Protein expression and purification
The HormR and GAIN domains of rat CL1 (residues 460–849) and human BAI3 (residues 498–868) were cloned into the Xba1–Not1 and the BamH1–Not1 sites of the pAcGP67A vector, respectively (CL1=43 kDa plus 5 carbohydrates; BAI3=41.7 kDa plus 3 carbohydrates). A 8 × His tag was added at the C-terminus for affinity purification. Spodoptera frugiperda (Sf9) cells (Invitrogen) were transfected with pAcGP67A carrying the gene and linearized AcNPV DNA (Sapphire Baculovirus DNA; Orbigen) using Cellfectin (Invitrogen). Baculovirus was amplified in Sf9 cells in 10% (v/v) fetal bovine serum containing SF900-II medium (Gibco).
Large-scale protein expression of CL1 and BAI3 was performed by infection of Trichoplusia ni (High Five) cells in Insect-Xpress medium (Biowhitaker) at a cell density of 2 × 106 cells/ml with an infection course of 72 h. The media containing the secreted, glycosylated, recombinant protein was concentrated and dialysed using a tangential flow filtration system (PALL) and subsequently purified with Ni-NTA resin (Qiagen) at 4°C. The proteins were eluted with 200 mM imidazole (pH 7.2). CL1 and BAI3 were further purified by size-exclusion chromatography using a Superdex 200 column (Amersham Pharmacia Biotech) in buffer containing 10 mM Hepes (pH 7.2) and 150 mM NaCl. The peak corresponding to the proteins was then concentrated to 10.7 mg/ml (for CL1) and 17 mg/ml (for BAI3) in 30 kDa Centricon (Millipore) concentrators and used for crystallization trials.
α-Latrotoxin (residues 413–1080) with N-terminal GST and Myc tags in a pGEX-KG vector was expressed in Origami cells. The expression and purification were described in Li et al (2005)
. The latrotoxin construct used for native gel experiments corresponds to the 20 ankyrin repeats of latrotoxin with a C-terminal His tag. The protein was expressed in High Five cells and secreted protein was purified by affinity chromatography followed by size-exclusion chromatography.
The GAIN domain of rat CL1 (residues Thr533–Ile849) was cloned into a pCMV5 vector and fused to Ig (26 kDa) at the C-terminus and carried an HA tag at the N-terminus. The GAIN domain of mouse GPR56 (residues Q115–E395) was cloned into the pDisplay vector with N-terminal HA and C-terminal myc tags and has the PDGFR single-spanning transmembrane domain at the C-terminus. The full-length human PKD construct carried N-terminal GFP and C-terminal Flag tags. Full-length human CL3 was synthesized by codon optimization and carried Flag and HA tags at identical positions as in CL1.
Crystallization of CL1 and BAI3
Initial screens for crystallization of CL1 were carried out using 96-well format kits (Hampton Index, Salt and Crystal Screen HT, Hampton Research) on a Phoenix crystallization robot (Art Robbins Instruments). Automatic plate scanning by a CrystalPro 2 imaging system (TriTek Corporation) identified initial hits in the Wizard 1–2 screen (Emerald Biosystems). After optimization, crystals of CL1 were grown using hanging drop vapour diffusion at 20°C in 24-well format. In all, 1 μl of 10.7 mg/ml protein in 10 mM HEPES pH 7.2, 150 mM NaCl was mixed with equal volume of mother liquor consisting of 1.26 M (NH4)2SO4, 0.1 M sodium acetate pH 4.5, 0.2 M NaCl, and equilibrated against 1 ml mother liquor. Crystals grew to full size within 3 days. Crystals were cryoprotected by transferring the crystals into mother liquor with 10, 20, and 28% glycerol gradually. The best crystals diffracted to dmin=1.85 Å. Crystals of CL1 formed in space group P3121 with one molecule per asymmetric unit and 67% solvent content.
