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[email protected] Protein & Peptide Letters, 2013, 20, 1211-1216
1211
Human Cardiac Ryanodine Receptor: Preparation, Crystallization and Preliminary X-ray Analysis of the N-terminal Region ubomír Borkoa, Július Ko anb, Alexandra Zahradníkováa,c, Vladimír Pevalaa, Juraj Gaperíka, Eva Hostinováa, ubica Urbánikováa, Kristina Djinovi -Carugob,d, Vladena Bauerová-Hlinkováa* and Jozef ev íka* a
Department of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovakia; bDepartment of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria; cDepartment of Muscle Cell Research, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia and dDepartment of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Akereva 5, SI-1000 Ljubljana, Slovenia. Abstract: Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation-contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating. The N-terminal region of hRyR2 has been implicated in regulating basal channel activity by interaction with the central hRyR2 region. This paper reports preparation, crystallization and preliminary X-ray analysis of recombinant hRyR21-606 N-terminal fragment. Soluble hRyR21-606 was expressed in Escherichia coli. Purification conditions were optimized using thermal shift assay. The quality and stability of the sample was probed by dynamic light scattering. A monomeric protein showing over 95% purity was obtained. The protein was crystallized by the hanging drop vapor-diffusion method. Diffraction data with resolution 2.39 Å were collected and processed.
Keywords: Crystallization, dynamic light scattering, human cardiac ryanodine receptor (hRyR2), protein purification, thermal shift assay. 1. INTRODUCTION Ryanodine receptors (RyRs) are homotetrameric proteins that reside in the membrane of endo/sarcoplasmic reticulum and form calcium permeable ion channels [1,2]. Each subunit contains 5000 amino acid (aa) residues [3]. The three known RyR isoforms, RyR1, RyR2, and RyR3, are differentially expressed: RyR1 is predominantly found in skeletal muscle cells, RyR2 in cardiac myocytes, and RyR3 is present in a variety of tissues [4]. In cardiac myocytes, the RyR2 isoform mediates the release of Ca2+ ions from the sarcoplasmic reticulum to the cytosol in response to electrical stimulation of the cell during excitation-contraction coupling [5]. A small amount of Ca2+ leaks out of the sarcoplasmic reticulum through the RyR2 channels in the absence of stimulation as well, resulting in diastolic calcium release [6]. Proper function of RyR2 channels is essential for the calcium homeostasis of the cardiac cells. The function of human RyR2 (hRyR2) channels is compromised in most cardiac diseases. In heart failure, calcium release in response to the action potential is decreased while diastolic calcium release is enhanced [7,8]. Some forms of the genetic diseases CVPT *Address correspondence to these authors at the Institute of Molecular Biology SAS, Dúbravská cesta 21, 845 51 Bratislava; Tel: +421 (2) 59307435; +421 (2) 59307423; E-mails:
[email protected];
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(catecholaminergic polymorphic ventricular tachycardia) and ARVD2 (arrhythmogenic right ventricular dysplasia) that lead to life-threatening arrhythmias are caused by hRyR2 mutations (http://www.fsm.it/cardmoc/). These mutations alter activity of the hRyR2 channel and may result in premature calcium release in the absence of the action potential [7,8]. Out of the 150 hRyR2 mutations and single nucleotide polymorphisms associated with cardiac arrhythmias, 31 are clustered within the first 655 N-terminal aa (http://www.fsm.it/cardmoc/). This region consists of three domains conserved in hRyR and IP3R channels – the Pfam domains INS145_P3_rec, MIR, and RIH [9,10,11] and is located in the large cytoplasmic part of the channel [12,13]. Structures of the rabbit RyR1 isoform, encompassing aa 1210 [14], 9-205 [15], and 1-559 [12] and of the mouse RyR2 aa 1-217 [15] have been determined. So far there are no data on the structure of the human cardiac N-terminal region. Because of the high amino acid identity between hRyR2 and rabbit RyR1 N-terminal regions, it is expected that the structure of hRyR2 in the N-terminal region will be closely similar to that of RyR1. However, there are several segments with major sequence differences, and further 97 amino acids (17%) not resolved in 2XOA. We expect that solving the structure of hRyR21-606 will increase the understanding of the structural basis of the effect of CPVT and ARVD mutations on RyR2 function. Here we report the preparation, crystalli© 2013 Bentham Science Publishers
1212 Protein & Peptide Letters, 2013, Vol. 20, No. 11
zation and preliminary X-ray analysis data of hRyR1-606. To obtain high-quality protein, the purification procedure was optimized by thermal shift assay. The crystallization procedure and conditions had to be considerably different from those used in crystallization of the RyR1 equivalent (aa 1559, 2XOA [12]).
