Structural Analysis of Glutamine Synthetase from Helicobacter pylori

Glutamine synthetase (GS) is an enzyme that regulates nitrogen metabolism and synthesizes glutamine via glutamate, ATP, and ammonia. GS is a homo-oligomeric protein of eight, ten, or twelve subunits, and each subunit-subunit interface has its own active site. GS can be divided into GS I, GS II, and GS III. GS I and GS III form dodecamer in bacteria and archaea, whereas GS II form decamer in eukaryotes. GS I can be further subdivided into GS I-α and GS I-β according to its sequence and regulatory mechanism. GS is an essential protein for the survival of Helicobacter pylori which its infection could promote gastroduodenal diseases. Here, we determined the crystal structures of the GS from H. pylori (Hpy GS) in its apo- and substrate-bound forms at 2.8 Å and 2.9 Å resolution, respectively. Hpy GS formed a dodecamer composed of two hexameric rings stacked face-to-face. Hpy GS, which belongs to GS I, cannot be clearly classified as either GS I-α or GS I-β based on its sequence and regulatory mechanism. In this study, we propose that Hpy GS could be classified as a new GS-I subfamily and provide structural information on the apo- and substrate-bound forms of the protein.


Structure determination and model quality.
To determine the structure of Hpy GS, crystals of the apoand substrate-bound forms in complex with ATP and phosphinothricin (PPT) were obtained. The apo structure of Hpy GS (Hpy GS apo ) was determined at 2.8 Å by molecular replacement using the GS model from S. typhimurium (1F52) and refined to crystallographic R work and R free values of 18.99% and 26.01%, respectively. The refined model (PDB entry 5ZLI) contained 2,846 amino acid residues of six identical subunits in the asymmetric unit, which could be used to generate a dodecamer. In each subunit, N-terminal residues (Met1-Asn7 in subunit A, E, and F; Met1-Gln6 in subunit B, C, and D) and internal residues (Leu408-Gly418 in all subunits) were disordered.
The substrate-bound form of Hpy GS (Hpy GS sub ) was obtained in the presence of ATP and PPT, which is a structural analogue of glutamate. Hpy GS sub was determined at 2.9 Å and refined to crystallographic R work and R free values of 16.45% and 24.27%, respectively. The refined model (PDB entry 5ZLP) contained 5,704 amino acid residues of twelve identical subunits in the asymmetric unit. In each subunit, N-terminal residues (Met1-Thr5 in subunit A, D, G, and L; Met1-Gln6 in subunit B, F, H, and K; Met1-Asn7 in subunit C; Met1-Ser8 in subunit E; Met1-Ile2 in subunit I; Met1-Val3 in subunit J) and internal residues (Leu408-Gly418 in all subunit) were disordered. Model qualities and refinement statistics are summarized in Table 1.
Overall structure of Hpy GS. The monomeric structure of Hpy GS containing 481 amino acid residues was composed of 14 α-helices and 15 β-strands, which could be divided into the N-terminal domain (residues 1-113) and C-terminal domain (residues 114-481) (Fig. 1a). The N-terminal domain was exposed to the solvent, whereas the C-terminal helix, called the 'helical thong' , was inserted into a hydrophobic hole in the opposite subunit of the other hexameric ring (Fig. 1b). The catalytic and regulatory loops of GS were highly conserved, which included the Glu loop (flap, PGYE 337 AP), Asp loop (latch, D 60 -D 74 ), Asn loop (residues F 265 -N 274 ), Tyr loop (S 163 -Y 190 -M 199 ), and adenylation loop (NLF 407 KLT) 9,12 (Fig. 2a,b). These loops completed the classical 'bifunnel' structure of the active site. In general, the adenylation loop is located near the active site and regulates GS activity by adenylation 13 . Although the location of the adenylation loop of Hpy GS was conserved with that of other GS enzymes, the tyrosine residue (NLYDLP) of the adenylation site was replaced with phenylalanine (NLF 407 KLT) (Fig. 2a). As a result, Hpy GS could not be regulated by adenylation which is a unique feature among GS I-β subfamily members.