Crystallization hits for BAI3 were obtained with a 96-well format kit (Protein Complex, Nextal) at 20°C. In all, 17 mg/ml protein in 10 mM HEPES, 150 mM NaCl pH 7.2 was mixed with equal volume of mother liquor consisting of 15% PEG6000, 0.1 M sodium citrate pH 5.5. Crystals grew to full size within 2 days and were cryoprotected with 25% glycerol. Crystals diffracted to dmin=2.3 Å. Crystals of BAI3 formed in space group C2221 with two molecules per asymmetric unit and 63% solvent content.
Structure determination and refinement of CL1
For the CL1 structure, experimental phase information was obtained from sulphur SAD data. This structure was the first structure that was determined at the Stanford Synchrotron Radiation Lightsource by sulphur SAD phasing using an optimized data collection strategy for a long wavelength at 2 Å, and one of a few de-novo
structures that has been determined by sulphur SAD at this resolution. More than 40 crystals were screened to find the best diffracting large crystal. A test radiation damage experiment was performed on a different crystal to determine the effect of radiation damage and to optimize the parameters for the experiment. Three data sets, each comprising 360° and offset by 20°, were collected on a single long crystal by translating the beam along the long edge of the crystal. The crystal was placed in a helium stream operating at 65 K to minimize background scatter at long wavelength and to further limit radiation damage. Data collection was carried out in inverse beam mode with a wedge angle of 0.5° and an exposure time of 8 s. Total data collection lasted 9 h. The data were collected at a wavelength of 2 Å, which limited the resolution to 2.3 Å (diffraction was observed to 1.8 Å resolution at a wavelength of 1 Å). The resolution limit decreased from 2.3 to 2.7 Å at the end of one 360° data collection as a result of radiation damage. All data sets were indexed, integrated, scaled, and combined in HKL2000 using the scale anomalous and high absorption options (Otwinowski, 1997
). The error model was not optimized during scaling. In all, 25 sulphur sites were found (17 sites within the protein and 8 in sulphate ions from the crystallization condition) by SHELXD (Schneider and Sheldrick, 2002
). SHELXD used a high-resolution cutoff of 2.7 Å, at which the I
/σ was ~50. The sulphur sites were refined using SHARP (Vonrhein et al, 2007
). Density modification was performed by DM (Cowtan and Zhang, 1999
). Among all space groups tested, only space group P31
21 yielded easily interpretable electron density maps. The density modified electron density maps showed good connectivity of secondary structure elements and cleavage at the GPS site (Supplementary Figure S7A
). The sulphur sites corresponded to the expected side chains when compared within the density modified electron density map. Automatic model building was done with Phenix Resolve (Terwilliger, 2003
) for two thirds of the structure. The rest of the model building was carried out manually using Coot (Emsley and Cowtan, 2004
), and refinement performed with Phenix.refine (Adams et al, 2010
) using the MLHL target function with experimental phases. The resulting model was then refined against the higher resolution native data set at 1.85 Å resolution. The subsequent refinement consisted of alternating rounds of positional minimization, individual restrained thermal factor refinement, both using the MLF maximum likelihood target function, and model building. Water molecules and N-linked carbohydrates were added. A summary of the data collection and refinement statistics is given in Supplementary Table S1
(PDB accession code 4DLQ
Structure determination and refinement of BAI3
BAI3 crystals were soaked into three solutions containing the crystallization condition, 0.25 M sodium iodide, and 10, 20, and 30% glycerol immediately before they were frozen in liquid nitrogen. The total soak time was about 1–2 min. Only one in ten crystals survived this treatment. The presence of increased glycerol, thus viscosity, in the soak solutions prevented the crystals from cracking. Data collection was done in inverse beam mode with a wedge angle of 25° and an exposure time of 5.5 s. The derivative crystals were not isomorphous to the native crystals, so the native data set could not be used for isomorphous phasing. The diffraction data were collected at a wavelength of 1.6 Å, resulting in diffraction data to 2.7 Å resolution. Data processing was similar to that of CL1. The BAI3 structure was independently determined with experimental phases obtained by iodine-SAD data (a density modified map of the iodine-SAD data around the GPS site is shown in Supplementary Figure S7B
, clearly indicating lack of cleavage in contrast to the CL1 structure). SHELXD used a high-resolution cutoff of 4 Å, at which the I
/σ was ~50, and it found 6 iodine sites. There were two molecules in the asymmetric unit and non-crystallographic symmetry (NCS) restraints were used during initial refinement cycles using Phenix.refine but were removed at later stages. A summary of the data collection and refinement statistics is given in Supplementary Table S1
(PDB accession code 4DLO
Cleavage and plasma membrane localization experiments
Antibodies. Mouse monoclonal anti-HA antibody was from Covance and rabbit polyclonal anti-Flag antibody was from Sigma.
