Structure, Mechanism, and Specificity of a Eukaryal trna Restriction Enzyme

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1 Supplemental Information Structure, Mechanism, and Specificity of a Eukaryal trna Restriction Enzyme Involved in Self-Nonself Discrimination Anupam K. Chakravarty, Paul Smith, Radhika Jalan, and Stewart Shuman Molecular Biology Program, Sloan-Kettering Institute, New York, NY USA

2 Figure S1. Kinetics of single-turnover cleavage and stimulation by TMAO. Related to Figure 1. (A) PaT titration. Reaction mixtures (10 µl) containing 0.1 µm 5 32 P-labeled 27-mer stem-loop RNA, increasing concentrations of PaT as specified, and either 2 M TMAO or no additive (control) were incubated for 60 min at 30 C. The products were analyzed by urea- PAGE. The extents of RNA cleavage are plotted as a function of PaT concentration. Each datum in the graph is the average of three separate experiments ±SEM. (B) Single-turnover kinetics. A reaction mixture (70 µl) containing 0.1 µm 5 32 P-labeled 27-mer stem-loop RNA, 2 µm PaT, and either 2 M TMAO ( ) or no additive ( ) was incubated at 30 C. Aliquots (10 µl) were withdrawn at the times specified times and quenched with formamide, EDTA. The products were analyzed by urea-page. The extents of RNA cleavage are plotted as a function of time. Each datum in the graph is the average of three separate experiments ± SEM. The kinetic data were fit by non-linear regression in Prism to a one-phase association. The apparent rate constants derived thereby are shown. The chemical structure of TMAO is illustrated at right.

3 Figure S2. PaT cleavage leaves a 2,3 -cyclic phosphate end. Related to Figure 1. (A) Reaction mixtures (10 µl) containing 0.1 µm 5 32 P-labeled 17-mer stem-loop RNA (depicted at left), 2 µm PaT, and either no additive or 2 M TMAO as specified were incubated at 30 C for 60 min. The products were analyzed by urea-page in parallel with an alkaline hydrolysis ladder of the 5 -labeled 17-mer RNA (lane OH). An autoradiograph of the gel is shown. The identities of the 3 terminal nucleotides are indicated next to the ladder. At right is a slightly enlarged image of a portion of the gel highlighting the separation of short alkaline hydrolysis products of the same nucleotide sequence into doublets in which the upper species has a 2,3 -cyclic-phosphate end and the lower species has a monophosphate end. (B) PaT acts via a transesterification mechanism in which the wobble ribose O2 make a nucleophilic attack on the scissile phosphodiester to form a 2,3-cyclic phosphodiester and expel a 5 -OH leaving strand.

4 Figure S3. The residual N-Ser-Met tag occludes the PaT active site. Related to Figure 4. Stereo view of the superimposed folds and active site regions of catalytically active PaT-His 6 (in gray) and catalytically inert N(Ser) PaT (in magenta; with the residual N-terminal Ser-Met tag shown as a stick model). The N-terminal Ser tag residue is tethered in place via hydrogen bonds (dashed lines) to Oγ and the main-chain carbonyl from Arg172 and from the Ser tag Oγ and N- terminal amino nitrogen to Asp286. With the N-tag docked in this conformation, the Asn2 side chain occupies the same position as the chloride anion (green sphere) the PaT-His 6 active site.

5 Figure S4. Comparison of PaT structure to other transesterifying ribonucleases. Related to Figure 3. The tertiary structures of PaT, colicin E5 (pdb 2DJH), colicin D (pdb 1V74), trna splicing endonuclease (pdb 2GJW), MazF (pdb 4MDX), restrictocin (pdb 1JBR), RNase A (pdb 1RUV), RNase T1 (pdb 1GSP) and Barnase (pdb 1BRN) are rendered as ribbon traces with cyan helices and magenta strands. Whereas PaT is predominantly helical, the other ribonucleases have predominant β character, especially in their active sites.