Borko et al.
throughput screening are given in Fig. 1. A 1:1000 dilution of the 5000 SYPRO Orange (Invitrogen) protein dye solution was used. The initial and final holding steps were set to 10 s at 25°C and 95°C, respectively, the ramp increment was set to 1°C and the scanning time to 15 s. 2.3. Dynamic Light Scattering
2. MATERIALS AND METHODS 2.1. Cloning, Expression and Purification The N-terminal fragment of the hRyR2, aa 1-606 [10, 11] was used in this study. In brief, it was prepared using the BT4 template [16] carrying the N-terminal cDNA sequence (aa 1-759) of hRyR2 and amplified using a standard PCR protocol with Platinum Pfx DNA polymerase (Invitrogen). The His-Tag was introduced to the C-terminus via reverse primer as an uncleavable fusion. The amplified product was inserted into the pET28a vector (Merck Millipore) using the restriction sites NcoI and BamHI upstream and downstream of the coding sequence, as previously described [11]. The resulting plasmid constructs were introduced to Escherichia coli TG1 cells for plasmid amplification. The integrity of the hRyR21-606 insert was verified by DNA sequencing. For heterologous expression, Escherichia coli BL21 (DE3) cells were transformed with the pET28a hRyR21-606 construct, expression was induced with IPTG in a concentration of 10 M, and carried out at 18°C for 12-16 hours. The bacterial cells were harvested by centrifugation (3000 g for 20 min at 4°C), resuspended in lysis solution (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM imidazole, 7 mM 2mercaptoethanol, 0.1 % Tween 20) and disrupted by sonication at 4°C. The lysate was centrifuged (100,000 g for 30 min at 4°C) and the obtained supernatant was applied to a gravity-flow immobilized metal ion chromatography (IMAC) column filled with His-Select nickel affinity gel (Sigma-Aldrich). The contaminant proteins were washed away by 20 volumes of wash solution (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 7 mM 2mercaptoethanol, 10 % glycerol, 1 mM CHAPS and 0.1 % betaine). The target hRyR21-606 was eluted by 20 mM TrisHCl pH 8, 150 mM NaCl, 400 mM imidazole, 7 mM 2mercaptoethanol, 10 % glycerol, 1 mM CHAPS and 0.1 % betaine. Eluted, partially purified protein was further subjected to size-exclusion chromatography (SEC) using Superose12 HR 10/30 column (GE Healthcare) pre-equilibrated with the storage buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 7 mM 2-mercaptoethanol, 10 % glycerol, 1 mM CHAPS and 0.1 % betaine). The whole purification procedure was performed at 4°C. Individual SEC fractions were analyzed by SDS-PAGE. The fractions with target protein were pooled and concentrated to 5 mg.ml-1 by Microsep Advance and Nanosep (Pall Corporation) centrifugal devices with 10 kDa cutoffs. All chemicals were from Sigma-Aldrich if not noted otherwise. 2.2. Thermal Shift Assay Thermal shift assay [17, 18] was used to improve purification conditions by finding a suitable buffer system, pH and additives. It was performed with iCycler IQ5 PCR Thermal Cycler (Bio Rad) operated with IQ5 software Ver. 2.1, using 5 g of protein in 25 l reaction. The conditions for high-
Dynamic light scattering (DLS), was used to analyze the polydispersity and stability of the hRyR21-606 sample. It was carried out on a Zetasizer Nano-S device (Malvern Instruments) at a concentration of 2-4 mg.ml-1 of protein in the storage buffer (composition as above). All DLS measurements were performed at 10°C in a UV microcuvette (70 μl, Brand). Samples were filtered before measurements through a 0.45 μm filter (Millipore) to remove dust particles. 2.4. Crystallization Purified protein samples concentrated to 5 mg.ml-1 were used for crystallization. Screening of the crystallization conditions was performed with a nanodrop-dispensing robot (Phoenix™ DT, Art Robbins Instruments) using the sitting drop vapor diffusion method in 96-well MRC2 plates (SWISSCI) and mixing the solution of hRyR21-606 in the storage buffer with the reservoir solution (see below) in a volume ratio of 1:1 and 2:1. The droplet volume was 200400 nl. Crystallization screens were performed at 22°C with the following crystallization kits: PACT Premiere, Morpheus, MIDAS HT-96 (all from Molecular Dimensions), JCSG (Qiagen), Crystal Screen, SaltRx and PEG/Ion HT (all from Hampton Research). Small tetragonal crystals were obtained after 5-day incubation in 200 mM ammonium formate, 20 % PEG 3350 from the PEG/Ion HT kit. Because of the observed decrease of crystal quality in time, the buffering conditions were further optimized. The buffers Tris-HCl, pH 7.0-7.5; HEPES, pH 6.7-7.3; Bis-Tris, pH 6.4-7.4 were tested. The best results were obtained with HEPES, which was used for fine optimization in the concentration range of 85-100 mM and in the pH range of 6.6-7.3. Other varied components were PEG 3350 (12-24 %) and ammonium formate (160-200 mM). Fine optimization was performed by the hanging drop vapor diffusion method using 48-well (VDX) and 24-well (Linbro) plates (Hampton Research). The droplet volume was 2-3 l. Final crystallization conditions are given in Table 1. 