www.sciencemag.org/cgi/content/full/312/5782/1954/DC1

Supporting Online Material for

Ammonia Channel Couples Glutaminase with Transamidase Reactions in GatCAB

Akiyoshi Nakamura, Min Yao, Sarin Chimnaronk, Naoki Sakai, Isao Tanaka*

*To whom correspondence should be addressed. E-mail: tanaka@castor.sci.hokudai.ac.jp

Published 30 June 2006, Science 312, 1954 (2006) DOI: 10.1126/science.1127156

This PDF file includes:

Materials and Methods Figs. S1 to S6 Table S1 References

Supporting Online Material for

Ammonia Channel Couples Glutaminase

with Transamidase Reactions in GatCAB

Akiyoshi Nakamura,* Min Yao,* Sarin Chimnaronk,

Naoki Sakai, and Isao Tanaka†

*These authors contributed equally to this work.

†To whom correspondence should be addressed.

E-mail: tanaka@castor.sci.hokudai.ac.jp

This PDF file includes:

Materials and Methods

Table. S1

Figures. S1 to S6

References

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Supporting Online Material Materials and Methods Protein expression and purification

The cassette of gatCAB operon from Staphylococcus aureus Mu50 was amplified by polymerase chain reaction (PCR), and cloned into NcoI/XhoI site of a pET-28b (+) vector (Novagen), fused with the hexahistidine tag at the C-terminus of GatB. Heterotrimeric complex of GatCAB was co-overexpressed in Escherichia coli B834 strain. Cells were grown at 298 K in LB medium containing 25 µg/mL kanamycin, and proteins expression were induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and proceeded for another 15 hours at 298 K. Cells were harvested (4,000 × g, 15 min at 277 K) and disrupted using sonication in buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM MgCl2, 1 mM DTT, 10 % (v/v) glycerol, and 1 mg/mL DNase I). All the following purification processes were carried out at 277 K. Cell debris was removed by centrifugation (40,000 × g, 1 hour), and clarified supernatant was applied to a HisTrap HP column (Amersham Biosciences) pre-equilibrated with buffer B (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM MgCl2, 1 mM DTT, and 10 % (v/v) glycerol). The column was washed with buffer B contained 20 mM imidazole, and proteins were eluted as a step-wise manner with buffer B contained 500 mM imidazole. Pooled fractions were dialyzed against buffer B for overnight prior to be loaded onto a HiLoad 26/60 Superdex 200 pg column (Amersham Biosciences) equilibrated with buffer C (20 mM Tris-HCl pH 8.0, 20 mM MgCl2, 100 mM NaCl, 1 mM DTT, and 10 % (v/v) glycerol). Fractions containing the intact GatCAB as jugged by SDS-PAGE were pooled and subsequently subjected to a RESOURCE Q anion-exchange column (Amersham Biosciences) pre-equilibrated with buffer C, and proteins were eluted with a linear gradient of 100 - 500 mM NaCl. GatCAB complex eluted at approximately 150 mM NaCl, and was collected and dialyzed against buffer D (20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM DTT, and 10 % (v/v) glycerol) for overnight, and concentrated by ultrafiltration using Vivaspin devices (VIVASCIENCE) to a final concentration of ~12 mg/ml. For the production of selenomethionine (Se-Met) derivative, cells were grown in the minimal medium containing 25 mg/L Se-Met. The purification procedure for Se-Met derivative was essentially the same as that for native enzyme.

All of GatCAB deletion variants were generated using the QuikChangeTM site-directed mutagenesis kit according to the manufacture’s protocol (Stratagene), and purified to homogeneity as abovementioned.

Crystallization and data collection

Crystals of Se-Met derivative were grown by hanging drop vapor diffusion against 50 mM HEPES-NaOH pH 7.0, 25 % (v/v) PEG 550 monomethyl ether and 5 mM MgCl2 at 293 K within a few days. The crystal of Se-Met derivative was rapidly soaked through the reservoir containing 12.5 % (v/v) glycerol as a cryoprotectant, and then a data set was collected to 2.8 Å resolution at SPring-8 beamline 41XU (Hyogo, Japan) at the selenium peak wavelength under cryogenic condition (100 K). During building the initial model of GatCAB, the high quality native crystals were obtained using the streak-seeding technique equilibrated with the reservoir containing 50 mM HEPES-NaOH pH 7.0, 20 % (v/v) PEG 550 monomethyl ether, 5 mM MgCl2, and 10 % (v/v) glycerol. The native data set was collected under the same condition with Se-Met derivative at a wavelength of 0.9 Å at Photon Factory beamline ARNW12 (Ibaraki, Japan). For the determination of GatCAB−ligand complex structures, the native crystal was presoaked in the reservoir containing 5 mM L-glutamine or L-asparagine, or 10 mM MnCl2 for 3 ~ 12 hours before