Mutagenesis and expression constructs.
CL1 deletion constructs were engineered by PCR oligonucleotide amplification. pCMV-CL1 HA Flag was generated as follows: The Flag epitope containing constructs were obtained by subcloning Hin
I PCR fragment from pCMV-CL1 into the same site of pCMV-NL1-Flag. The Flag epitope was fused at Leu25 of CL1. A single HA epitope was inserted by PCR between Tyr918 and Glu919 in the second extracellular loop of CL1. Deletion constructs were generated using pCMV-CL1 HA Flag construct as a backbone. Deletion constructs are as follow: pCMV-CL1-ΔHormR HA Flag was generated by deleting Phe479–Cys532. pCMV-CL1-GPS HA Flag was generated by deleting Phe479–Glu792. pCMV-CL1-GPS_HormR HA Flag was generated by deleting Pro535–Glu792. pCMV-CL1-ΔSubA HA Flag was generated by deleting Pro535–Val666. pCMV-IgCL1 LEC was generated by amplifying Met1–Tyr131 PCR fragment and subcloning into Eco
I of pCMV-Ig. pCMV-IgCL1 GAIN was generated by subcloning the PCR-amplified fragment of Thr533–Ile849 into Sac
I of pDisplay then by excising Eco
I fragment and subcloning into the same sites of pCMV-Ig. Therefore, the resulting pCMV-IgCL1 GAINR construct also bears an N-terminal HA epitope. pCMV IgN1a-1 and pCMVN1b were described elsewhere (Ushkaryov et al, 1994
; Sugita et al, 2001
). pCMV-CL1 T838A and pCMV-CL1 T838P were generated by PCR mutagenesis. PKD mutagenesis was performed by cutting out the wild-type human PKD with Bsi
WI restriction enzyme, pasting the small fragment into a carrier plasmid. After site-directed mutagenesis was performed, the mutated fragments were subcloned back into the original plasmid. CL3 and CL1 mutagenesis was performed by the Qiagen site-directed mutagenesis kit.
Cell culture and transfection. For immunoblotting and immunocytochemistry experiments, HEK293T cells were cultured in 6-well plates until they reached 70–80% confluency. The cells were then transfected using 4 μl of Fugene 6® (Roche Diagnostics) and 2 μg of respective CL1 DNA constructs. For immunoblotting experiments, cells were harvested 48 h post transfection. For immunocytochemistry experiments, the cells were trypsinized 24 h post transfection and splitted into 12-well plates containing coverslips that have been previously coated with 0.5 mg/ml poly-L-lysine in borate buffer. The cells were grown on the coverslips for an additional 24 h before further experiments were done. For recombinant Ig-protein expression, HEK cells were transfected using calcium phosphate with chloroquine and 20 μg of cDNA corresponding to the various Ig proteins. Media was then harvested 4 days post transfection.
Immunocytochemistry and image acquisition. Cells transfected with CL1 constructs were washed once with PBS and fixed with 4% paraformaldehyde for 10 min on ice. Cells were washed again three times with cold PBS and incubated at room temperature for 30 min in a blocking solution containing 3% BSA in PBS with or without 0.1% Triton X-100 meaning that cells were permeabilized or not, respectively. Mouse anti-HA antibody was then added (1:500 ratio) and the incubation was prolonged for another 2 h. Cells were washed three times with blocking solution and incubated for 1 h at room temperature with anti-mouse Alexa-488 fluorescent antibody to label CL1 receptors. Cells were finally washed again three times with blocking solution and once with water before mounting on slides using media containing DaPI for nuclear staining. Slides were then analysed by confocal microscopy. Images were acquired using the confocal microscope Leica TCS2. The same confocal acquisition settings were applied to all samples of the experiment. Collected z-section images were analysed blindly using Leica confocal software.