2.5. Data Collection and Preliminary X-ray Diffraction Analysis For testing and data collection, flash-frozen crystals in liquid nitrogen and 15 % ethylene glycol as a cryoprotectant were used. Diffraction data to 2.39 resolution were collected at the Synchrotron Radiation Source BESSY II, Berlin, Germany, using MX Beamline 14.1 and temperature -196°C. The wavelength of radiation was 0.918 Å, the crystal-to-detector distance was 276.76 mm and the crystal rotation angle per image was 0.5°. The data were processed with XDS [19] and iMosflm [20] and scaled by SCALA [21]. Crystals belonged to space group P42212. The crystal packing density [22] was calculated from the unit-cell volume (1,407,622 Å3), the molecular mass of the protein (67 kDa) and the space group (P42212), giving VM = 2.64 Å3.Da-1, and
Human Cardiac Ryanodine Receptor N-terminal Region
53.5 % solvent content for one protein molecule in the asymmetric unit. The details on diffraction data collection and processing are given in Table 2. 3. RESULTS AND DISCUSSION In our previous work we reported cloning, expression and production of several recombinant cardiac ryanodine receptor (RyR2) N-terminal protein fragments with and without fusion tags (NusA protein and thioredoxin) [11]. The Nterminal domain of hRyR2 has 63% identity and 77% similarity with the published rabbit RyR1 structure. The alignment of the two sequences has been previously reported and used to build a model of hRyR21-543 [10]. As the main predicted difference, hRyR2 contains an extra -helix (aa 91108), not present in the rabbit RyR1 isoform. In this study we have chosen the hRyR21-606 fragment because of its high expression level, stability and sample homogeneity. The N-terminal part of hRyR2 (aa 1-655) has
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been implicated in regulating basal RyR2 channel activity by interaction with the central hRyR2 domain [23]. Moreover, currently there are more than 25 known distinct mutations in this region (http://www.fsm.it/cardmoc/) that participate in the etiology of inherited arrhythmias (catecholaminergic polymorphic ventricular tachycardia, CPVT, and arrhythmogenic right ventricular dysplasia, ARVD; see [24] for review. To validate initial purification conditions and to find stabilizing additives for improved protein purification conditions, required for successful crystallization, high-throughput screening using thermal shift assay was performed. Detailed conditions of the screening are shown in Fig. 1. Screening confirmed a higher stability of hRyR21-606 in neutral-basic pH (7.0 – 8.5) with TM of 42-45°C in comparison to acidic pH (5.0 – 6.2) with TM 36°C [10]. The Tris-HCl buffer (pH 8.0) was chosen for further purification. Stability of hRyR21-606 was improved by 10% glycerol while imidazole
Figure 1. Thermal shift assay of the hRyR21-606 fragment. Higher melting temperatures indicate a positive effect of buffers/additives on protein stability. The columns represent an average of three independent measurements.
Figure 2. Purification of hRyR1-606. A. Purification profile of hRyR1-606 (thick line) and of the molecular weight standard BSA (thin line) at 280 nm. Monomers of hRyR1-606 and of BSA (MW 67 kDa) were eluted at 12.90 ml and BSA dimers were eluted at 11.67 ml. Fraction numbers (above the x-axis) correspond to the SDS-PAGE gel in panel B. B. 10 % Tris-glycine SDS-PAGE gel with fractions after SEC on Superose12 HR10/30 column.
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Figure 3. The DLS profile of hRyR1-606 at 10°C one hour (solid line), two days (dashed line) and four days (crosses) after preparative size exclusion chromatography. Each curve represents an average of three independent measurements. A. Intensity size distribution. B. Volume size distribution.
