Slow luminescence kinetics of semi-synthetic aequorin: expression, purification and structure determination of cf3-aequorin

Slow luminescence kinetics of semi-synthetic aequorin: expression, purification and structure... Abstract cf3-Aequorin is one of the semi-synthetic aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3-CTZ) and the histidine-tagged apoaequorin expressed in Escherichia coli cells. The purified cf3-aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native aequorin, and its luminescence capacity was estimated to be 72.8% of that of native aequorin. The crystal structure of cf3-aequorin was determined at 2.15 Å resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p-hydroxy moiety at the C6-phenyl group. In native aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3-aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3-CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3-aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3-aequorin. calcium-binding protein, EF-hand motif, photoprotein, van der Waals interaction, water molecules Aequorin is a Ca2+-binding photoprotein that was isolated from the luminous jellyfish Aequorea victoria and emits light by an intramolecular reaction upon binding with Ca2+ (1). The primary structure of aequorin consists of 189 amino acid residues (21.4 kDa) with three EF-hand motifs characteristic of a Ca2+-binding site (2, 3). Crystal structure analysis of recombinant native aequorin (Protein Data Bank [PDB] ID: 1EJ3) revealed that aequorin is a complex of apoaequorin (an apoprotein) and noncovalently bound 2-peroxycoelenterazine [CTZ-OOH; molecular oxygen-activated coelenterazine (CTZ)] and has three EF-hand motifs with a helix–loop–helix structure (4, 5). Upon the addition of Ca2+ to aequorin, CTZ-OOH stabilized in the hydrophobic core structure of apoaequorin decomposes into coelenteramide and CO2 to produce blue light (λmax = ∼470 nm) (Fig. 1A) (6–9). After the Ca2+-triggered luminescence reaction, apoaequorin can be regenerated to active aequorin by incubation with CTZ under reducing conditions in the presence of a calcium-chelating reagent (Fig. 1A) (10–12). Furthermore, semi-synthetic aequorin was prepared by replacing CTZ with a chemically synthesized CTZ analog, and more than 50 kinds of semi-synthetic aequorins have been characterized (13–17). Among the semi-synthetic aequorins with C2-modified CTZ analogs, the crystal structures of i-, br- and n-aequorin have been determined (PDB ID: 1UHH, 1UHJ and 1UHK, respectively). The structures of these semi-synthetic aequorins were almost identical to native aequorin (Fig. 2), but they showed luminescence kinetics differing from that of native aequorin (5). Fig. 1 View largeDownload slide Bioluminescence reaction of aequorin triggered with Ca2+ and regeneration to aequorin from blue fluorescent protein (BFP) with coelenterazine (A) and the chemical structure of C2-modified coelenterazine analogs for semi-synthetic aequorins (B). The Ca2+-binding sites of three EF-hands are shown as I, III, and IV, respectively. CTZ, coelenterazine; CTZ-OOH, 2-peroxycoelenterazine; EDTA, ethylenediaminetetraacetic acid. Fig. 1 View largeDownload slide Bioluminescence reaction of aequorin triggered with Ca2+ and regeneration to aequorin from blue fluorescent protein (BFP) with coelenterazine (A) and the chemical structure of C2-modified coelenterazine analogs for semi-synthetic aequorins (B). The Ca2+-binding sites of three EF-hands are shown as I, III, and IV, respectively. CTZ, coelenterazine; CTZ-OOH, 2-peroxycoelenterazine; EDTA, ethylenediaminetetraacetic acid. Fig. 2 View largeDownload slide Primary structure of native aequorin from Aequorea victoria. The loop regions of EF-hands I, III, and IV are labelled with [I], [III], and [IV], respectively. The helices are shown as cylinders with italic letters A–H, and the β-sheets are shown as arrows. The conserved amino acid residues of His16, Tyr82, Trp86, Ile105, Tyr132, Thr166, His169, and Tyr184 for interacting with 2-peroxycoelenterazine among various Ca2+-binding proteins are labelled. The C-terminal proline residue (Pro189), which interacts with Arg15 at helix A, is boxed. Fig. 2 View largeDownload slide Primary structure of native aequorin from Aequorea victoria. The loop regions of EF-hands I, III, and IV are labelled with [I], [III], and [IV], respectively. The helices are shown as cylinders with italic letters A–H, and the β-sheets are shown as arrows. The conserved amino acid residues of His16, Tyr82, Trp86, Ile105, Tyr132, Thr166, His169, and Tyr184 for interacting with 2-peroxycoelenterazine among various Ca2+-binding proteins are labelled. The C-terminal proline residue (Pro189), which interacts with Arg15 at helix A, is boxed. Previously, we reported a C2-modified CTZ analog (Fig. 1B), cf3-coelenterazine (cf3-CTZ) (16), which is more stable in aqueous solutions than CTZ and h-coelenterazine (h-CTZ) (17). Semi-synthetic aequorin with cf3-CTZ (cf3-aequorin) showed slow decay of luminescence pattern with a half-decay time of ∼6 s which is slower than that of native aequorin and h-aequorin with ∼0.8 s and ∼0.7 s, respectively (16, 17). In the cell-based G-protein-coupled receptor (GPCR) assay using Chinese hamster ovary K1 (CHO-K1) cells expressing apoaequorin, h-aequorin has generally been used as a Ca2+ indicator because of the high sensitivity to Ca2+. However, in some cases, it is difficult to determine the accurate value of half maximal effective concentration (EC50) using h-aequorin with fast luminescence kinetics. In contrast, cf3-aequorin regenerated in CHO-K1 cells showed lower coefficient of variation values of luminescence intensity than h-aequorin. Thus, the slow reaction kinetics of cf3-aequorin makes it more advantageous in an aequorin-based GPCR assay than h-aequorin (16). To understand the slow luminescence kinetics of cf3-aequorin with Ca2+, we determined the crystal structure of cf3-aequorin and discussed the relationships between its protein structure and luminescence properties by comparison to that of native aequorin. Materials and Methods Materials The following materials were obtained from commercial sources: cf3-coelenterazine (cf3-CTZ), coelenterazine (CTZ), and recombinant aequorin (JNC Co., Tokyo, Japan); 2-mercaptoethanol, imidazole, dithiothreitol (DTT), ethylenediaminetetraacetic acid disodium salt (EDTA·2Na), and imidazole (Wako Pure Chemicals, Osaka, Japan); Q-Sepharose Fast Flow and Butyl-Sepharose 4 Fast Flow (GE Healthcare, Piscataway, NJ, USA); Disform CE475 (NOF Co., Tokyo, Japan); and bovine serum albumin (BSA; Sigma, St. Louis, MO, USA). Expression and purification of recombinant cf3-aequorin from E. coli cells To express histidine-tagged apoaequorin in the periplasmic space of E. coli, the bacterial strain WA802 carrying an expression vector, piP-His-HE (9), was used. A seed culture of E. coli cells was grown in 10 ml of Luria–Bertani (LB) broth containing ampicillin (50 μg/ml) at 25°C for 18 h and then transferred into 400 ml of LB broth containing 50 μl of antifoam (Disform CE475) in a 2-L Sakaguchi flask and cultured with reciprocal shaking (130 rpm) at 37°C for 18 h. After harvesting the cells from 2 L of the culture medium, the cells were suspended in 100 ml of 50 mM Tris-HCl (pH 7.6) and disrupted by sonication for 15 min (3 min × 5) in an ice–water bath using a Branson (Danbury, CT, USA) model 250 sonifier. The soluble fractions obtained by centrifugation at 12,000 g for 20 min were applied on a nickel chelate column (ø2.5 × 5.5 cm) equilibrated with 50 mM Tris-HCl (pH 7.6). After washing with 300 ml of 50 mM Tris-HCl (pH 7.6), the proteins adsorbed on a gel were eluted with 100 mM imidazole in 50 mM Tris-HCl (pH 7.6). From 2 L of cultured cells, 390 mg of proteins with over 90% purity was obtained, added to a final concentration of 5 mM DTT, and stored at −80°C. To purify cf3-aequorin, the partially purified histidine-tagged apoaequorin was regenerated to cf3-aequorin by incubation with cf3-CTZ and applied to a Q-Sepharose column, followed by a Butyl-Sepharose column, as previously described in the purification of recombinant native aequorin (11, 12). The regeneration mixture contained histidine-tagged apoaequorin (138 mg protein), cf3-CTZ (2 mg dissolved in 2 ml of ethanol), and DTT (20 mg) in 250 ml of 50 mM Tris-HCl (pH 7.6)–10 mM EDTA (TE). After incubating at 4°C for 12 h, the regenerated mixture was applied on a Q-Sepharose Fast Flow column (ø2.5 × 7 cm) equilibrated with TE and washed with 200 ml of 0.1 M NaCl in TE, followed by eluting with 0.4 M NaCl in TE. The eluted fraction (30 ml, 84.5 mg protein) was adjusted immediately to a final concentration of 2 M (NH4)2SO4 in the total volume of 50 ml of TE and applied on a Butyl-Sepharose 4 Fast Flow column (ø1.5 × 7.5 cm) equilibrated with 2 M (NH4)2SO4 in TE. After washing with 50 ml of 2 M (NH4)2SO4 in TE, cf3-aequorin (47.6 mg protein) was eluted with 1.2 M (NH4)2SO4 in TE. The purified cf3-aequorin was over 95% purity and stored at −80°C. All column chromatography was performed at 23°C–25°C. Protein analysis Protein concentration was determined by the dye-binding method using a commercially available kit (Bio-Rad, Richmond, CA, USA) and BSA as a standard (Pierce, Rockford, IL, USA). Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis was performed under reducing conditions using a 12% separation gel (TEFCO, Tokyo, Japan), and the gels were stained with a colloidal CBB staining kit (TEFCO). Determination of luminescence activity and luminescence pattern of cf3-aequorin In the steps for purifying histidine-tagged apoaequorin, the luminescence activity was determined by regeneration to native aequorin with 1 µg of CTZ (1 µg/µl in ethanol) in 1 ml of 30 mM TE containing 1 µl of 2-mercaptoethanol at 4°C for 2 h. For the preparation of cf3-aequorin, cf3-CTZ was used for the assay instead of CTZ. The maximum intensities of luminescence (Imax) of aequorin and cf3-aequorin were determined by injection with 100 µl of 50 mM CaCl2 in 50 mM Tris-HCl (pH 7.6) using an ATTO (Tokyo, Japan) model AB2200 luminometer in 0.1-s intervals for 10 s. The luminescence patterns of purified aequorin and cf3-aequorin were determined by injection of 100 µl of 50 mM CaCl2 solution in 50 mM Tris-HCl (pH 7.6) into 3 µl of the protein solution (0.3 ng protein) dissolved in 50 mM Tris-HCl (pH 7.6) containing 0.01 mM EDTA, 0.1% BSA, and 150 mM NaCl using a luminometer Centro LB960 (Berthold, Bad Wildbad, Germany) for 30 s in 0.1-s intervals. The luminescence activity was determined as the mean value (n = 6). Measurements of absorbance, bioluminescence emission and fluorescence emission spectra of cf3-aequorin The absorption spectra were measured in 10 mM TE containing 1.2 M (NH4)2SO4 using a Jasco (Tokyo, Japan) V-560 spectrophotometer (bandwidth, 0.5 nm; response, medium; scan speed, 100 nm/min) at 22°C–25°C with a quartz cuvette (10-mm light path). The bioluminescence emission spectra were measured on a Jasco FP-6500 fluorescence spectrophotometer (emission bandwidth, 20 nm; response, 0.5 s; sensitivity, medium; scan speed, 2000 nm/min) at 22°C–25°C with the excitation light source turned off. The reaction mixtures (0.5 ml) contained 21.5 µg of cf3-aequorin in TE, and the luminescence reaction was initiated by the addition of 100 μl of 10 mM CaCl2 in 50 mM Tris-HCl (pH 7.6). For the determination of the fluorescence spectra, the fraction eluted from a Butyl-Sepharose column with TE was measured using a fluorescence spectrophotometer (excitation at 330 nm, emission bandwidth, 5 nm; response, 0.5 s; sensitivity, medium; scan speed, 100 nm/min; scan times, 3). The corrected luminescence spectrum was obtained in accordance with the manufacturer’s protocol. Crystallization, data collection and structure determination The crystallization was performed by the method of hanging drop vapour diffusion. The purified cf3-aequorin was concentrated using an Amicon Ultra centrifugal filter unit (MWCO 10,000), and the crystals were grown in a mixture of 2 μl of cf3-aequorin (21 mg/ml) and 2 μl of the precipitant solution [10 mM Bis-Tris (pH 7.3)–2 mM EDTA containing 2 M (NH4)2SO4] at 20°C. After a few months of incubation for equilibration against the precipitant solution, single yellow crystals with dimensions of 150 × 50 × 50 µm were obtained (Supplementary Fig. S1). The crystals were cryoprotected in the reservoir solution supplemented with 15% (v/v) glycerol prior to flash cooling in liquid nitrogen. An X-ray diffraction data set was collected to 2.15 Å at a wavelength of 1.0 Å on beamline BL26B2 at SPring-8. The diffraction data were processed using the XDS programs (18), and the structure was solved by molecular replacement using the Phaser program (19) from the PHENIX programs (20), with the aequorin coordinates (PDB ID: 1EJ3) as the search model (4). The structural model was built into the electron density map using Coot (21) and refined using the PHENIX program (20). Structure figures were prepared using the PyMOL Molecular Graphics System, version 2.07. Results and Discussion Purification and characterization of cf3-aequorin The histidine-tagged apoaequorin expressed in the periplasmic space of E. coli cells was partially purified with Ni-chelate affinity chromatography, and the protein yield was 390 mg with over 90% purity from 2 L of cultured cells (Fig. 3A). An eluted fraction of the Ni-chelate column (138 mg protein) was regenerated to cf3-aequorin by incubation with cf3-CTZ, and highly purified cf3-aequorin (47.6 mg protein) was obtained by the column chromatography of a Q-Sepharose gel, followed by a Butyl-Sepharose gel (Fig. 3B and C; Table I). The purified cf3-aequorin showed absorbance peaks at 280, 292 and 459 nm, which were almost the same as those for native aequorin (Fig. 4). The absorbance around 460 nm was derived from CTZ-OOH, and the absorbance ratio of 460 to 280 nm was 0.0320, which was close to the value of 0.0315 from the purified recombinant native aequorin [Inouye, unpublished]. This result suggested that the circumstances for the binding pocket of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) were almost the same as those of CTZ-OOH in native aequorin. Table I. Purification of cf3-aequorin from 2 L of cultured E. coli cells Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 a Luminescence activity was obtained from aequorin regenerated from apoaequorin and CTZ. b Luminescence activity was obtained from cf3-aequorin regenerated from apoaequorin and cf3-CTZ. c Not determined. Table I. Purification of cf3-aequorin from 2 L of cultured E. coli cells Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 a Luminescence activity was obtained from aequorin regenerated from apoaequorin and CTZ. b Luminescence activity was obtained from cf3-aequorin regenerated from apoaequorin and cf3-CTZ. c Not determined. Fig. 3 View largeDownload slide Sodium dodecyl sulphate polyacrylamide gel electrophoresis analyses of proteins at various chromatographic steps of cf3-aequorin. (A) Ni-chelate column. M, molecular weight markers (TEFCO); Sup, 12,000 g supernatant (57 µg protein/5 µl); FT, flow-through fraction (5 µl); E3–5, fractions eluted with 0.1 M imidazole (5 µl); E3, 14.7 µg protein; E4, 147 µg protein; E5, 33.0 µg protein. (B) Q-Sepharose column. M, molecular weight markers (TEFCO); E, fraction eluted with 0.4 M NaCl (85.5 µg protein/5 µl). (C) Butyl-Sepharose column. E2–5, fractions eluted with 1.2 M (NH4)2SO4 (10 µl); E2, 6.1 µg protein; E3, 15.6 µg protein; E4, 21.5 µg protein; E5, 8.9 µg protein. Fig. 3 View largeDownload slide Sodium dodecyl sulphate polyacrylamide gel electrophoresis analyses of proteins at various chromatographic steps of cf3-aequorin. (A) Ni-chelate column. M, molecular weight markers (TEFCO); Sup, 12,000 g supernatant (57 µg protein/5 µl); FT, flow-through fraction (5 µl); E3–5, fractions eluted with 0.1 M imidazole (5 µl); E3, 14.7 µg protein; E4, 147 µg protein; E5, 33.0 µg protein. (B) Q-Sepharose column. M, molecular weight markers (TEFCO); E, fraction eluted with 0.4 M NaCl (85.5 µg protein/5 µl). (C) Butyl-Sepharose column. E2–5, fractions eluted with 1.2 M (NH4)2SO4 (10 µl); E2, 6.1 µg protein; E3, 15.6 µg protein; E4, 21.5 µg protein; E5, 8.9 µg protein. Fig. 4 View largeDownload slide Absorption spectrum of purified cf3-aequorin. The protein concentration of purified cf3-AQ is 0.5 mg/ml in 10 mM Tris-HCl (pH 7.6)–2 mM EDTA containing 1.2 M (NH4)2SO4. Fig. 4 View largeDownload slide Absorption spectrum of purified cf3-aequorin. The protein concentration of purified cf3-AQ is 0.5 mg/ml in 10 mM Tris-HCl (pH 7.6)–2 mM EDTA containing 1.2 M (NH4)2SO4. The different properties between cf3-aequorin and native aequorin were as follows: The bioluminescence emission peak of cf3-aequorin triggered by Ca2+ was observed at 487 nm. The emission peak was red shifted to 17 nm from that of native aequorin with 470 nm, but the value of full width at half maximum (FWHM) of cf3-aequorin of 92 nm was the same as that of native aequorin (Fig. 5A). The luminescence decay pattern of cf3-aequorin triggered by Ca2+ was slower than that of native aequorin. The half-decay times of Imax for cf3-aequorin and native aequorin were 5.0 and 0.9 s, respectively (Fig. 5B). The luminescence capacity of cf3-aequorin was estimated to be 72.8% of that of native aequorin (Table II). After the calcium-triggered reaction of native aequorin, blue fluorescence protein (BFP, a complex of Ca2+-bound apoaequorin and coelenteramide; λmax = ∼470 nm, excited at 330 nm) was formed and was converted to a greenish fluorescence protein (gFP, a complex of Ca2+-unbound apoaequorin and coelenteramide; λmax = ∼485 nm, excited at 330 nm) by removing Ca2+ in the presence of EDTA (7–9). In cf3-aequorin, the bioluminescence emission spectrum was identical to the fluorescence emission spectrum of a complex of Ca2+-bound apoaequorin and cf3-coelenteramide, similar to the case of native aequorin (7, 8). The differences in bioluminescence spectra between cf3-aequorin and native aequorin might be explained by the distinct emitter species between cf3-coelenteramide and coelenteramide in an apoaequorin molecule. Table II. Luminescence activities of native aequorin and cf3-aequorin Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 a The maximum intensity of luminescence. b The luminescence capacity was obtained by integrating for 30 s in 0.1-s intervals. Table II. Luminescence activities of native aequorin and cf3-aequorin Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 a The maximum intensity of luminescence. b The luminescence capacity was obtained by integrating for 30 s in 0.1-s intervals. Fig. 5 View largeDownload slide Comparison of luminescence properties between cf3-aequorin and native aequorin. (A) Bioluminescence emission spectra of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. (B) Luminescence patterns of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. Each protein (0.3 ng) was used for the assay. Fig. 5 View largeDownload slide Comparison of luminescence properties between cf3-aequorin and native aequorin. (A) Bioluminescence emission spectra of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. (B) Luminescence patterns of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. Each protein (0.3 ng) was used for the assay. Unexpectedly, in a Butyl-Sepharose column step in the presence of EDTA, over 30% of cf3-aequorin was converted to a complex of Ca2+-unbound apoaequorin and cf3-coelenteramide. The fraction eluted with TE from a Butyl-Sepharose column showed the fluorescence emission spectrum with λmax = 487 nm (excited at 330 nm, FWHM = 115 nm). From these observations, the binding environment of cf3-coelenteramide in an apoaequorin molecule might be identical in the presence and absence of Ca2+. The instability of cf3-aequorin in the Butyl-Sepharose gel chromatography might be explained by the hydrophilic interactive conditions of cf3-aequorin with a butyl gel in the presence of 1.2 M (NH4)2SO4. Similar instability in the butyl gel was observed in an aequorin mutant, AM20 (22), and a clytin mutant, CLI-ESNA (23). Structure of cf3-aequorin The crystal structure of cf3-aequorin was determined at 2.15 Å resolution (PDB ID: 5ZAB) (Fig. 6A and B). The statistical values of data collection and structure refinement are summarized in Table III. The asymmetric unit of the crystal contained 16 molecules of cf3-aequorin (Fig. 6C). The overall structure of cf3-aequorin was basically the same as that of native aequorin (PDB ID: 1EJ3). The structure of cf3-aequorin showed four EF-hand motifs arranged in pairs to form a globular molecule, but the functional motifs having calcium-binding ability might be EF-hands I, III and IV (3, 4). The C2-peroxide of cf3-CTZ (cf3-CTZ-OOH) for a light source was present in a similar conformation to that of CTZ-OOH in native aequorin and interacted with the same amino acid residues (Figs 7 and 8). The structure of cf3-aequorin was also similar to those of other semi-synthetic aequorins, including i-, br- and n-aequorins, except for the number of water molecules (Table IV). Table III. Statistics of data collection and structure refinement Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB a Statistics for the highest resolution shell are given in parentheses. b Rsym = (∑h∑i|Ihi–<Ih>|/∑h∑i|Ihi|) where h indicates unique reflection indices and i indicates symmetry equivalent indices. c Rwork = ∑|Fobs–Fcalc|/∑Fobs for all reflections and Rfree was calculated using randomly selected reflections (6%). Table III. Statistics of data collection and structure refinement Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB a Statistics for the highest resolution shell are given in parentheses. b Rsym = (∑h∑i|Ihi–<Ih>|/∑h∑i|Ihi|) where h indicates unique reflection indices and i indicates symmetry equivalent indices. c Rwork = ∑|Fobs–Fcalc|/∑Fobs for all reflections and Rfree was calculated using randomly selected reflections (6%). Table IV. Water molecules in semi-synthetic aequorins Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d a The presence and absence of water molecules are shown as plus (+) and minus (−), respectively. b Data taken from ref. 16. c Data taken from ref. 13. d Data taken from ref. 26. Table IV. Water molecules in semi-synthetic aequorins Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d a The presence and absence of water molecules are shown as plus (+) and minus (−), respectively. b Data taken from ref. 16. c Data taken from ref. 13. d Data taken from ref. 26. Fig. 6 View largeDownload slide Crystal structure of cf3-aequorin. (A) Ribbon diagram of cf3-aequorin, A form and B form. The calcium-binding loop residues at 153–160 in B form is shown as dashed circle. (B) The superposition of cf3-aequorin of A form and B form. (C) A complex of 16 molecules of cf3-aequorin (A–P). A form: A, C, D, E, G, H, I, J, K, M, N, and O; B form: B, F, L, and P. Fig. 6 View largeDownload slide Crystal structure of cf3-aequorin. (A) Ribbon diagram of cf3-aequorin, A form and B form. The calcium-binding loop residues at 153–160 in B form is shown as dashed circle. (B) The superposition of cf3-aequorin of A form and B form. (C) A complex of 16 molecules of cf3-aequorin (A–P). A form: A, C, D, E, G, H, I, J, K, M, N, and O; B form: B, F, L, and P. Fig. 7 View largeDownload slide Omit electron density map of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) in cf3-aequorin and interaction of the C2-benzyl group with helices E and H. (A) Stereo view of the structure of cf3-aequorin with omit electron density map of 2-peroxytrifluoromethylcoelenterazine contoured at 3.0 σ. Helix G is omitted for the visibility of the ligand. Helices A-H are shown as A-H with circle. The amino terminus and the carboxyl terminus of cf3-aequorin are indicated by “N” and “C”, respectively. (B) Close-up view of the binding site of cf3-CTZ-OOH. Dashed lines depict hydrogen bonds. Fig. 7 View largeDownload slide Omit electron density map of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) in cf3-aequorin and interaction of the C2-benzyl group with helices E and H. (A) Stereo view of the structure of cf3-aequorin with omit electron density map of 2-peroxytrifluoromethylcoelenterazine contoured at 3.0 σ. Helix G is omitted for the visibility of the ligand. Helices A-H are shown as A-H with circle. The amino terminus and the carboxyl terminus of cf3-aequorin are indicated by “N” and “C”, respectively. (B) Close-up view of the binding site of cf3-CTZ-OOH. Dashed lines depict hydrogen bonds. Fig. 8 View largeDownload slide Comparison of the hydrogen-bonding networks in the 2-peroxycoelenterazine binding cavity between cf3-aequorin (A) and native aequorin (B). Three water molecules assigned to Water 1, Water 2, and Water 3 in native aequorin are represented by W1, W2, and W3, respectively. Fig. 8 View largeDownload slide Comparison of the hydrogen-bonding networks in the 2-peroxycoelenterazine binding cavity between cf3-aequorin (A) and native aequorin (B). Three water molecules assigned to Water 1, Water 2, and Water 3 in native aequorin are represented by W1, W2, and W3, respectively. Although the structures of cf3-aequorin among the 16 molecules in the crystallographic asymmetric unit were almost identical, they can be divided into two structurally distinguishable forms: A and B (4 molecules for the A form and 12 molecules for the B form) (Fig. 6A). The major difference between the two structural forms was a conformational difference in the calcium-binding loop residues at 153–160 in the EF-hand IV motif (shown as dashed circle in Fig. 6A). Similar alternative loop conformations were also observed in the native aequorin structure. Presumably, the calcium-binding loop of EF-hand IV can adopt multiple conformations in aqueous solutions, and the flexibility might influence the calcium-binding affinity (Supplementary Fig. S2). Hydrogen-bonding networks in the C2-peroxide of cf3-coelentrazine (cf3-CTZ-OOH) and water molecules A schematic representation of the hydrogen-bonding networks around cf3-CTZ-OOH in cf3-aequorin is shown in Fig. 8A (Supplementary Table S1). In native aequorin (4), the amino acid residues of His16, Tyr82, Tyr132, His169 and Tyr184 are close enough to the atoms of the CTZ-OOH molecule to undergo hydrogen bonding with it (Fig. 8B). The amino acid triad of His16, Tyr82 and Trp86 