6 Table S1: Crystallographic data and refinement statistics Native N(Ser) PaT SeMet N(Ser) PaT PaT His 6 Space group P P P Unit cell 130K (Å) Crystallographic data quality a = 61.87, b = 77.43, c = a = 61.95, b = 77.57, c = a = 61.88, b = 77.46, c = Number of crystals Resolution, Å ( ) ( ) ( ) Wavelength Å Å Å R a sym, % 3.3 (25.7) 4.3 (23.1) 9.3 (32.3) [ ] 17.7 (87.5) [ ] R a cryst, % 11.2 (42.6) Unique reflections b (3880) (3455) (2448) Mean redundancy 11.4 (10.0) 35.7 (35.3) 7.6 (5.8) Completeness, % (100.0) (100.0) 98.8 (94.4) Phasing Statistics Mean I/σI 11.7 (2.8) 20.4 (7.4) 6.5 (2.6) Phasing method N/A Se- SAD (6 sites) N/A Resolution, Å Signal to noise c Ano = Figures of merit d Refinement and model statistics (F>0) 0.44 (Phaser) 0.78 (Resolve) Resolution, Å ( ) N/A ( ) Completeness, % 99.7 (98.9) 98.6 (95.9) R free e / R work, % 19.7 / 16.0 (34.2/25.2) 24.7/18.8 (32.8/24.3) RMS deviation: bonds / angles Å/ / Ramachandran plot 96.9% favored, 1 outlier f 98.1% favored, 1 outlier f B- factors, Overall/Wilson 32.5/20.5 Å /38.9 Å 2 TLS Groups Mean Anisotropy 0.38 (10 TLS Groups) 0.58 (8 TLS Groups) Model Contents Protomers in ASU 2 2 Protein amino acids Heteroatoms 27 sulfates, 2 citrates, 738 waters 524 waters 19 sulfates, 2 chlorides, 164 waters PDB ID 4O87 4O88 Diffraction data were collected at NSLS X25 beamline. Standard definitions are used for all parameters. Figures in parentheses refer to data in highest resolution bin. Diffraction data statistics are from MOSFLM/AIMLESS. The refinement and geometric statistics are from PHENIX. a R sym and R cryst are defined as R pim (as computed in SCALA) from a single crystal and from all merged data respectively. The figures within brackets denote the ranges of resolution used in case of each individual crystal. b F + and F - were treated as independent for SeMet and equivalent for native datasets. c Signal to noise for anomalous phasing (Ano) of the SeMet N- tag PaT dataset is the fraction of data for which D ano 3σ D ano for data to 3.0 Å, as calculated in XTRIAGE. d Figure of Merit (FOM) values as output following initial phasing by PHASER and following density modification by RESOLVE. e R free sets for cross validation consisted of 5% of data selected at random against which structures were not refined. f. Outliers: Thr77A in N(Ser) PaT; Gly96A, in PaT His 6.

7 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Genetic tests of PaT toxicity. Versions of PaT lacking a putative N-terminal signal peptide were expressed in haploid S. cerevisiae in a glucose-repressible, galactose-inducible manner by inserting the PaT open reading frames in a yeast 2µ LEU2 plasmid (prs425) under the transcriptional control of a GAL1 promoter and terminator. We initially varied the translation start site by placing a methionine start codon at phased one-amino-acid intervals, as depicted in Fig. 1A. Subsequently, alanine mutations were introduced, by two-stage overlap extension PCR, into the biologically active version of PaT with the N-terminal sequence MPTTCLNE. The PaT gene inserts in the expression plasmids were sequenced to verify that no unwanted coding changes were acquired. The pgal-pat plasmids were transformed into yeast. Single Leu + transformants were selected on glucose-containing SD-Leu agar, grown in liquid cultures at 30 C in medium lacking leucine. The cultures were adjusted to A 600 of 0.1, and then diluted in water in serial 10- fold increments. Aliquots (2 µl) of three serial 10-fold dilutions were spotted in parallel on Leu agar medium containing 2% glucose or 2% galactose. The plates were photographed after incubation at 30 C for 2 d (glucose) or 3 d (galactose). Purification of PaT His 6. Plasmid pet23-pat-his 6 encoding PaT (with N-terminal sequence MPTTCLNE) fused to a C-terminal His 6 tag was transformed into E. coli BL21(DE3) codon plus cells. An 8-liter culture derived from a single transformant was grown at 37 C in Luria-Bertani medium containing 100 µg/ml ampicillin and 12.5 µg/ml chloramphenicol until the A 600 reached 0.6. The culture was then adjusted to 0.1 mm IPTG and 2% ethanol and incubated for 16 h at 17 C with continuous shaking. Cells were harvested by centrifugation, and the pellet was stored at 80 C. All subsequent procedures were performed at 4 C. Thawed cell pellets were resuspended in 100 ml of buffer A (50 mm Tris-HCl, ph 7.4, 250 mm NaCl, 10% sucrose). Lysozyme was added to a final concentration of 0.2 mg/ml. After mixing for 1 h, the lysate was sonicated to reduce viscosity and insoluble material was removed by centrifugation for 45 min at 30,000g. The soluble extract was mixed for 1 h with 12 ml of a 50% slurry of Ni-NTA resin (Qiagen) that had been equilibrated in buffer A. The resin was recovered by centrifugation and resuspended in 15 ml of buffer B (50 mm Tris-HCl, ph 7.4, 150 mm NaCl, 10% glycerol) containing 25 mm imidazole. The cycle of centrifugation and resuspension of the resin was repeated thrice, after which the resin (6 ml) was poured into a column. The column was washed serially with 12 ml of buffer C (50 mm Tris-HCl, ph 7.4, 2 M KCl) and 10 ml of buffer B containing 50 mm imidazole. The bound proteins were eluted step-wise in 12 ml aliquots of 100, 200, and 300 mm imidazole in buffer B. The elution profile was monitored by SDS-PAGE. The

