Materials and Methods
Cloning, expression and purification
The S. pyogenes cas1 and cas9 genes were cloned into pET28a vectors with a C-terminal (His)6 tag (Supplementary Table S1). The S. pyogenes cas2 and csn2 genes were cloned into pET28a and pHMGWA vectors, respectively, each of which contain an N-terminal (His)6-maltose binding protein (MBP) tag and a tobacco etch virus (TEV) protease cleavage site (Supplementary Table S1). E. coli BL21 (DE3) cells transformed with each individual construct were cultured in lysogeny broth (LB) medium at 37°C until the optical density at 600 nm reached 0.7. Protein expression was induced by the addition of 0.3 mM isopropyl-β-d-thiogalactopyranoside, followed by incubation at 17°C for 16 h. Cells were harvested by centrifugation and resuspended in lysis buffer (300 mM NaCl, 10% (w/v) glycerol, 5 mM β-mercaptoethanol (BME) and 20 mM Tris–HCl (pH 8.0)).
After sonication and centrifugation, the supernatant was loaded onto a 5 ml HisTrap HP column (GE Healthcare, USA) that was pre-equilibrated with elution buffer (300 mM NaCl, 10% (w/v) glycerol, 5 mM BME, 30 mM imidazole, 20 mM Tris–HCl (pH 8.0)). After washing the column with elution buffer, the bound protein was eluted by applying a linear gradient of imidazole (up to 450 mM). The (His)6-MBP tag of Csn2 was cleaved with TEV protease and separated by using a 5 ml HisTrap HP column (GE Healthcare), whereas that of Cas2 was maintained due to the solubility issue. Cas1 was further purified using a 5 ml HiTrap Heparin HP column (GE Healthcare). All proteins were finally purified using a HiLoad 16/60 Superdex200 column (GE Healthcare) equilibrated with SEC buffer (200 mM NaCl, 10% (w/v) glycerol, 2 mM dithiothreitol (DTT), 20 mM Tris–HCl (pH 8.0)).
The Cas1–(His)6-MBP-Cas2 complex was generated by co-expression. The genes of Cas1 and Cas2 were cloned into pET21a, which does not contain a tag, and pET28a, which includes an N-terminal (His)6-MBP tag, respectively. Cas1 and (His)6-MBP-Cas2 were co-expressed in E. coli BL21 (DE3) cells containing both constructs. The expression was induced as described above for the individual Cas proteins. The Cas1–(His)6-MBP-Cas2 complex was purified in a three-step procedure using a HisTrap HP column, a HiTrap Heparin HP column and a HiLoad 16/60 Superdex200 column (GE Healthcare).
Crystallization and structure determination of Cas2
To determine the crystal structure of S. pyogenes Cas2, we generated a truncated cas2 construct, in which the first N-terminal five residues were replaced with a Ser-Gly-Ser-Gly-Ser segment, and the C-terminal tail (residues 92–113) was removed. This construct was cloned into a pET28a vector containing an N-terminal (His)6-MBP tag and a TEV protease cleavage site (Supplementary Table S1). The protein was expressed and purified as described above for Csn2 without the (His)6-MBP tag. Crystals were grown at 20°C by the sitting-drop vapor diffusion method from 14 mg/ml protein solution in buffer (300 mM NaCl, 5% (w/v) glycerol, 5 mM BME, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.0)) mixed with an equal amount of reservoir solution (22.0% (w/v) polyethylene glycol (PEG) 4000, 325 mM ammonium sulfate, 0.1 M sodium acetate (pH 4.6)). To determine the crystal structure using single-wavelength anomalous diffraction, the selenomethionyl protein was expressed in E. coli BL21 (DE3) cells grown in M9 medium supplemented with SeMet, as described previously (42). The selenomethionyl protein was purified as described above for the native protein, and its crystals were grown under similar conditions (26.5% (w/v) PEG 4000, 225 mM ammonium sulfate, 0.1 M sodium acetate (pH 3.6)). The native and selenomethionyl crystals were flash-frozen in liquid nitrogen without additional cryo-protective reagents.
Diffraction data were collected at the beamline 7A of the Pohang Accelerator Laboratory at 100 K. Diffraction images were processed with HKL2000 (43). Determinations of selenium positions, density modification and initial modeling of the selenomethionyl protein were done using PHENIX (44). The initial model of the selenomethionyl structure was used for phasing of the native structure in PHASER (45). The structure of the native protein was completed using alternate cycles of manual fitting in COOT (46) and refinement in PHENIX (44). The stereochemical quality of the final model was assessed using MolProbity (47).
Analytical SEC was performed on Superdex 200 10/300 GL columns (GE Healthcare). The columns were equilibrated with buffer (150 mM NaCl, 2 mM DTT, 20 mM Tris–HCl (pH 8.0)). To test complex formation, protein samples were incubated together in buffer (200 mM NaCl, 2 mM DTT, 20 mM Tris–HCl (pH 8.0)) at 4°C for 1 h and loaded onto one of the columns at a flow rate of 0.5 ml/min. Control experiments for single protein components were performed as references in the same column as their mixtures. A standard curve for the SEC column is provided in Supplementary Figure S1. Experiments with varying salt concentrations were performed by applying different amounts of NaCl to the column. Elution fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie staining.
The ITC experiments were performed at 25°C using a MicroCal iTC200 system (GE Healthcare). Proteins in a 200 μl sample cell were titrated with 20 consecutive 2 μl injections in buffer (150 mM NaCl, 10% (w/v) glycerol, 20 mM Tris–HCl (pH 8.0)). Origin software (OriginLab) was used for processing and analysis of the ITC titration data with a fitting model assuming multiple identical independent binding sites.
The SPR binding assays were performed using a CM5 chip on a Biacore T200 (GE Healthcare). The amine coupling ligand immobilization procedure was performed at a flow rate of 5 μl/min. The CM5 chip was activated with a mixture of 0.1 M N-hydroxysuccinimide and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride at a ratio of 1:1 for 400 s. Subsequently, 60 μg/ml of Cas9 dissolved in 10 mM sodium acetate (pH 5.0) was injected for 30 s. The remaining activated carboxyl groups on the sensor chip surface were deactivated with 1 M ethanolamine (pH 8.5) for 400 s. The multi-cycle analysis was performed at a flow rate of 30 μl/min. Each concentration of Csn2 in running buffer (150 mM NaCl, 10 mM Tris–HCl (pH 8.0)) was injected for 600 s, followed by dissociation for 1200 s in a separate analysis cycle. The sensor chip surface was regenerated with 10 mM NaOH between cycles. Data were fit with the bivalent analyte model. The equilibrium dissociation constant (_K_d) was determined based on kinetic rate constants calculated using Biacore T200 evaluation software (GE Healthcare).