Oligomeric structure of Hpy GS. Although a hexamer of Hpy GS apo was present in each asymmetric unit of the crystal, it could be used to generate a dodecamer with 62 symmetry by stacking two hexameric rings face-to-face (Fig. 1b). The hexameric structure of Hpy GS was formed mainly by the Tyr loop of each subunit interacting through hydrogen bonds, hydrophobic interactions, and salt bridges. The solvent-accessible surface area (SAS) buried at the interface between the subunits in the hexameric structure of Hpy GS apo and Hpy GS sub were calculated to 3,698 Å 2 and 3,672 Å 2 (~18% of the monomer surface area), respectively (Protein-Protein Interaction Server PDBePISA at http://www.ebi.ac.uk/msd-srv/prot_int/). The hexameric interface of the dodecamer was composed of C-terminal helices 13 and 14 (residues 461-481), which extended to a hydrophobic hole of the opposite hexameric ring. The C-terminal helices 13 (Fig. S1). The SAS buried at the interface between the hexameric rings in the dodecameric structure of Hpy GS apo and Hpy GS sub were calculated to 2,875 Å 2 and 2,870 Å 2 (~13% of the monomer surface area), respectively. These results demonstrated the dodecameric form of Hpy GS.  (Table S1.). The adenylation loops had higher r.m.s.d. values, which may be attributed to the disorder of the loop. These results indicated that Hpy GS does not undergo a large conformational change upon substrate binding.
Active site of Hpy GS. The active site and substrate-binding residues of Hpy GS were examined using ATP and PPT as substrates. The active site was located at the centre of the bifunnel contributed by the Glu loop (flap, residues 334-339), Asn loop (residues 265-277), and Tyr loop (residues 163-199) of each subunit and the Asp loop (latch, residues 60-74) of the neighboring subunit (Fig. 2b). As shown in other GS structures, the entrance for ATP was located at the top of the bifunnel opening to the external surface, and glutamate could enter through the bottom of the bifunnel facing the hexameric interface (Fig. 1a). In general, PPT is phosphorylated in GS in the presence of ATP and forms an intermediate state analogue, phosphinothricin phosphate (P3P), and ADP 24,25 . However, although ATP and PPT were added to Hpy GS, ATP/ADP substrates were clearly identified in all subunits, whereas PPT/P3P substrates were not identified in most of the subunits of Hpy GS. Among twelve subunits, PPT was not phosphorylated, resulting in a substrate-bound form with PPT and ATP in three subunits. In only one subunit, PPT was phosphorylated by ATP hydrolysis, resulting in an intermediate state with P3P and ADP (Fig. 3a). Thereafter, we described the Hpy GS bound to P3P and ADP as intermediate state (Hpy GS int ).
All known 19 active site and catalytic residues in bacterial GS 9 were mostly well conserved in Hpy GS except for a serine residue (S53 in S. typhimurium and S57 in M. tuberculosis), which was replaced with cysteine (C63 in H. pylori). This suggests the similarity of the overall catalytic mechanism of the Hpy GS with that of other bacterial GS proteins. The adenine ring of ATP/ADP was oriented similarly to Mtb GS 26 , bound by hydrogen bonds (N1 to S283 Oγ, and N6 to N362 O), and stacked in the hydrophobic patch by residues F235 and R365. The phosphate group of ATP/ADP was bound by ionic interactions (O1α to R365 NH2, and O2β to R349 NH2). PPT was bound by six hydrogen bonds with Hpy GS (NP to E141 Oε1/G275 O, OTP to R331 Nε, OP to H279 Nε2/R331 NH2, and OεB to R369 NH2), and P3P was bound by seven hydrogen bonds, where the O13 atom additionally formed a hydrogen bond with R349 NH2 (Fig. 3a,b). The possible ammonium binding site was blocked by the methyl group of P3P, acting as an inhibitor.
Two or three divalent metal ions have been reported to be involved in the active site of GS in other organisms. Two metal ions (n1 and n2 metal-binding sites) were identified per subunit in the substrate-bound GS from S. typhimurium, and three metal ions (n1, n2, and n3 metal-binding sites) were identified in the substrate-bound GS from M. tuberculosis. In both the Hpy GS int and Hpy GS sub structures, one magnesium ion was identified in the n1 metal binding site. The magnesium ion was coordinated with P3P (OεB and O15), E141 (Oε1), E230 (Oε2), and E223 (Oε1) (Fig. S2).
Comparison with other GS proteins. The structural similarity of Hpy GS with other known bacterial GS proteins was determined by DALI server 27 12,19 . However, the Glu flap in both the Hpy GS apo and Hpy GS sub structures showed a similar closed conformation with little differences. The side chain of E337 in the Hpy GS apo structure was disordered, demonstrating the flexibility of the Glu flap, whereas the side chain of E337 in the Hpy GS sub structure formed strong hydrogen bonds with the N274 Oδ1 and D60 Nδ2 of the adjacent subunit (Fig. 4a,b). This finding suggests that the Glu flap in Hpy GS apo could exist in the open or closed form in the absence of substrates.