Figure 4. A. Tetragonal crystals of hRyR1-606 with dimensions of 100 100 40 m B. Example of a diffraction image collected from hRyR1-606 crystals.
was found to have destabilizing effect (Fig. 1). The IMAC purification step requires the usage of imidazole; however, the simultaneous presence of glycerol compensated its deleterious effect. The zwitterionic detergent CHAPS was used below critical micellar concentration and was complemented with 0.1-1% betaine. The resulting conditions for purification and storage of hRyR21-606 were as follows: 20 mM TrisHCl, pH 8.0, 150 mM NaCl, 7 mM 2-mercaptoethanol, 10 % glycerol, 1 - 4 mM CHAPS, 0.1-1 % betaine. The purification profile of hRyR21-606 is shown in Fig. 2. Pooled fractions 15 – 17 were concentrated to 5 mg.ml-1 and used for crystallization trials. The polydispersity and stability of the sample was tested by dynamic light scattering (DLS). Intensity size distribution demonstrated multiple peaks, however, volume size distribution confirmed that the amount of large aggregates was negligible and the main component of the sample were particles with mode hydrodynamic radius of 3.765 nm corresponding to protein monomer with MW = 75.4 kDa. The hRyR21-606 MW is 67 kDa. The MW discrepancy emerging from DLS
measurements is probably caused by the composition of the storage buffer and the shape of the hRyR21-606 molecule. The polydispersity index of the sample was 0.355 with protein monomer polydispersity of 31.2 %. The sample remains stable for at least four days when kept in the storage buffer at 610°C (Fig. 3). No effect of several cycles of freezing to 196°C and thawing on the stability of hRyR21-606 was observed (data not shown). After initial high-throughput screening and consequent scaled-up crystallization, diffraction quality tetragonal crystals (with dimensions of 100 100 40 m) were obtained within 4-6 days (Fig. 4A). The crystallization procedure and conditions had to be considerably different from those used in crystallization of the RyR1 equivalent (aa 1559, 2XOA [12]). The optimized crystallization conditions are shown in detail in Table 1. A complete diffraction data set was collected to 2.39 Å resolution (Fig. 4B). Data collection statistics are shown in Table 2. Structure determination and refinement of the hRyR21-606 is in progress.
Human Cardiac Ryanodine Receptor N-terminal Region
Table 1.
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Final crystallization conditions of the recombinant hRyR21-606 fragment. hRyR1-606 Method
Hanging drop
Plate
24-well Linbro plate
Temperature
22°C
Protein concentration
5 mg.ml-1
Protein sample buffer composition
20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 % glycerol, 7 mM 2-mercaptoethanol, 1 mM CHAPS, 0.1 % betaine
Reservoir solution composition
100 mM HEPES, pH 6.9, 200 mM ammonium formate, 21 % PEG 3350
Drop volume and ratio
3 l, 1:2
Reservoir volume
400 l
Table 2.
Data collection statistics. Values in parentheses correspond to the highest resolution shell. Beamline
MX Beamline 14.1
Wavelength ( )
0.918
Detector
MarMosaic 225
Temperature (K)
100
Resolution range ( )
44.77 – 2.39 (2.52-2.39)
Space group
P42212
Unit-cell parameters a,b,c ( ); , , (°)
75.45, 75.45, 248.84; 90, 90, 90
Rmeas
0.16 (1.54)
*R(I)merge
0.15 (1.44 )
Observed reflections
406071 (58253)
Unique reflections
29469 (4205)
Redundancy
13.8 (13.9)
I/(I)
12.2 (1.9)
Completeness (%)
100 (100)
2
B (Wilson plot) ( ) 3
50.4 -1
Matthews coefficient (Å Da )
2.64
Molecules in asymmetric unit
1
Mosaicity (°)
1.14
Solvent content (%)
53.5
*R(I)merge = h,k,l j Ihkl,j - ‹ Ihkl,j › / h,k,l j Ihkl,j ‹ Ihkl,j › is the average of symmetry (or Friedel) related observations of a unique reflection.
CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS This work was supported by the research grants from the Slovak Grant Agency VEGA No. 2/0131/10 and Slovak Re-
search and Development Agency APVV-0628-10. The authors wish to thank Dr. Eva Kutejová, Dr. Jacob Bauer for helpful discussions and Dr. Bjoern Sjoeblom for help with thermal shift assay. The use of the plasmid BT4 from the laboratory of F. A. Lai, Cardiff University in previously published work [10, 11], in which the RyR21-606 fragment was constructed, is gratefully acknowledged.
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Received: March 20, 2013
Revised: May 29, 2013
Accepted: May 30, 2013
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