interacts with the p-hydroxy moiety at the C6-phenyl group in CTZ-OOH. His169 and Tyr184 stabilize the C3-carbonyl moiety and the C2-peroxy moiety of CTZ-OOH, respectively. Furthermore, three water molecules inside native aequorin contribute to stabilizing CTZ-OOH (Fig. 8B). The p-hydroxy moiety at the C2-benzyl group of CTZ-OOH interacts with Thr166 and Ile105 through a water molecule (Water 1), and Tyr132 hydrogen bonds with the N1 position of CTZ-OOH and interacts with His58 through a water molecule (Water 2). Further, His58 interacts with Ala55 through a water molecule (Water 3) (Fig. 8B). Similar to native aequorin, His169 and Tyr184 in cf3-aequorin stabilized the C3-carbonyl and C2-peroxy moieties of cf3-CTZ-OOH, respectively. In addition, His16, Tyr82 and Trp86 interacted with the p-hydroxy moiety at the C6-phenyl group of cf3-CTZ-OOH (Fig. 8A). Therefore, the hydrogen-bonding networks formed around the C2-peroxy moiety and the C6-hydoroxyphenyl moiety were conserved between cf3-CTZ-OOH and CTZ-OOH (Fig. 8; Supplementary Table S1). In contrast, cf3-aequorin lacked two water molecules (Water 1 and Water 2), which were present around CTZ-OOH in native aequorin (Fig. 8B). The trifluoromethyl group at the C2-benzyl group of cf3-CTZ-OOH interacted with helices E and H through van der Waals interactions (Fig. 7), but a water molecule (Water 2) interacting with Tyr132 in native aequorin was not observed in cf3-aequorin. In a previous report (5), it was described that the structures of semi-synthetic aequorins with C2-modified CTZ, such as i-, br-, and n-coelenterazine, lacked one water molecule (Water 1) around the C2-benzyl group. However, other water molecules of Water 2 and Water 3 related to Tyr132 and His58 remained (Table IV). Among other Ca2+-binding photoproteins, the crystal structure of recombinant obelin (PDB ID, 1EL4) is quite similar to that of native aequorin (24). The presence of CTZ-OOH in obelin was confirmed (PDB ID, 1QV1), and it was stabilized by the amino acid residues His175 and Tyr190, which correspond to His169 and Tyr184 in native aequorin (25). Recently, Natashin et al. have proposed that a water molecule (Water 2) in obelin takes part in the decarboxylation reaction of CTZ-OOH by protonation of a dioxeteanone anion, based on the structures of the Y138F obelin mutant (PDB ID, 4MRX) and the Ca2+-discharged Y138F obelin mutant (PDB ID, 4MRY) (26). However, there is no direct evidence for their proposal, and our observations did not support this hypothesis. When Ca2+ binds to aequorin, the luminescence reaction of aequorin will start to release the hydrogen bonds of His169 and Tyr184 from the C2-peroxy moiety, followed by cleavage of the C2–C3 bond of the pyrazine ring to give the excited coelenteramide and CO2. The light emission is produced from the excited state of coelenteramide in apoaequorin. In general, CTZ-OOH is unstable in aqueous solutions, and the decomposition of CTZ through CTZ-OOH proceeds spontaneously without significant light emission. Thus, the conformational changes by Ca2+ to cleave the hydrogen bonds from the C2-peroxy moiety of CTZ are crucial for the emission of light from the complex of coelenteramide and Ca2+-bound apoaequorin. Luminescence kinetics of aequorin Based on the luminescence mechanism of aequorin by Ca2+, the luminescence kinetics could be explained by the binding affinity of Ca2+ to the EF-hand motifs of aequorin, followed by the speed of conformational changes to cleave the hydrogen bonds between amino acid residues and CTZ-OOH. Thus, slow conformational changes of the cf3-aequorin molecule by Ca2+ could give slow decay luminescence kinetics with a long half-decay time of Imax. Some semi-synthetic aequorins with CTZ analogs possessing a bulky C2-moiety, such as i-, br-, and n-aequorin, showed slow luminescence kinetics (Table IV) (13–15). A bulky moiety at the C2-benzyl group might interfere with the smooth conformational changes to cleave the hydrogen bonds of CTZ-OOH. In other cases, EF-hand I loop mutants of native aequorin with a positively charged amino acid such as AM20 also showed a slow decay luminescence pattern with a long half-decay time, as previously reported (22). This slow decay luminescence kinetics might be explained by the low Ca2+-binding affinity to EF-hands, resulting in a slow conformational change to emit light. In contrast, fast luminescence kinetics was observed in the semi-synthetic aequorin of cp-aequorin, which was prepared using cp-CTZ possessing a cyclopentylmethyl (cp) group replaced with a benzyl group at the C8 position of CTZ, the crystal structure of which had previously been determined (5). In the structure of native aequorin, the benzyl group at the C8 position of CTZ-OOH was involved in a π–π interaction with Trp108 of EF-hand III and a stacking interaction with Lys39 of EF-hand I. Thus, the fast luminescence kinetics of cp-aequorin was due to the absence of the stacking and π–π interactions of the cp group that induces the conformational changes faster than native aequorin to emit light. Conclusion Semi-synthetic aequorin of cf3-aequorin was prepared by incubation with cf3-CTZ and apoaequorin expressed in E. coli cells. Highly purified cf3-aequorin was obtained by ion exchange and hydrophobic column chromatography and showed slow luminescence kinetics with a long half-decay time of Imax. The crystal structure of cf3-aequorin, including 16 molecules per asymmetric unit, was determined, showing virtually the same protein structure as that of native aequorin. The major difference between cf3-aequorin and native aequorin is that two of the three water molecules that stabilize CTZ-OOH in native aequorin are missing in cf3-aequorin. The bulky trifluoromethyl group of cf3-CTZ-OOH interacted with helices E and H of apoaequorin and might interfere with the smooth conformational changes to emit light by cleavage of the hydrogen bonds at the C2-peroxy moiety of cf3-CTZ-OOH after the binding of Ca2+. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements The authors thank Drs. H. Niwa, N. Sakai, and T. Umehara, and the beamline staffs for the synchrotron radiation experiments at SPring-8. Funding This work was supported in part by grants from Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP17am0101098 (Platform Project for Supporting Drug Discovery and Life Science Research). The synchrotron radiation experiments were performed at BL26B2 in SPring-8 with the approval of RIKEN (Proposal No. 20160018). Conflict of Interest None declared. References 1 Shimomura O. , Johnson F.H. , Saiga Y. ( 1962 ) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea . J. Cell. Comp. Physiol. 59 , 223 – 239 Google Scholar CrossRef Search ADS PubMed 2 Inouye S. , Noguchi M. , Sakaki Y. , Takagi Y. , Miyata T. , Iwanaga S. , Miyata T. , Tsuji F.I. ( 1985 ) Cloning and sequence analysis of cDNA for the luminescent protein aequorin . Proc. Natl. Acad. Sci. 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Crystallogr. 70 , 720 – 732 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations cf3-CTZ trifluoromethylcoelenterazine cf3-CTZ-OOH 2-peroxytrifluoromethylcoelenterazine CHO-K1 Chinese hamster ovary K1 CTZ coelenterazine CTZ-OOH 2-peroxycoelenterazine DTT dithiothreitol EDTA ethylenediaminetetraacetic acid FWHM full width at half maximum GPCR G-protein-coupled receptor Imax maximum intensity of luminescence LB Luria–Bertani; PDB, Protein Data Bank rlu relative light units TE 50 mM Tris-HCl (pH 7.6)–10 mM EDTA © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Slow luminescence kinetics of semi-synthetic aequorin: expression, purification and structure determination of cf3-aequorin

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0021-924X
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Abstract

