, was not significantly increased at high Gn-HCl concentration. The result showed that Gn-HCl at an appropriate concentration can increase the bioactivity of AnxBl. Analysis of AnxB1 Crystal Forms. Diffraction data of the native AnxB1 crystals were collected on an R-AXIS IVþ2 detector using a Rigaku FR-E SuperBright X-ray generator with a wavelength of 1.541 A˚ at the Institute of Biophysics, Chinese Academy of Science.12,13 The highest amino acid sequence similarity between the AnxB1 and other annexins (such as annexin A5, A6, and A8) is 35%, and attempts of phase determination by the molecular replacement method
were unsuccessful. We thus collected data sets with crystals with Sel-Met AnxB1 in order to carry out phase determination with SAD. The Sel-Met crystal data sets were collected at NSLS. The diffraction data was processed and scaled with Denzo/HKL, shown in Table 1. Structure determination of the AnxB1 crystal grown in the presence of Gn-HCl complex is underway. AnxB1 from Taenia solium cysticercus crystallizes in cubic space group P213 in the absence of Gn-HCl, while AnxB1 (selenomethionine-labeled) in the presence of Gn-HCl crystallized in orthorhombic space group P212121 with three molecules in the asymmetric unit. The latter crystals yielded
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Acknowledgment. We are grateful to other members of our group for assistance, Prof. Jian-Ping Ding for using the support in dynamic light scattering analysis in his laboratory, and Dr. Mausumi Mazundar for editing the manuscript. This investigation was supported by SIBS Structural Biology Project (to S.-X.L.). This study was funded by the national major special projects (No. 2009ZX09102-238) and 863 projects (No. 2009AA02Z113) (to S.-H.S.) for new drug creations of China. Cloning, expression, and bioactivity studies of AnxB1 were completed in S.-H.S.’s laboratory.
References
Figure 5. KPTT analysis of annexin B1 under different concentrations of Gn-HCl. Low concentration of Gn-HCl significantly increased anticoagulant activity which reached a maximum value at 60 mM Gn-HCl. But high concentration showed no significant change in anticoagulant activity. Data are expressed as the mean ( SD of five individual experiments. Table 1. Data Collection Statistics native AnxB1 in the absence of Gn-HCl
Sel-Met AnxB1 in the presence of Gn-HCl
space group unit cell parameters (A˚)
P213 a = b = c = 107.20, R = β = γ = 90°
wavelength (A˚) detector resolution (A˚)a Rmergeb unique reflections completeness (%)c redundancy I/σ mosaicity (°)
1.541 R-AXIS IVþ2 3.1 (3.11-3.00) 0.119 (0.478) 8480 100 (100) 15.2 12 0.37
P212121 a = 102.35, b = 103.59, c = 114.60, R = β = γ = 90° 0.979 CCD ADSC 1.9 (1.93-1.90) 0.08 (0.402) 104067 99 (98) 10.27 22.2 0.98
crystal
a Data in the highest-resolution shell are shown in parentheses. Rmerge = ΣhΣi/Ihi - /Σ, where Ihi is the intensity of the ith observation of reflection h, and is the average intensity of redundant measurements of the h reflections. c Data determined using three images separated by one degree oscillation. b
a high quality data set at 1.9 A˚ for structure determination. The KPTT assay showed that low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity. This may indicate that guanidine causes a conformational change of AnxB1, at least on its surface for packing interactions, coincident with crystal space group modification from cubic to orthorhombic. Phasing using selenomethionine SAD data has been successful and has produced good-quality electron-density maps. Model building is currently in progress.
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