8 100 and 200 mm imidazole eluate fractions containing PaT His 6 were pooled and dialyzed for 3 h against 4 liters of buffer E (50 mm Tris-HCl, ph 7.4, 20 mm NaCl, 10% glycerol). The dialysate was then mixed for 1 h with 4 ml of a 50% slurry of SP-Sepharose resin (GE) that had been equilibrated in buffer E. The resin (2 ml) was then poured into a column and eluted stepwise with buffer E containing 150 mm and 300 mm NaCl. PaT His 6 was recovered in the 150 mm NaCl fraction and concentrated by centrifugal ultrafiltration. This material was gel-filtered through a 120-ml HiLoad Superdex 200 column (GE Healthcare) equilibrated in buffer D (10 mm Tris-HCl, ph 8.0, 150 mm NaCl, 1 mm DTT), at a flow rate of 1 ml/min. The elution profile was monitored by A 280 and then by SDS-PAGE. The peak PaT His 6 fractions were pooled and concentrated by centrifugal ultrafiltration to 4.5 mg/ml. The yield of PaT His 6 was 1.8 mg. Alanine mutants of PaT His 6 (R172A, K175A, S283A, and H287A), and wild-type PaT His 6 in parallel, were purified from 4-liter cultures of IPTG-induced bacteria as described above, omitting the gel filtration step. The yields at the SP-Sepharose step were: WT (3.6 mg); R172A (17 mg); K175A (12.5 mg); H287A (18 mg); S283A (4.2 mg). Purification of N(Ser) PaT. Plasmid pet28-his 10 Smt3 PaT encoding PaT (with N-terminal sequence MNPTTCLNE) fused to an N-terminal His 10 Smt3 domain was transformed into E. coli BL21(DE3) codon plus cells. An 8-liter culture derived from a single transformant was grown at 37 C in Luria-Bertani medium containing 50 µg/ml kanamycin and 12.5 µg/ml chloramphenicol until the A 600 reached 0.6. IPTG induction and preparation of a soluble lysate fraction were performed as described above for PaT His 6. The soluble extract was mixed for 1 h with 10 ml of a 50% slurry of His60 Ni superflow resin (Clontech) that had been equilibrated in buffer A. The resin was recovered by centrifugation and resuspended in 25 ml of buffer B containing 25 mm imidazole. The cycle of centrifugation and resuspension of the resin was repeated thrice, after which the resin (5 ml) was poured into a column. The column was washed serially with 20 ml of buffer C and 10 ml of buffer B containing 50 mm imidazole. The bound proteins were eluted with 500 mm imidazole in buffer B while collecting 10 ml fractions. The elution profile was monitored by SDS-PAGE. Fractions containing His 10 Smt3 PaT were pooled and supplemented with Smt3- specific protease Ulp1 (to attain a Ulp1:His 10 Smt3 PaT ratio of 1:50). The mixture was dialyzed for 48 h against 4 liters of buffer B. The dialysate was then mixed for 1 h with 6 ml of a 50% slurry of His60 Ni superflow resin that had been equilibrated in buffer B containing 50 mm imidazole. The resin (3 ml) was poured into a column, washed with 50 mm imidazole in buffer B, and then eluted with 100 mm and 500 mm imidazole in buffer B. PaT protein with an N-terminal serine derived from the Smt3 fusion junction, N(Ser) PaT, was recovered in the flow-through;