Abstract cf3-Aequorin is one of the semi-synthetic aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3-CTZ) and the histidine-tagged apoaequorin expressed in Escherichia coli cells. The purified cf3-aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native aequorin, and its luminescence capacity was estimated to be 72.8% of that of native aequorin. The crystal structure of cf3-aequorin was determined at 2.15 Å resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p-hydroxy moiety at the C6-phenyl group. In native aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3-aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3-CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3-aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3-aequorin. calcium-binding protein, EF-hand motif, photoprotein, van der Waals interaction, water molecules Aequorin is a Ca2+-binding photoprotein that was isolated from the luminous jellyfish Aequorea victoria and emits light by an intramolecular reaction upon binding with Ca2+ (1). The primary structure of aequorin consists of 189 amino acid residues (21.4 kDa) with three EF-hand motifs characteristic of a Ca2+-binding site (2, 3). Crystal structure analysis of recombinant native aequorin (Protein Data Bank [PDB] ID: 1EJ3) revealed that aequorin is a complex of apoaequorin (an apoprotein) and noncovalently bound 2-peroxycoelenterazine [CTZ-OOH; molecular oxygen-activated coelenterazine (CTZ)] and has three EF-hand motifs with a helix–loop–helix structure (4, 5). Upon the addition of Ca2+ to aequorin, CTZ-OOH stabilized in the hydrophobic core structure of apoaequorin decomposes into coelenteramide and CO2 to produce blue light (λmax = ∼470 nm) (Fig. 1A) (6–9). After the Ca2+-triggered luminescence reaction, apoaequorin can be regenerated to active aequorin by incubation with CTZ under reducing conditions in the presence of a calcium-chelating reagent (Fig. 1A) (10–12). Furthermore, semi-synthetic aequorin was prepared by replacing CTZ with a chemically synthesized CTZ analog, and more than 50 kinds of semi-synthetic aequorins have been characterized (13–17). Among the semi-synthetic aequorins with C2-modified CTZ analogs, the crystal structures of i-, br- and n-aequorin have been determined (PDB ID: 1UHH, 1UHJ and 1UHK, respectively). The structures of these semi-synthetic aequorins were almost identical to native aequorin (Fig. 2), but they showed luminescence kinetics differing from that of native aequorin (5). Fig. 1 View largeDownload slide Bioluminescence reaction of aequorin triggered with Ca2+ and regeneration to aequorin from blue fluorescent protein (BFP) with coelenterazine (A) and the chemical structure of C2-modified coelenterazine analogs for semi-synthetic aequorins (B). The Ca2+-binding sites of three EF-hands are shown as I, III, and IV, respectively. CTZ, coelenterazine; CTZ-OOH, 2-peroxycoelenterazine; EDTA, ethylenediaminetetraacetic acid. Fig. 1 View largeDownload slide Bioluminescence reaction of aequorin triggered with Ca2+ and regeneration to aequorin from blue fluorescent protein (BFP) with coelenterazine (A) and the chemical structure of C2-modified coelenterazine analogs for semi-synthetic aequorins (B). The Ca2+-binding sites of three EF-hands are shown as I, III, and IV, respectively. CTZ, coelenterazine; CTZ-OOH, 2-peroxycoelenterazine; EDTA, ethylenediaminetetraacetic acid. Fig. 2 View largeDownload slide Primary structure of native aequorin from Aequorea victoria. The loop regions of EF-hands I, III, and IV are labelled with [I], [III], and [IV], respectively. The helices are shown as cylinders with italic letters A–H, and the β-sheets are shown as arrows. The conserved amino acid residues of His16, Tyr82, Trp86, Ile105, Tyr132, Thr166, His169, and Tyr184 for interacting with 2-peroxycoelenterazine among various Ca2+-binding proteins are labelled. The C-terminal proline residue (Pro189), which interacts with Arg15 at helix A, is boxed. Fig. 2 View largeDownload slide Primary structure of native aequorin from Aequorea victoria. The loop regions of EF-hands I, III, and IV are labelled with [I], [III], and [IV], respectively. The helices are shown as cylinders with italic letters A–H, and the β-sheets are shown as arrows. The conserved amino acid residues of His16, Tyr82, Trp86, Ile105, Tyr132, Thr166, His169, and Tyr184 for interacting with 2-peroxycoelenterazine among various Ca2+-binding proteins are labelled. The C-terminal proline residue (Pro189), which interacts with Arg15 at helix A, is boxed. Previously, we reported a C2-modified CTZ analog (Fig. 1B), cf3-coelenterazine (cf3-CTZ) (16), which is more stable in aqueous solutions than CTZ and h-coelenterazine (h-CTZ) (17). Semi-synthetic aequorin with cf3-CTZ (cf3-aequorin) showed slow decay of luminescence pattern with a half-decay time of ∼6 s which is slower than that of native aequorin and h-aequorin with ∼0.8 s and ∼0.7 s, respectively (16, 17). In the cell-based G-protein-coupled receptor (GPCR) assay using Chinese hamster ovary K1 (CHO-K1) cells expressing apoaequorin, h-aequorin has generally been used as a Ca2+ indicator because of the high sensitivity to Ca2+. However, in some cases, it is difficult to determine the accurate value of half maximal effective concentration (EC50) using h-aequorin with fast luminescence kinetics. In contrast, cf3-aequorin regenerated in CHO-K1 cells showed lower coefficient of variation values of luminescence intensity than h-aequorin. Thus, the slow reaction kinetics of cf3-aequorin makes it more advantageous in an aequorin-based GPCR assay than h-aequorin (16). To understand the slow luminescence kinetics of cf3-aequorin with Ca2+, we determined the crystal structure of cf3-aequorin and discussed the relationships between its protein structure and luminescence properties by comparison to that of native aequorin. Materials and Methods Materials The following materials were obtained from commercial sources: cf3-coelenterazine (cf3-CTZ), coelenterazine (CTZ), and recombinant aequorin (JNC Co., Tokyo, Japan); 2-mercaptoethanol, imidazole, dithiothreitol (DTT), ethylenediaminetetraacetic acid disodium salt (EDTA·2Na), and imidazole (Wako Pure Chemicals, Osaka, Japan); Q-Sepharose Fast Flow and Butyl-Sepharose 4 Fast Flow (GE Healthcare, Piscataway, NJ, USA); Disform CE475 (NOF Co., Tokyo, Japan); and bovine serum albumin (BSA; Sigma, St. Louis, MO, USA). Expression and purification of recombinant cf3-aequorin from E. coli cells To express histidine-tagged apoaequorin in the periplasmic space of E. coli, the bacterial strain WA802 carrying an expression vector, piP-His-HE (9), was used. A seed culture of E. coli cells was grown in 10 ml of Luria–Bertani (LB) broth containing ampicillin (50 μg/ml) at 25°C for 18 h and then transferred into 400 ml of LB broth containing 50 μl of antifoam (Disform CE475) in a 2-L Sakaguchi flask and cultured with reciprocal shaking (130 rpm) at 37°C for 18 h. After harvesting the cells from 2 L of the culture medium, the cells were suspended in 100 ml of 50 mM Tris-HCl (pH 7.6) and disrupted by sonication for 15 min (3 min × 5) in an ice–water bath using a Branson (Danbury, CT, USA) model 250 sonifier. The soluble fractions obtained by centrifugation at 12,000 g for 20 min were applied on a nickel chelate column (ø2.5 × 5.5 cm) equilibrated with 50 mM Tris-HCl (pH 7.6). After washing with 300 ml of 50 mM Tris-HCl (pH 7.6), the proteins adsorbed on a gel were eluted with 100 mM imidazole in 50 mM Tris-HCl (pH 7.6). From 2 L of cultured cells, 390 mg of proteins with over 90% purity was obtained, added to a final concentration of 5 mM DTT, and stored at −80°C. To purify cf3-aequorin, the partially purified histidine-tagged apoaequorin was regenerated to cf3-aequorin by incubation with cf3-CTZ and applied to a Q-Sepharose column, followed by a Butyl-Sepharose column, as previously described in the purification of recombinant native aequorin (11, 12). The regeneration mixture contained histidine-tagged apoaequorin (138 mg protein), cf3-CTZ (2 mg dissolved in 2 ml of ethanol), and DTT (20 mg) in 250 ml of 50 mM Tris-HCl (pH 7.6)–10 mM EDTA (TE). After incubating at 4°C for 12 h, the regenerated mixture was applied on a Q-Sepharose Fast Flow column (ø2.5 × 7 cm) equilibrated with TE and washed with 200 ml of 0.1 M NaCl in TE, followed by eluting with 0.4 M NaCl in TE. The eluted fraction (30 ml, 84.5 mg protein) was adjusted immediately to a final concentration of 2 M (NH4)2SO4 in the total volume of 50 ml of TE and applied on a Butyl-Sepharose 4 Fast Flow column (ø1.5 × 7.5 cm) equilibrated with 2 M (NH4)2SO4 in TE. After washing with 50 ml of 2 M (NH4)2SO4 in TE, cf3-aequorin (47.6 mg protein) was eluted with 1.2 M (NH4)2SO4 in TE. The purified cf3-aequorin was over 95% purity and stored at −80°C. All column chromatography was performed at 23°C–25°C. Protein analysis Protein concentration was determined by the dye-binding method using a commercially available kit (Bio-Rad, Richmond, CA, USA) and BSA as a standard (Pierce, Rockford, IL, USA). Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis was performed under reducing conditions using a 12% separation gel (TEFCO, Tokyo, Japan), and the gels were stained with a colloidal CBB staining kit (TEFCO). Determination of luminescence activity and luminescence pattern of cf3-aequorin In the steps for purifying histidine-tagged apoaequorin, the luminescence activity was determined by regeneration to native aequorin with 1 µg of CTZ (1 µg/µl in ethanol) in 1 ml of 30 mM TE containing 1 µl of 2-mercaptoethanol at 4°C for 2 h. For the preparation of cf3-aequorin, cf3-CTZ was used for the assay instead of CTZ. The maximum intensities of luminescence (Imax) of aequorin and cf3-aequorin were determined by injection with 100 µl of 50 mM CaCl2 in 50 mM Tris-HCl (pH 7.6) using an ATTO (Tokyo, Japan) model AB2200 luminometer in 0.1-s intervals for 10 s. The luminescence patterns of purified aequorin and cf3-aequorin were determined by injection of 100 µl of 50 mM CaCl2 solution in 50 mM Tris-HCl (pH 7.6) into 3 µl of the protein solution (0.3 ng protein) dissolved in 50 mM Tris-HCl (pH 7.6) containing 0.01 mM EDTA, 0.1% BSA, and 150 mM NaCl using a luminometer Centro LB960 (Berthold, Bad Wildbad, Germany) for 30 s in 0.1-s intervals. The luminescence activity was determined as the mean value (n = 6). Measurements of absorbance, bioluminescence emission and fluorescence emission spectra of cf3-aequorin The absorption spectra were measured in 10 mM TE containing 1.2 M (NH4)2SO4 using a Jasco (Tokyo, Japan) V-560 spectrophotometer (bandwidth, 0.5 nm; response, medium; scan speed, 100 nm/min) at 22°C–25°C with a quartz cuvette (10-mm light path). The bioluminescence emission spectra were measured on a Jasco FP-6500 fluorescence spectrophotometer (emission bandwidth, 20 nm; response, 0.5 s; sensitivity, medium; scan speed, 2000 nm/min) at 22°C–25°C with the excitation light source turned off. The reaction mixtures (0.5 ml) contained 21.5 µg of cf3-aequorin in TE, and the luminescence reaction was initiated by the addition of 100 μl of 10 mM CaCl2 in 50 mM Tris-HCl (pH 7.6). For the determination of the fluorescence spectra, the fraction eluted from a Butyl-Sepharose column with TE was measured using a fluorescence spectrophotometer (excitation at 330 nm, emission bandwidth, 5 nm; response, 0.5 s; sensitivity, medium; scan speed, 100 nm/min; scan times, 3). The corrected luminescence spectrum was obtained in accordance with the manufacturer’s protocol. Crystallization, data collection and structure determination The crystallization was performed by the method of hanging drop vapour diffusion. The purified cf3-aequorin was concentrated using an Amicon Ultra centrifugal filter unit (MWCO 10,000), and the crystals were grown in a mixture of 2 μl of cf3-aequorin (21 mg/ml) and 2 μl of the precipitant solution [10 mM Bis-Tris (pH 7.3)–2 mM EDTA containing 2 M (NH4)2SO4] at 20°C. After a few months of incubation for equilibration against the precipitant solution, single yellow crystals with dimensions of 150 × 50 × 50 µm were obtained (Supplementary Fig. S1). The crystals were cryoprotected in the reservoir solution supplemented with 15% (v/v) glycerol prior to flash cooling in liquid nitrogen. An X-ray diffraction data set was collected to 2.15 Å at a wavelength of 1.0 Å on beamline BL26B2 at SPring-8. The diffraction data were processed using the XDS programs (18), and the structure was solved by molecular replacement using the Phaser program (19) from the PHENIX programs (20), with the aequorin coordinates (PDB ID: 1EJ3) as the search model (4). The structural model was built into the electron density map using Coot (21) and refined using the PHENIX program (20). Structure figures were prepared using the PyMOL Molecular Graphics System, version 2.07. Results and Discussion Purification and characterization of cf3-aequorin The histidine-tagged apoaequorin expressed in the periplasmic space of E. coli cells was partially purified with Ni-chelate affinity chromatography, and the protein yield was 390 mg with over 90% purity from 2 L of cultured cells (Fig. 3A). An eluted fraction of the Ni-chelate column (138 mg protein) was regenerated to cf3-aequorin by incubation with cf3-CTZ, and highly purified cf3-aequorin (47.6 mg protein) was obtained by the column chromatography of a Q-Sepharose gel, followed by a Butyl-Sepharose gel (Fig. 3B and C; Table I). The purified cf3-aequorin showed absorbance peaks at 280, 292 and 459 nm, which were almost the same as those for native aequorin (Fig. 4). The absorbance around 460 nm was derived from CTZ-OOH, and the absorbance ratio of 460 to 280 nm was 0.0320, which was close to the value of 0.0315 from the purified recombinant native aequorin [Inouye, unpublished]. This result suggested that the circumstances for the binding pocket of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) were almost the same as those of CTZ-OOH in native aequorin. Table I. Purification of cf3-aequorin from 2 L of cultured E. coli cells Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 a Luminescence activity was obtained from aequorin regenerated from apoaequorin and CTZ. b Luminescence activity was obtained from cf3-aequorin regenerated from apoaequorin and cf3-CTZ. c Not determined. Table I. Purification of cf3-aequorin from 2 L of cultured E. coli cells Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 Steps Total volume Total protein Total activity Specific activity (ml) (mg) (%) (Imax, ×109 rlu) (%) (×109 rlu/mg) 1. Purification of His-tagged apoaequorin 1) Crude extracts (12,000 g sup) 100 1147 (100) 691 (100)a 0.60 2) Ni-chelate column (ø2.5 × 5.5 cm) Eluted fractions with 0.1 M imidazole 30 390 (34) 634 (92)a 1.63 2. Purification of cf3-aequorin 1) Regenerated mixture with cf3-CTZ 250 138 (100) 17.9 (100)b 0.14 2) Q-Sepharose column (ø2.5 × 7 cm) Eluted fractions and dissolved in 2 M (NH4)2SO4 50 84.5 (64) NDc ND 3) Butyl-Sepharose column (ø1.5 × 7.5 cm ) fr-1. Eluted fractions with 1.2 M (NH4)2SO4 35 47.6 (34) 8.7 (46)b 0.18 fr-2. Eluted fractions with TE (fluorescence) 10 47.1 (34) 0.013 (0.7)b 0.0003 a Luminescence activity was obtained from aequorin regenerated from apoaequorin and CTZ. b Luminescence activity was obtained from cf3-aequorin regenerated from apoaequorin and cf3-CTZ. c Not determined. Fig. 3 View largeDownload slide Sodium dodecyl sulphate polyacrylamide gel electrophoresis analyses of proteins at various chromatographic steps of cf3-aequorin. (A) Ni-chelate column. M, molecular weight markers (TEFCO); Sup, 12,000 g supernatant (57 µg protein/5 µl); FT, flow-through fraction (5 µl); E3–5, fractions eluted with 0.1 M imidazole (5 µl); E3, 14.7 µg protein; E4, 147 µg protein; E5, 33.0 µg protein. (B) Q-Sepharose column. M, molecular weight markers (TEFCO); E, fraction eluted with 0.4 M NaCl (85.5 µg protein/5 µl). (C) Butyl-Sepharose column. E2–5, fractions eluted with 1.2 M (NH4)2SO4 (10 µl); E2, 6.1 µg protein; E3, 15.6 µg protein; E4, 21.5 µg protein; E5, 8.9 µg protein. Fig. 3 View largeDownload slide Sodium dodecyl sulphate polyacrylamide gel electrophoresis analyses of proteins at various chromatographic steps of cf3-aequorin. (A) Ni-chelate column. M, molecular weight markers (TEFCO); Sup, 12,000 g supernatant (57 µg protein/5 µl); FT, flow-through fraction (5 µl); E3–5, fractions eluted with 0.1 M imidazole (5 µl); E3, 14.7 µg protein; E4, 147 µg protein; E5, 33.0 µg protein. (B) Q-Sepharose column. M, molecular weight markers (TEFCO); E, fraction eluted with 0.4 M NaCl (85.5 µg protein/5 µl). (C) Butyl-Sepharose column. E2–5, fractions eluted with 1.2 M (NH4)2SO4 (10 µl); E2, 6.1 µg protein; E3, 15.6 µg protein; E4, 21.5 µg protein; E5, 8.9 µg protein. Fig. 4 View largeDownload slide Absorption spectrum of purified cf3-aequorin. The protein concentration of purified cf3-AQ is 0.5 mg/ml in 10 mM Tris-HCl (pH 7.6)–2 mM EDTA containing 1.2 M (NH4)2SO4. Fig. 4 View largeDownload slide Absorption spectrum of purified cf3-aequorin. The protein concentration of purified cf3-AQ is 0.5 mg/ml in 10 mM Tris-HCl (pH 7.6)–2 mM EDTA containing 1.2 M (NH4)2SO4. The different properties between cf3-aequorin and native aequorin were as follows: The bioluminescence emission peak of cf3-aequorin triggered by Ca2+ was observed at 487 nm. The emission peak was red shifted to 17 nm from that of native aequorin with 470 nm, but the value of full width at half maximum (FWHM) of cf3-aequorin of 92 nm was the same as that of native aequorin (Fig. 5A). The luminescence decay pattern of cf3-aequorin triggered by Ca2+ was slower than that of native aequorin. The half-decay times of Imax for cf3-aequorin and native aequorin were 5.0 and 0.9 s, respectively (Fig. 5B). The luminescence capacity of cf3-aequorin was estimated to be 72.8% of that of native aequorin (Table II). After the calcium-triggered reaction of native aequorin, blue fluorescence protein (BFP, a complex of Ca2+-bound apoaequorin and coelenteramide; λmax = ∼470 nm, excited at 330 nm) was formed and was converted to a greenish fluorescence protein (gFP, a complex of Ca2+-unbound apoaequorin and coelenteramide; λmax = ∼485 nm, excited at 330 nm) by removing Ca2+ in the presence of EDTA (7–9). In cf3-aequorin, the bioluminescence emission spectrum was identical to the fluorescence emission spectrum of a complex of Ca2+-bound apoaequorin and cf3-coelenteramide, similar to the case of native aequorin (7, 8). The differences in bioluminescence spectra between cf3-aequorin and native aequorin might be explained by the distinct emitter species between