Immunoblotting procedures. Transfected HEK cells were harvested 2 days after transfection and solubilized for 1 h at 4°C in buffer containing 20 mM Hepes-NaOH pH 7.4, 0.1 mM EDTA, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 and 1% Triton. Insoluble material was removed by centrifugation. SDS–PAGE sample buffer was added to the supernatant and the resulting sample was loaded on 6% SDS–PAGE. Gels were then transferred onto nitrocellulose membranes and processed using standard procedures. Membranes were probed with a mouse monoclonal anti-HA antibody or a rabbit polyclonal anti-Flag antibody followed by Horseradish peroxidase-coupled secondary antibody, incubated with ECL reagents and revealed on X-ray films.
Ig-fusion protein purification and pull-down experiments
The soluble Neurexin and CL1 Ig-fusion proteins were affinity purified from transfected HEK293 cell supernatant using protein-A Sepharose 4 Fast Flow (Amersham Pharmacia Biotech AB, Sweden) to bind the human-IgG portion and washed to remove unbound proteins. The immobilized Ig-fusion proteins were then incubated 16 h at 4°C with the eluted 3 μg GST–myc–Latrotoxin in buffer containing 20 mM Hepes buffer, pH 7.4; 0.1 mM EDTA, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 and 1% (v/v) Triton X-100. The beads were then washed, incubated in boiling SDS–PAGE sample buffer and loaded on 6% SDS–PAGE. SDS–PAGE gels were either transferred onto nitrocellulose membrane and immunoblotted with anti-myc antibody or stained using Coomassie Brilliant Blue R-250 (Bio-Rad).
BAI cleavage experiments
Antibodies Goat antiserum 11509 and rabbit antisera 233C and 234C were generated against a synthetic peptide (N-CEKAGATIPLVGQDIIDLQTEV-C; mouse BAI1). Rabbit antisera A324 and A323 were raised against recombinant GST-fusion proteins (P1447 → V1582 of mouse BAI1 and R1151 → E1348 of human BAI3, respectively). T3743 was raised against GFP in rabbit.
Cell culture HEK293T cells were plated in 6-well plates. At 70–80% confluency, they were transfected with 1 μg expression plasmid (FuGENE 6 from Roche), and then cultivated for 2 days. Cells were lysed in extraction buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitors), homogenized, and the soluble fractions were collected after centrifugation.
Immunoprecipitation Two mouse brains were homogenized in 8 ml buffer (25 mM HEPES pH 7.2, 320 mM sucrose, protease inhibitors). After centrifugation at 1500 g for 10 min, supernatant S1 was centrifuged at 150 000 g for 60 min. Pellet P2 was then homogenized in 6 ml lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1% Triton X-100, protease inhibitors), and centrifuged again at 150 000 g for 60 min. In all, 3 ml of supernatant S3 was incubated with 18 μl antiserum 11509 for 60 min. Then, 180 μl protein G sepharose (GE Healthcare) was added, and reactions were incubated for 3 h. The beads were washed four times with lysis buffer, and the proteins were eluted in SDS buffer.
Immunoblotting Proteins were resolved on 6% acrylamide gels and transferred onto nitrocellulose membranes (Whatman). After a blocking step, membranes were incubated overnight with the primary antibody diluted in blocking solution (5% milk powder, 2.5% goat or rabbit serum, 0.1% Tween-20, in TBS). After washing with TBS containing 0.1% Tween-20, blots were incubated with a peroxidase-conjugated secondary antibody (Cappel) for 45 min. After washing, immunoreactive bands were detected by ECL (GE Healthcare).