Materials and Methods

The structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites

MATERIALS AND METHODSCloning, expression and purificationThe synthetic AcrIE4-F7 gene was cloned into pET28a containing either N-terminal (His)6 or (His)6-maltose binding protein (MBP) tags with a tobacco etch virus (TEV) protease cleavage site. Mutant AcrIE4-F7 genes were generated using polymerase chain reaction (PCR) with mutagenic primers. Each construct was transformed into Escherichia coli BL21(DE3) cells, which were grown in LB medium at 37°C to an optical density at 600 nm of 0.6. Protein expression was induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 17°C for 16 h. The cells were then harvested by centrifugation and resuspended in lysis buffer (20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0, 300 mM NaCl, 5 mM β-mercaptoethanol (BME), 10% (w/v) glycerol, 30 mM imidazole, 0.3 mM phenylmethylsulfonyl fluoride and 0.02% (w/v) Triton X-100). After sonication and centrifugation, the resulting supernatant was loaded onto a 5-mL HisTrap HP column (GE Healthcare) pre-equilibrated with binding buffer A (20 mM MOPS, pH 7.0, 300 mM NaCl, 5 mM BME, 10% (w/v) glycerol and 30 mM imidazole). After washing with the same buffer, the protein was eluted with a linear gradient of imidazole (up to 500 mM). The N-terminal (His)6-MBP tag was cleaved by TEV protease and separated using the 5-ml HisTrap HP column (GE Healthcare). AcrIE4-F7 was further purified by size-exclusion chromatography (SEC) using a HiLoad 16/60 Superdex 75 column (GE Healthcare) pre-equilibrated with SEC buffer A (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0, 150 mM NaCl and 2 mM 1,4-dithiothreitol (DTT)).The genetic fragments encoding the N-terminal and C-terminal domains of AcrIE4-F7 were amplified using PCR from its full-length gene and cloned into pET21a with a C-terminal (His)6 tag and the N-terminal (His)6-MBP tag with a TEV protease cleavage site, respectively. The resulting constructs were transformed into E. coli BL21(DE3) cells and expressed as described above for the full-length AcrIE4-F7. The proteins were loaded onto a 5-ml HisTrap HP column (GE Healthcare) pre-equilibrated with binding buffer A. After washing the column with the same buffer, the bound proteins were eluted with a linear gradient of imidazole (up to 500 mM). The (His)6-MBP tag of the C-terminal domain was cleaved by TEV protease and separated with the 5-ml HisTrap HP column (GE Healthcare). Finally, the proteins were purified by SEC using a HiLoad 16/60 Superdex 75 column (GE healthcare) pre-equilibrated with SEC buffer A.To produce the Cas8f:Cas5f heterodimer, a subunit of the type I-F Cascade complex, synthetic Cas8f and Cas5f genes from Xanthomonas albilineans were cloned, respectively, into pET28a with an N-terminal (His)6-MBP tag and a TEV protease cleavage site and into pET21a without a tag. Both constructs were co-transformed into E. coli BL21(DE3) cells and co-expressed with 0.5 mM IPTG at 17°C for 16 h. The (His)6-MBP-tagged Cas8f:Cas5f heterodimer was loaded onto a 5-ml HisTrap HP column (GE Healthcare) pre-equilibrated with binding buffer B (20 mM tris(hydroxymethyl)aminomethane (Tris)–HCl, pH 7.5, 300 mM NaCl, 5 mM BME, 10% (w/v) glycerol and 30 mM imidazole). After washing the column with the same buffer, the protein sample was eluted with a linear gradient of imidazole (up to 500 mM). The N-terminal (His)6-MBP tag was cleaved by TEV protease and separated on a 5-ml HisTrap HP column (GE Healthcare). The Cas8f:Cas5f heterodimer was finally purified by SEC using a HiLoad 16/60 Superdex 200 column (GE Healthcare) pre-equilibrated with SEC buffer B (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM DTT and 5% (w/v) glycerol).The genes of type I-E Cas proteins (i.e. Cas5e, Cas6e, Cas7e, Cas8e and Cas11) were amplified by PCR from P. aeruginosa PRD-10 and E. coli DH5α genomic DNAs. They were cloned into pET28a with an N-terminal (His)6-MBP tag and a TEV protease cleavage site. Mutant Cas8e genes were generated by site-directed mutagenesis using mutagenic PCR primers. The resulting wild-type (WT) and mutant constructs were transformed into E. coli BL21(DE3) cells. The type I-E Cas proteins were expressed individually as described above for the expression of AcrIE4-F7. The protein samples were purified without cleaving the (His)6-MBP tag because we found removal of the N-terminal tag destabilized the individual Cas proteins in our experimental conditions. The Cas proteins were then loaded onto a 5-mL HisTrap HP column (GE Healthcare) pre-equilibrated with binding buffer C (20 mM HEPES, pH 7.0, 500 mM NaCl, 5 mM BME, 20% (w/v) glycerol and 30 mM imidazole). After washing the column with the same buffer, the bound proteins were eluted with a linear gradient of imidazole (up to 500 mM). Finally, the proteins were purified by SEC using a HiLoad 16/60 Superdex 200 column (GE Healthcare) pre-equilibrated with SEC buffer A and 10% (w/v) glycerol.Analytical SECAnalytical SEC was performed using a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with buffer (20 mM HEPES, pH 7.0, 150 mM NaCl, 2 mM DTT and 5% (w/v) glycerol). Proteins (20 μM each) were mixed and incubated at 4°C for 1 h, and then 