cf3-coelenteramide and coelenteramide in an apoaequorin molecule. Table II. Luminescence activities of native aequorin and cf3-aequorin Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 a The maximum intensity of luminescence. b The luminescence capacity was obtained by integrating for 30 s in 0.1-s intervals. Table II. Luminescence activities of native aequorin and cf3-aequorin Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 Purified proteins Luminescence activity (rlu/μg protein) Half-decay time of Imax (s) Imaxa (%) Capacityb(%) Native aequorin 4.86 ×108 (100) 5.65 × 109 (100) 0.9 cf3-aequorin 0.58× 108 (12.0) 4.11 × 109 (72.8) 5.0 a The maximum intensity of luminescence. b The luminescence capacity was obtained by integrating for 30 s in 0.1-s intervals. Fig. 5 View largeDownload slide Comparison of luminescence properties between cf3-aequorin and native aequorin. (A) Bioluminescence emission spectra of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. (B) Luminescence patterns of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. Each protein (0.3 ng) was used for the assay. Fig. 5 View largeDownload slide Comparison of luminescence properties between cf3-aequorin and native aequorin. (A) Bioluminescence emission spectra of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. (B) Luminescence patterns of cf3-aequorin (solid line) and native aequorin (dashed line), triggered by Ca2+. Each protein (0.3 ng) was used for the assay. Unexpectedly, in a Butyl-Sepharose column step in the presence of EDTA, over 30% of cf3-aequorin was converted to a complex of Ca2+-unbound apoaequorin and cf3-coelenteramide. The fraction eluted with TE from a Butyl-Sepharose column showed the fluorescence emission spectrum with λmax = 487 nm (excited at 330 nm, FWHM = 115 nm). From these observations, the binding environment of cf3-coelenteramide in an apoaequorin molecule might be identical in the presence and absence of Ca2+. The instability of cf3-aequorin in the Butyl-Sepharose gel chromatography might be explained by the hydrophilic interactive conditions of cf3-aequorin with a butyl gel in the presence of 1.2 M (NH4)2SO4. Similar instability in the butyl gel was observed in an aequorin mutant, AM20 (22), and a clytin mutant, CLI-ESNA (23). Structure of cf3-aequorin The crystal structure of cf3-aequorin was determined at 2.15 Å resolution (PDB ID: 5ZAB) (Fig. 6A and B). The statistical values of data collection and structure refinement are summarized in Table III. The asymmetric unit of the crystal contained 16 molecules of cf3-aequorin (Fig. 6C). The overall structure of cf3-aequorin was basically the same as that of native aequorin (PDB ID: 1EJ3). The structure of cf3-aequorin showed four EF-hand motifs arranged in pairs to form a globular molecule, but the functional motifs having calcium-binding ability might be EF-hands I, III and IV (3, 4). The C2-peroxide of cf3-CTZ (cf3-CTZ-OOH) for a light source was present in a similar conformation to that of CTZ-OOH in native aequorin and interacted with the same amino acid residues (Figs 7 and 8). The structure of cf3-aequorin was also similar to those of other semi-synthetic aequorins, including i-, br- and n-aequorins, except for the number of water molecules (Table IV). Table III. Statistics of data collection and structure refinement Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB a Statistics for the highest resolution shell are given in parentheses. b Rsym = (∑h∑i|Ihi–<Ih>|/∑h∑i|Ihi|) where h indicates unique reflection indices and i indicates symmetry equivalent indices. c Rwork = ∑|Fobs–Fcalc|/∑Fobs for all reflections and Rfree was calculated using randomly selected reflections (6%). Table III. Statistics of data collection and structure refinement Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB Data collection and processing Beamline BL26B2 Space group P1 Unit-cell parameter a, b, c (Å) 91.8, 97.6, 121.8 α, β, γ (Å) 77.6, 73.1, 75.2 Wavelength (Å) 1.000 Resolution range (Å) 50–2.15 (2.29–2.15) Redundancy 4.0 (3.9) Completeness (%) a 97.6 (94.8) Rsymb (%) a 2.7 (76.2) I/σ (I) a 12.1 (2.0) No. monomers/asymmetric unit 16 Model refinement No. of reflections 205732 No. of protein atoms 24426 No. of water molecules 1468 Rwork/Rfreec (%) 20.7/22.9 r.m.s.d. for bond length (Å) 0.002 r.m.s.d. for bond angles (˚) 0.463 Residues in the Ramachandran plot Favored region (%) 99.17 Allowed regions (%) 0.83 PDB entry 5ZAB a Statistics for the highest resolution shell are given in parentheses. b Rsym = (∑h∑i|Ihi–<Ih>|/∑h∑i|Ihi|) where h indicates unique reflection indices and i indicates symmetry equivalent indices. c Rwork = ∑|Fobs–Fcalc|/∑Fobs for all reflections and Rfree was calculated using randomly selected reflections (6%). Table IV. Water molecules in semi-synthetic aequorins Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d a The presence and absence of water molecules are shown as plus (+) and minus (−), respectively. b Data taken from ref. 16. c Data taken from ref. 13. d Data taken from ref. 26. Table IV. Water molecules in semi-synthetic aequorins Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d Photoproteins Coelenterazine analogs PDB ID Water 1a Water 2a Water 3a Half-decay time of Imax (s) aequorin CTZ 1EJ3 + + + 0.8b cp-aequorin cp-CTZ 1UHH + + + 0.15–0.3c i-aequorin i-CTZ 1UHI − + + 11.4b br-aequorin br-CTZ 1UHJ − + + 2.4c n-aequorin n-CTZ 1UHK − + + 4.7b cf3-aequorin cf3-CTZ 5ZAB − − + 6.0b obelin CTZ 1EL4 + + + 0.06d Y138F obelin CTZ 4MRX + − + 11.9d a The presence and absence of water molecules are shown as plus (+) and minus (−), respectively. b Data taken from ref. 16. c Data taken from ref. 13. d Data taken from ref. 26. Fig. 6 View largeDownload slide Crystal structure of cf3-aequorin. (A) Ribbon diagram of cf3-aequorin, A form and B form. The calcium-binding loop residues at 153–160 in B form is shown as dashed circle. (B) The superposition of cf3-aequorin of A form and B form. (C) A complex of 16 molecules of cf3-aequorin (A–P). A form: A, C, D, E, G, H, I, J, K, M, N, and O; B form: B, F, L, and P. Fig. 6 View largeDownload slide Crystal structure of cf3-aequorin. (A) Ribbon diagram of cf3-aequorin, A form and B form. The calcium-binding loop residues at 153–160 in B form is shown as dashed circle. (B) The superposition of cf3-aequorin of A form and B form. (C) A complex of 16 molecules of cf3-aequorin (A–P). A form: A, C, D, E, G, H, I, J, K, M, N, and O; B form: B, F, L, and P. Fig. 7 View largeDownload slide Omit electron density map of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) in cf3-aequorin and interaction of the C2-benzyl group with helices E and H. (A) Stereo view of the structure of cf3-aequorin with omit electron density map of 2-peroxytrifluoromethylcoelenterazine contoured at 3.0 σ. Helix G is omitted for the visibility of the ligand. Helices A-H are shown as A-H with circle. The amino terminus and the carboxyl terminus of cf3-aequorin are indicated by “N” and “C”, respectively. (B) Close-up view of the binding site of cf3-CTZ-OOH. Dashed lines depict hydrogen bonds. Fig. 7 View largeDownload slide Omit electron density map of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) in cf3-aequorin and interaction of the C2-benzyl group with helices E and H. (A) Stereo view of the structure of cf3-aequorin with omit electron density map of 2-peroxytrifluoromethylcoelenterazine contoured at 3.0 σ. Helix G is omitted for the visibility of the ligand. Helices A-H are shown as A-H with circle. The amino terminus and the carboxyl terminus of cf3-aequorin are indicated by “N” and “C”, respectively. (B) Close-up view of the binding site of cf3-CTZ-OOH. Dashed lines depict hydrogen bonds. Fig. 8 View largeDownload slide Comparison of the hydrogen-bonding networks in the 2-peroxycoelenterazine binding cavity between cf3-aequorin (A) and native aequorin (B). Three water molecules assigned to Water 1, Water 2, and Water 3 in native aequorin are represented by W1, W2, and W3, respectively. Fig. 8 View largeDownload slide Comparison of the hydrogen-bonding networks in the 2-peroxycoelenterazine binding cavity between cf3-aequorin (A) and native aequorin (B). Three water molecules assigned to Water 1, Water 2, and Water 3 in native aequorin are represented by W1, W2, and W3, respectively. Although the structures of cf3-aequorin among the 16 molecules in the crystallographic asymmetric unit were almost identical, they can be divided into two structurally distinguishable forms: A and B (4 molecules for the A form and 12 molecules for the B form) (Fig. 6A). The major difference between the two structural forms was a conformational difference in the calcium-binding loop residues at 153–160 in the EF-hand IV motif (shown as dashed circle in Fig. 6A). Similar alternative loop conformations were also