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back-soaking in the cryoprotectant. Crystals with ADP/AlF4–/Mg2+ were obtained only by

the cross streak-seeding technique equilibrated with the reservoir containing 50 mM HEPES-NaOH pH 7.0, 25 % (v/v) PEG 550 monomethyl ether, 10 mM MgCl2, 0.33 mM AlCl3, 5 mM NaF and 1 mM ADP. The all data sets of GatCAB−ligand complexes were collected at SPring-8 beamline 41XU or BL44B2. The all data sets were processed and scaled using the HKL2000 package (1). All crystals belonged to the space group P212121, and the asymmetric unit was estimated to contain one GatCAB heterotrimeric molecule. Structure determination

The phases were obtained by the single wavelength anomalous diffraction (SAD) method at 2.8 Å resolution with 23 of 28 Se sites identified by using the program SOLVE/RESOLVE (2). The initial model could not be built automatically, and was manually built to 70 % at a resolution of 2.8 Å by using the program O (3) with the use of the peptide amidase structure (PDB code; 1M21 (4)) as a good reference for GatA. Subsequently the initial phases were improved by averaging of multiple crystals between Se-Met and native crystal and extended to 2.5 Å by using the program DMMULTI (5), then the completion of model building was achieved. Several rounds of further manual refinement using program CNS (8) with O followed by automatic rebuilding using the program LAFIRE (6, 7) with CNS (8), yielded the final model with the crystallographic R factor of 23.8 % and Rfree factor of 27.5 %. The refined structure of GatCAB was validated by the program PROCHECK (9) and WHATIF (10). The structures of ligand-bound complexes were solved by molecular replacement using AMoRe in the CCP4 suite (11), with the refined model of the native structure as a search model. The summary of data statistics is presented in Table S1. Secondary structure elements of proteins were assigned with the program WHATIF (10), and all figures were generated by PyMol (12). tRNAs preparation

Wild type Sav tRNAGln and tRNAGlu, as well as a series of tRNAGln variants were prepared by in vitro transcription using T7 RNA polymerase. Double-strand DNA transcription templates were obtained by PCR cycles with three overlapping primers, and were purified by a Sephacryl 16/60 gel filtration column (Amersham Biosciences) equilibrated with TE buffer. The in vitro transcription was performed in a solution containing 40 mM HEPES-NaOH pH 7.8, 8 mM MgCl2, 1 mM spermine, 5 mM DTT, 100 mM KCl, 0.05 mg/mL BSA, 0.01 % TritonX-100, 1 mM NTPs, 10 mM UMP or GMP, 4 µg/ml transcription template and 0.02 mg/ml T7 RNA polymerase at 310 K for 12 hour. The reaction mixture was subsequently isopropanol-precipitated, and purified by denaturing urea-polyacrylamide gel electrophoresis. tRNAs were extracted and refolded simultaneously with 500 mM NH4 acetate pH 6.0, 5 mM Mg acetate, 0.1 mM EDTA, 0.1 % SDS at 310 K for 12 hour. Gel-shift assay

Gel-shift assay were performed in a 10 µL reaction mixture containing 50 mM HEPES-KOH pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 50 pmol enzyme and 150 pmol tRNA. Reaction mixtures were incubated at 310 K for 30 min, and were immediately load on a 5 % non-denaturing polyacrylamide gel composed of 50 mM Tris-MES pH 6.8, 0.5 mM DTT, 10 mM magnesium acetate, and 50 mM ammonium acetate, and run at 277 K for 4 hour at a constant 60 V.

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aValues in parentheses are for the outermost resolution shell. bRmerge = ΣhΣj | <Ι>h - Ih,j | /ΣhΣjΙh,j, where <Ι>h is the mean intensity of symmetry-equivalent reflections. cRwork = Σ | Fobs - Fcal | /ΣFobs, where Fobs and Fcal are observed and calculated structure factor amplitudes. dRfree value was calculated for R factor, using only an unrefined subset of reflections data. eRamachandran plot was calculated by PROCHECK (9).

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Figure. S1. The interfacial interactions between GatA and GatB. Interface view form GatB side (A) and GatA side (B). A putative ammonia channel (black) is running through in the middle of the interface, which is surrounded by inner interactive layer constructed hydrogen-bonding bridges (red, negative charged residues; blue, positive charged residues). The outer hydrophobic layer in the interfacial interactions is depicted in yellow.