700 μl of the mixture was loaded onto the SEC column at a flow rate of 0.5 ml/min. The eluted SEC fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie staining.Isothermal titration calorimetry (ITC)ITC experiments were performed at 25°C using an iTC200 Calorimeter (Malvern). Samples in 200-μl cells were titrated with nineteen 2-μl injections. To analyze the binding of AcrIE4-F7 with the Cas8f:Cas5f heterodimer, AcrIE4-F7 (250 μM) was injected into a sample cell containing Cas8f:Cas5f (35 μM) in 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP). For the interaction of AcrIE4-F7 (or its mutants) with Cas8e, we placed 20 μM of either AcrIE4-F7 or (His)6-MBP tagged Cas8e in the cell and titrated with 150–200 μM of the partner protein in 20 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM TCEP and 5% (w/v) glycerol. The titrations were conducted in both directions, and the data were analyzed using the Origin software provided with the instrument.Multi-angle light scattering (MALS)Static light scattering data were obtained using a Superdex 75 Increase 10/300 GL column (GE Healthcare) coupled with a miniDAWN (3-angle) light scattering detector (Wyatt Technology) and an Optilab T-rEX refractive index detector (Wyatt Technology). The column was equilibrated with 20 mM HEPES, pH 7.0 and 150 mM NaCl. Then, 100 μl of AcrIE4-F7 (150 μM) was loaded onto the column at a flow rate of 0.5 ml/min at 25°C. The results were analyzed using the ASTRA 8 software (Wyatt Technology).NMR spectroscopyThe NMR sample was prepared as 0.6 mM 13C,15N-labeled AcrIF4-F7 in 10 mM sodium phosphate, pH 7.0, 100 mM NaCl, 1 mM benzamidine and 10% (v/v) D2O. NMR spectra were obtained at 25°C on Bruker AVANCE III 800 MHz and AVANCE NEO 900 MHz spectrometers equipped with an xyz-shielded gradient triple resonance cryoprobe. NMR data were processed using the NMRPipe program (17) and analyzed using the PIPP/CAPP/STAPP (18) and NMRView (19) programs. Sequential backbone assignments were performed using 3D triple resonance through-bond scalar correlation experiments, which included HNCO, HN(CA)CO, HNCACB, CBCA(CO)NH and HBHA(CO)NH experiments. Side chain assignments were performed using HCCH-TOCSY, H(CCO)NH, and C(CO)NH experiments. Distance restraints were obtained using 13C-seperated NOESY and 15N-seperated NOESY experiments with a mixing time of 120 ms. {1H}–15N heteronuclear NOE measurements were acquired using 3 s of 120° 1H pulses separated by 5 ms intervals using a previously employed pulse program (20). Residual 1DNH dipolar couplings were obtained by taking the difference in the 1JNH splitting values measured in aligned (11.5 mg/ml of pf1 phage, ASLA Biotech) and isotropic media using 2D in-phase/antiphase 1H–15N HSQC spectra.Structure calculationInterproton distance restraints were derived from the NOE spectra and classified into distance ranges according to peak intensity. Backbone φ/ψ torsion angle restraints were derived from backbone chemical shifts using the program TALOS+ (21). Structures were calculated by simulated annealing in torsion angle space using the Xplor-NIH program (22). The target function for simulated annealing included covalent geometry, a quadratic van der Waals repulsion potential, square-well potentials for interproton distance and torsion angle restraints, hydrogen bonding, harmonic potentials for 13Cα/13Cβ chemical shift restraints (23), and a multidimensional torsion angle database potential of mean force (24).Multiple sequence alignmentHomologous sequences of AcrIE4-F7 were retrieved using the PSI-BLAST program (25), and redundant sequences (90% identity) were clustered using the CD-HIT program (26). The curated sequences were then aligned using the Clustal Omega program (27), and the multiple sequence alignment was analyzed and visualized using the Jalview program (28).Molecular dockingThe model of the AcrIE4-F7:Cas8e complex was obtained using the HADDOCK 2.4 web server (29). We used the structural coordinates of the N-terminal domain of AcrIE4-F7 (from this study) and P. aeruginosa Cas8e (modeled from PDB code 5U07 and chain C; see the Results section). Key interfacial residues identified by SEC and ITC were used as ambiguous restraints for molecular docking. Active interfacial residues were defined as follows: Glu19, Tyr20, Asp22, Asp30 and Glu31 for the Acr proteins; Lys176, Lys183 and Lys357 for P. aeruginosa Cas8e. Passive interfacial residues were defined as those within 6.5 Å of the active residues. One thousand structures were generated via rigid body docking and energy minimization from random initial states, and the 200 lowest energy structures were selected for subsequent semi-flexible simulated annealing and explicit water refinement. The structure with the best HADDOCK score was displayed using the PyMOL software (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.).

Article TitleThe structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites

Abstract

The atomic coordinates of the AcrIE4-F7 solution structure and NMR restraints have been deposited in the Protein Data Bank (PDB code 7VZM) and the Biological Magnetic Resonance Bank (accession code 36454), respectively.


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