9 the cleaved His 10 Smt3 protein and residual intact His 10 Smt3 PaT were recovered in the 500 mm imidazole fraction. The flow-through containing N(Ser) PaT was concentrated by centrifugal ultrafiltration and gel-filtered through a 120 ml Superdex 200 column (as described above for PaT His 6 ). The peak N(Ser) PaT fractions were pooled and concentrated by centrifugal ultrafiltration to 8 mg/ml. The yield of N(Ser) PaT protein was 80 mg. Selenomethionine (SeMet) substituted N(Ser) PaT was prepared by transforming pet28- His 10 Smt3 PaT into E. coli B834, a methionine auxotroph. A single transformant was inoculated into 5 ml of LB medium containing 50 µg/ml kanamycin and incubated for 7 h at 37 C. The bacteria were harvested by centrifugation and then resuspended in 200 ml of complete LeMaster medium containing 50 µg/ml kanamycin. This step was repeated twice and the cells were finally resuspended in 200 ml of complete LeMaster medium (with kanamycin) containing 50 µg/ml SeMet (racemic). After overnight incubation, the culture volume was increased to 4 liters with fresh LeMaster medium containing kanamycin and SeMet, and growth was continued at 37 C with constant shaking until the A 600 reached 0.6. The culture was adjusted to 0.1 mm IPTG and 2% ethanol and then incubated for 16 h at 17 C with continuous shaking. The SeMetsubstituted His 10 Smt3 PaT fusion protein was purified from soluble extract by Ni-affinity chromatography, then cleaved with Ulp1 during dialysis to generate SeMet N(Ser) PaT, which was recovered in the flow-through during a second Ni-affinity step and finally purified by gel filtration following the procedures described above for the unmodified N(Ser) PaT protein. Crystallization of PaT. Aliquots (2 µl) of a 6.5 mg/ml solution of N(Ser) PaT or SeMetsubstituted N(Ser) PaT were mixed on a cover slip with an equal volume of precipitant solution containing 100 mm sodium citrate (ph 5.6), M (NH 4 ) 2 SO 4, 100 mm magnesium acetate. Aliquots (2 µl) of a 4.5 mg/ml solution of PaT His 6 were mixed on a cover slip with an equal volume of precipitant solution containing 100 mm sodium citrate (ph 5.6), M (NH 4 ) 2 SO 4, 200 mm LiCl. Crystals were grown at 22 C by hanging drop vapor diffusion against reservoirs of the precipitant solutions. Crystals appeared after 2 days; single crystals were cryoprotected with precipitant solution saturated with (NH 4 ) 2 SO 4 before freezing in liquid nitrogen. Diffraction data collection and structure determination. X-ray diffraction was performed at the National Synchrotron Light Source (Brookhaven, NY) beamline X25 equipped with a Pilatus 6M detector. Diffraction data at 2.1 Å resolution for SeMet-substituted N(Ser) PaT were collected with high redundancy from a single crystal at Å (the energy of the K-edge adsorption for selenium) in three separate sweeps of 730 continuous increments of 0.5 each with 0.5 s, 2 s and 6 s exposures per frame in the three sweeps, respectively. Data from all the

10 images were indexed and integrated using MOSFLM and scaled using AIMLESS/SCALA. The diffraction statistics are compiled in Table S1. The SeMet-N(Ser) PaT crystals belonged to orthorhombic space group P Substructure solution and initial SAD phase calculations were performed in PHENIX.AUTOSOL (Adams et al., 2002). Six selenium sites were located. Density modification was performed in PHENIX.AUTOSOL assuming two protomers per asymmetric unit and 50% solvent content, after which ~90% of the amino acids in both the protomers were placed by the auto-build function. The model was adjusted in COOT (Emsley and Cowtan, 2004) and subjected to two rounds of refinement in PHENIX without using noncrystallographic symmetry (NCS) restraints. Diffraction data at 1.8 Å for a single native N(Ser) PaT crystal were collected at Å in two separate sweeps of 370 continuous increments of 0.5 each with 2 s exposures per frame. Data from all the images were indexed and integrated in MOSFLM and scaled in AIMLESS/SCALA. The native N(Ser) PaT crystal was isomorphous to the SeMet-N(Ser) PaT crystal and the structure of N(Ser) PaT was determined by refinement of the diffraction data against the modeled SAD structure of SeMet-N(Ser) PaT from which all non-protein atoms had been removed. The N(Ser) PaT model was iteratively adjusted in COOT and refined in PHENIX. TLS B-factor refinements were implemented in PHENIX. The final model, comprising two N(Ser) PaT protomers, had R work /R free values of 0.160/0.197, excellent geometry (one Ramachandran outlier out of 637 amino acids), and no large F o -F c difference Fourier peaks (Table S1). Diffraction data at 2.9 Å and 3.0 Å for PaT His 6 were collected from two different crystals at a wavelength of Å in 185 continuous increments of 1.0 each with 2 s exposures per frame. Data from each crystal were individually indexed and integrated using MOSFLM. Data from 125 images (from a crystal diffracting to 2.9 Å) and 140 images (from a crystal diffracting to 3.0 Å) were subsequently merged and scaled using AIMLESS/SCALA to generate a composite dataset. The PaT His 6 crystals were isomorphous to the N(Ser) PaT crystal. The PaT His 6 structure was determined by refining the diffraction data against the native N(Ser) PaT model from which all non-protein atoms had been removed. The PaT His 6 model was iteratively rebuilt in COOT and refined in PHENIX using TLS B-factor refinements and NCS restraints. During the final rounds of the refinement, additional restraint against the high resolution native N(Ser) PaT structure was applied. The rmsd values between the A protomers of PaT His 6 and N(Ser) PaT structures, before and after applying reference model restraints, were 0.6 Å and 0.55 Å at 313 Cα atoms, respectively. The final PaT His 6 model, comprising two N(Ser) PaT protomers, had