Conclusions
We determined the apo-and substrate-bound forms of Hpy GS by X-ray crystallography. A comparison of Hpy GS with Sty GS, Mtb GS, and Bsu GS also revealed high similarities in the structure and regulatory amino acid residues. However, the open form of GS was not found in Hpy GS, and the Glu flap was well ordered with a closed conformation in Hpy GS apo in the absence of substrates. This suggests that Hpy GS could exist in either the open or closed form regardless of the presence of substrates. Based on the sequence and structural information of Hpy GS, this protein cannot be clearly classified as GS I-α or GS I-β. Therefore, we may classify Hpy GS as a new member of the GS I subfamily, which has a specific 25-amino acid sequence and lacks the adenylation regulatory

Methods
Expression and purification. The gene encoding GS (glnA) was amplified by polymerase chain reaction using the genomic DNA of H. pylori as a template. It was inserted into the Nde1/Xho1-digested expression vector pET28b(+) (Novagen, Germany), containing a hexahistidine tag at its N-terminus. The recombinant Hpy GS was transformed and expressed in E.coli BL21 (DE3) star pLysS cells (Invitrogen, USA). The cells harbouring the glnA gene were grown at 310 K to an OD 600 of ~0.5 in Luria-Bertani medium supplemented with 30 µg mL −1 kanamycin and chloramphenicol. Overexpression of the recombinant protein was induced with 1.0 mM isopropyl β-D-thiogalactopyranoside (IPTG), and cell growth was continued at 303 K for 4 h. The cells were harvested by centrifugation at 4,200 g for 10 min at 277 K and immediately frozen at 193 K. Cell pellets were resuspended in lysis buffer [20 mM Tris-HCl pH 8.0/0.5 M NaCl/10% (v/v) glycerol/1 mM phenylmethylsulfonylfluoride] and lysed using an ultra sonicator (Sonics TM Vibra Cell VCX 750; Sonics, USA). The insoluble fractions were removed by centrifugation at 31,000 g for 1 h at 277 K.
The recombinant Hpy GS in the supernatant fraction was loaded on a nickel-charged His-trap immobilized metal affinity chromatography (IMAC) column (GE Healthcare, UK). The hexahistidine tagged Hpy GS was Crystallization. Purified Hpy GS was concentrated to 41 mg mL −1 using Centricon YM-10 (Millipore, USA) for crystallization trials. Initial screening was performed by the sitting-drop vapour diffusion method using 96-well CrystalQuick plates (Greiner Bio-One, Germany) with various commercial screens (Hampton Research, USA; Qiagen, Germany; Axygen, USA; Emerald Biosystems, USA). Each sitting drop was prepared by mixing  0.75 µl protein solution and 0.75 µl reservoir solution and incubated at room temperature. The initial crystals of Hpy GS were grown after four weeks of incubation under several conditions with polyethylene glycol (PEG) 6,000. The crystals of Hpy GS were further optimized, and the best crystals were grown in 10% (v/v) PEG 6,000, 100 mM HEPES pH 7.0, and 50 mM choline. To obtain the cocrystals with substrates, Hpy GS was concentrated to 19 mg mL −1 and mixed with 5 mM ATP, 5 mM PPT, and 5 mM magnesium chloride before crystallization. It was stored at 277 K for 1 h. The crystals of substrate-bound Hpy GS were grown in 2.0 M sodium formate and 100 mM sodium citrate at pH 5.0. X-ray data collection. Crystals of the apo-and substrate-bound Hpy GS were transferred to a cryoprotectant solution containing 30% PEG 6,000 and 30% glycerol in reservoir solution, respectively, and immediately flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K on an ADSC Quantum 315 CCD image-plate detector using synchrotron radiation on beamline 5 C of the Pohang Accelerator Laboratory, Pohang, Republic of Korea. Data were collected with 1° oscillation per image and a crystal-to-detector distance of 650 mm, and a total of 180 frames were recorded. Data were processed and scaled using the HKL-2000 program suite 28 .
Structure determination and refinement. The crystal structure of the apo form of Hpy GS was solved by molecular replacement using the GS structure from S. typhimurium as a template (1F52) with PHASER from the CCP4 program suite. Hpy GS in complex with ATP and PPT was solved using the apo form of Hpy GS as a template in the same program suite. Both structures were refined with REFMAC from the CCP4 program suite with intensity based twin refinement. Further refinement was carried out by the COOT and PHENIX program package. The refined model was finally evaluated by MolProbity. Data collection and refinement statistics are presented in Table 1.