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Figure. S2. (A) Side view of GatA, further rotated by roughly 90° around the horizontal axis compared to Fig. 1A for clarity. Glutamines are shown as ball-and-stick models, whereas the residues building the active site in stick representations. (B) Asparagine in the glutaminase center of GatA. Soaking GatCAB crystals with asparagines (green stick) showed the identical recognition of the main chain as glutamine; however, the side chain of asparagine could not reach the distance of covalent bond with Ser178. Thus, asparagine may not be a good amide donor for the glutaminase reaction of GatCAB (13). The Fo-Fc electron density map (contoured at 3σ, magenta mesh) calculated after molecular replacement. (C) Partial sequence alignments for representative members of the amidase signature family. Conserved amino acids are in white in red-filled rectangles. Similar residues are in red, surrounded by blue boxes. Sav_GatA indicates GatA from S. aureus; PAM_1M21 refers to a peptide amidase from Stenotrophomonas maltophilia (PDB code: 1M21 (4)); MAE2_1OCK is malonamidase E2 from Bradyrhizobium japonicum (PDB code: 1OCK (14)), and FAAH_1MT5 as a fatty acid amide hydrolase from Rattus norvegicus (PDB code: 1MT5 (15)). Stars mark residues contributing to the Ser-cis-Ser-Lys catalytic scissors, while residues involved in forming the oxyanion hole and hydrogen-bonded network with the catalytic scissors are indicated by filled circles and triangles, respectively. Each protein sequence was aligned by CLUSTALW (16) and figure was prepared with the program ESPript (17).

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Figure. S3. Sequence alignments of bacterial GatB, archaeal GatE and Yqey protein. STAAM indicates Staphylococcus aureus Mu50; BACSU stands for Bacillus subtilis; AQUAE is Aquifex aeolicus; HELPJ is Helicobacter pylori J99; PSEAE, Pseudomonas aeruginosa; ARCFU, Archaeoglobus fulgidus; AERPE, Aeropyrum pernix; METJA, Methanococcus jannaschii; METMA, Methanosarcina mazei, and PYRAB refers Pyrococcus abyssi. AspRS insertion domain and interfacial insertion of GatE are shown as blue line and green line, respectively. The two Mn2+s (or Mg2+s) are coordinated to invariant residues marked with stars. Conserved residues involved in ATP recognition and likely in ammonia channel gating are indicated with filled triangles and circles, respectively.

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Figure. S4. (A) Superposition of GatB from the native (gold) and glutamine-soaked (green) crystals in the front view. The flexible C-terminal helical domains are rotated by 14.4° between the two structures, due to differences in the crystal-packing. (B) Two manganese ion binding sites in the cradle domain of GatB. Blue mesh indicates the strong peaks of the manganese ions (magenta spheres) seen in the Fobs-Fcal electron density map contoured at 4σ level. Coordinate sites are drawn as stick models and labeled. Mn2+

1 is in the same position as Mg2+ site in the native or ADP/AlF4–-bound complex

structures. Mn2+2 implies the secondary transient metal binding site in the kinase center of

GatB.

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Figure. S5. Comparison of the D-loops and the acceptor stems among tRNAGln, tRNAGlu, tRNAAsn and tRNAAsp from various organisms. Our identified identity elements (the first U1-A72 base pair and an insertion in the D-loop) for discrimination of tRNAGln by GatCAB are emphasized by red boxes, which are well conserved through evolution. Base pairs in the D-stem are indicated by bold characters.

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Figure. S6. Proposed model for the overall reaction cycle of GatCAB. GatCAB recognizes two remote identity elements on the body of Glu-tRNAGln. The docking model suggests that the first U1-A72 base pair of tRNAGln is denatured by colliding with the cradle domain of GatB, whereas the D-loop makes specific interactions with the distal C-terminal helical bundle of GatB (right panel). Accommodation of Glu-tRNAGln in the correct position could induce the movement of the Glu125 gate to open the ammonia channel (as discussed in the text), resulting in activation of the glutaminase reaction of GatA. Subsequently, ATP enters the active site and coordinates its γ-phosphate group to a newly-created transient Mg2+-binding site. At this stage, the active center should be shielded from water enabling the carboxylic group of Mg2+-bound glutamate to attack the hydroxyl group of γ-phosphate (lower panel), which is in turn, instantaneously transamidated with the ammonia guided by the invariant Lys79 residue located at the exit of the ammonia channel (left panel).

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