11 R work /R free values of 0.188/0.247, excellent geometry (one Ramachandran outliers out of 630 amino acids) and no large F o -F c difference Fourier peaks (Table S1). Purification of ImmPaT His 6. Plasmid pet23-immpat-his 6 encoding the 363-amino acid PaOrf4 toxin immunity factor ImmPaT (Genbank accession CAE84959) fused to a C-terminal His 6 tag was transformed into E. coli BL21(DE3). A 4-liter culture derived from a single ampicillin-resistant transformant was grown at 37 C in Luria-Bertani medium containing 100 µg/ml ampicillin until the A 600 reached 0.6. IPTG-induction, preparation of a soluble lysate, and purification of ImmPaT-His 6 by sequential Ni-NTA chromatography and Superdex gel filtration steps were performed essentially as described above for PaT His 6. The peak Superdex fractions containing ImmPaT-His 6 were pooled and dialyzed for 3 h against 4 liters of buffer E. The dialysate was concentrated by centrifugal ultrafiltration and then applied to a 4-ml column of SP-Sepharose that had been equilibrated with buffer E. ImmPaT His 6 was recovered in the flow-through and re-concentrated by centrifugal ultrafiltration (to 1.75 mg/ml). The yield of ImmPaT protein was 1.5 mg. Preparation of anticodon stem-loop substrates. RNA oligonucleotides used as substrates in anticodon nuclease assays were purchased from Dharmacon. The RNAs were deprotected according to the vendor s instructions and 5 32 P-labeled by reaction with T4 polynucleotide kinase and [γ 32 P]ATP. The labeled RNAs were purified free of ATP by denaturing PAGE, eluted from an excised gel slice, ethanol-precipitated, resuspended in 10 mm Tris-HCl, ph 6.8, 1 mm EDTA and stored at 20 C. Assay of anticodon nuclease activity in vitro. Reaction mixtures (10 µl) containing 10 mm Tris-HCl, ph 7.5, 50 mm NaCl, 1 mm DTT, 0.1 µm 5 32 P-labeled stem-loop RNA, and PaT His 6 as specified were incubated at 30 C for 60 min. The mixtures were then supplemented with 10 µl of 90% formamide, 50 mm EDTA. The reaction products were analyzed by electrophoresis through a 40-cm 20% polyacrylamide gel containing 7.5 M urea in 45 mm Tris-borate, 1 mm EDTA. The radiolabeled RNAs were visualized by autoradiography and the extents of RNA cleavage were quantified by scanning the gel with a Fuji BAS2500 imager. The RNA markers used to identify the cleavage sites were prepared by: (i) partial alkaline hydrolysis of the 5 32 P- labeled stem-loops, by incubation for 12 min at 95 C in a solution of 40 mm NaHCO 3, 60 mm Na 2 CO 3 (ph 10.2); or (ii) treatment of the 5 32 P-labeled stem-loop with RNase T1 (7 U; Fermentas) for 20 min at 37 C.

12 SUPPLEMENTAL REFERENCES Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002) PHENIX: Building new software for automated crystallographic structure determination. Acta Crystallogr. D58, Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D60,

Six genes, Lsm1, Lsm2, Lsm3, Lsm5, Lsm6, and Lsm7, were amplified from the

Supplementary information, Data S1 Methods Clones and protein preparation Six genes, Lsm1, Lsm2, Lsm3, Lsm5, Lsm6, and Lsm7, were amplified from the Saccharomyces cerevisiae genomic DNA by polymerase chain