observed in the native aequorin structure. Presumably, the calcium-binding loop of EF-hand IV can adopt multiple conformations in aqueous solutions, and the flexibility might influence the calcium-binding affinity (Supplementary Fig. S2). Hydrogen-bonding networks in the C2-peroxide of cf3-coelentrazine (cf3-CTZ-OOH) and water molecules A schematic representation of the hydrogen-bonding networks around cf3-CTZ-OOH in cf3-aequorin is shown in Fig. 8A (Supplementary Table S1). In native aequorin (4), the amino acid residues of His16, Tyr82, Tyr132, His169 and Tyr184 are close enough to the atoms of the CTZ-OOH molecule to undergo hydrogen bonding with it (Fig. 8B). The amino acid triad of His16, Tyr82 and Trp86 interacts with the p-hydroxy moiety at the C6-phenyl group in CTZ-OOH. His169 and Tyr184 stabilize the C3-carbonyl moiety and the C2-peroxy moiety of CTZ-OOH, respectively. Furthermore, three water molecules inside native aequorin contribute to stabilizing CTZ-OOH (Fig. 8B). The p-hydroxy moiety at the C2-benzyl group of CTZ-OOH interacts with Thr166 and Ile105 through a water molecule (Water 1), and Tyr132 hydrogen bonds with the N1 position of CTZ-OOH and interacts with His58 through a water molecule (Water 2). Further, His58 interacts with Ala55 through a water molecule (Water 3) (Fig. 8B). Similar to native aequorin, His169 and Tyr184 in cf3-aequorin stabilized the C3-carbonyl and C2-peroxy moieties of cf3-CTZ-OOH, respectively. In addition, His16, Tyr82 and Trp86 interacted with the p-hydroxy moiety at the C6-phenyl group of cf3-CTZ-OOH (Fig. 8A). Therefore, the hydrogen-bonding networks formed around the C2-peroxy moiety and the C6-hydoroxyphenyl moiety were conserved between cf3-CTZ-OOH and CTZ-OOH (Fig. 8; Supplementary Table S1). In contrast, cf3-aequorin lacked two water molecules (Water 1 and Water 2), which were present around CTZ-OOH in native aequorin (Fig. 8B). The trifluoromethyl group at the C2-benzyl group of cf3-CTZ-OOH interacted with helices E and H through van der Waals interactions (Fig. 7), but a water molecule (Water 2) interacting with Tyr132 in native aequorin was not observed in cf3-aequorin. In a previous report (5), it was described that the structures of semi-synthetic aequorins with C2-modified CTZ, such as i-, br-, and n-coelenterazine, lacked one water molecule (Water 1) around the C2-benzyl group. However, other water molecules of Water 2 and Water 3 related to Tyr132 and His58 remained (Table IV). Among other Ca2+-binding photoproteins, the crystal structure of recombinant obelin (PDB ID, 1EL4) is quite similar to that of native aequorin (24). The presence of CTZ-OOH in obelin was confirmed (PDB ID, 1QV1), and it was stabilized by the amino acid residues His175 and Tyr190, which correspond to His169 and Tyr184 in native aequorin (25). Recently, Natashin et al. have proposed that a water molecule (Water 2) in obelin takes part in the decarboxylation reaction of CTZ-OOH by protonation of a dioxeteanone anion, based on the structures of the Y138F obelin mutant (PDB ID, 4MRX) and the Ca2+-discharged Y138F obelin mutant (PDB ID, 4MRY) (26). However, there is no direct evidence for their proposal, and our observations did not support this hypothesis. When Ca2+ binds to aequorin, the luminescence reaction of aequorin will start to release the hydrogen bonds of His169 and Tyr184 from the C2-peroxy moiety, followed by cleavage of the C2–C3 bond of the pyrazine ring to give the excited coelenteramide and CO2. The light emission is produced from the excited state of coelenteramide in apoaequorin. In general, CTZ-OOH is unstable in aqueous solutions, and the decomposition of CTZ through CTZ-OOH proceeds spontaneously without significant light emission. Thus, the conformational changes by Ca2+ to cleave the hydrogen bonds from the C2-peroxy moiety of CTZ are crucial for the emission of light from the complex of coelenteramide and Ca2+-bound apoaequorin. Luminescence kinetics of aequorin Based on the luminescence mechanism of aequorin by Ca2+, the luminescence kinetics could be explained by the binding affinity of Ca2+ to the EF-hand motifs of aequorin, followed by the speed of conformational changes to cleave the hydrogen bonds between amino acid residues and CTZ-OOH. Thus, slow conformational changes of the cf3-aequorin molecule by Ca2+ could give slow decay luminescence kinetics with a long half-decay time of Imax. Some semi-synthetic aequorins with CTZ analogs possessing a bulky C2-moiety, such as i-, br-, and n-aequorin, showed slow luminescence kinetics (Table IV) (13–15). A bulky moiety at the C2-benzyl group might interfere with the smooth conformational changes to cleave the hydrogen bonds of CTZ-OOH. In other cases, EF-hand I loop mutants of native aequorin with a positively charged amino acid such as AM20 also showed a slow decay luminescence pattern with a long half-decay time, as previously reported (22). This slow decay luminescence kinetics might be explained by the low Ca2+-binding affinity to EF-hands, resulting in a slow conformational change to emit light. In contrast, fast luminescence kinetics was observed in the semi-synthetic aequorin of cp-aequorin, which was prepared using cp-CTZ possessing a cyclopentylmethyl (cp) group replaced with a benzyl group at the C8 position of CTZ, the crystal structure of which had previously been determined (5). In the structure of native aequorin, the benzyl group at the C8 position of CTZ-OOH was involved in a π–π interaction with Trp108 of EF-hand III and a stacking interaction with Lys39 of EF-hand I. Thus, the fast luminescence kinetics of cp-aequorin was due to the absence of the stacking and π–π interactions of the cp group that induces the conformational changes faster than native aequorin to emit light. Conclusion Semi-synthetic aequorin of cf3-aequorin was prepared by incubation with cf3-CTZ and apoaequorin expressed in E. coli cells. Highly purified cf3-aequorin was obtained by ion exchange and hydrophobic column chromatography and showed slow luminescence kinetics with a long half-decay time of Imax. The crystal structure of cf3-aequorin, including 16 molecules per asymmetric unit, was determined, showing virtually the same protein structure as that of native aequorin. The major difference between cf3-aequorin and native aequorin is that two of the three water molecules that stabilize CTZ-OOH in native aequorin are missing in cf3-aequorin. The bulky trifluoromethyl group of cf3-CTZ-OOH interacted with helices E and H of apoaequorin and might interfere with the smooth conformational changes to emit light by cleavage of the hydrogen bonds at the C2-peroxy moiety of cf3-CTZ-OOH after the binding of Ca2+. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements The authors thank Drs. H. Niwa, N. Sakai, and T. Umehara, and the beamline staffs for the synchrotron radiation experiments at SPring-8. Funding This work was supported in part by grants from Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP17am0101098 (Platform Project for Supporting Drug Discovery and Life Science Research). The synchrotron radiation experiments were performed at BL26B2 in SPring-8 with the approval of RIKEN (Proposal No. 20160018). Conflict of Interest None declared. References 1 Shimomura O. , Johnson F.H. , Saiga Y. ( 1962 ) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea . J. Cell. Comp. Physiol. 59 , 223 – 239 Google Scholar CrossRef Search ADS PubMed 2 Inouye S. , Noguchi M. , Sakaki Y. , Takagi Y. , Miyata T. , Iwanaga S. , Miyata T. , Tsuji F.I. ( 1985 ) Cloning and sequence analysis of cDNA for the luminescent protein aequorin . Proc. Natl. Acad. Sci. 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( 2003 ) Atomic resolution structure of obelin: soaking with calcium enhances electron density of the second oxygen atom substituted at the C2-position of coelenterazine . Biochem. Biophys. Res. Commun . 311 , 433 – 439 Google Scholar CrossRef Search ADS PubMed 26 Natashin P.V. , Ding W. , Eremeeva E.V. , Markova S.V. , Lee J. , Vysotski E.S. , Liu Z.J. ( 2014 ) Structures of the Ca2+-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction . Acta Crystallogr. D Biol. Crystallogr. 70 , 720 – 732 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations cf3-CTZ trifluoromethylcoelenterazine cf3-CTZ-OOH 2-peroxytrifluoromethylcoelenterazine CHO-K1 Chinese hamster ovary K1 CTZ coelenterazine CTZ-OOH 2-peroxycoelenterazine DTT dithiothreitol EDTA ethylenediaminetetraacetic acid FWHM full width at half maximum GPCR G-protein-coupled receptor Imax maximum intensity of luminescence LB Luria–Bertani; PDB, Protein Data Bank rlu relative light units TE 50 mM Tris-HCl (pH 7.6)–10 mM EDTA © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: May 23, 2018

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