Title: Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase
Abstract: Article23 November 2006free access Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase Chien-Hua Pai Chien-Hua Pai Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan Search for more papers by this author Bing-Yu Chiang Bing-Yu Chiang Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Tzu- Ping Ko Tzu- Ping Ko Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Search for more papers by this author Chia-Cheng Chou Chia-Cheng Chou Genomics Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Cheong-Meng Chong Cheong-Meng Chong Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Search for more papers by this author Fang-Jiun Yen Fang-Jiun Yen Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Search for more papers by this author Shoujun Chen Shoujun Chen Departments of Medicinal Chemistry & Chemistry, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author James K Coward James K Coward Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Andrew H-J Wang Corresponding Author Andrew H-J Wang Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Genomics Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chun-Hung Lin Corresponding Author Chun-Hung Lin Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Genomics Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chien-Hua Pai Chien-Hua Pai Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan Search for more papers by this author Bing-Yu Chiang Bing-Yu Chiang Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Tzu- Ping Ko Tzu- Ping Ko Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Search for more papers by this author Chia-Cheng Chou Chia-Cheng Chou Genomics Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Cheong-Meng Chong Cheong-Meng Chong Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Search for more papers by this author Fang-Jiun Yen Fang-Jiun Yen Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Search for more papers by this author Shoujun Chen Shoujun Chen Departments of Medicinal Chemistry & Chemistry, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author James K Coward James K Coward Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Andrew H-J Wang Corresponding Author Andrew H-J Wang Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Genomics Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chun-Hung Lin Corresponding Author Chun-Hung Lin Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Genomics Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Author Information Chien-Hua Pai1,2, Bing-Yu Chiang1,3, Tzu- Ping Ko1, Chia-Cheng Chou4, Cheong-Meng Chong1, Fang-Jiun Yen1, Shoujun Chen5, James K Coward3, Andrew H-J Wang 1,3,4 and Chun-Hung Lin 1,3,4 1Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan 2Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan 3Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan 4Genomics Research Center, Academia Sinica, Taipei, Taiwan 5Departments of Medicinal Chemistry & Chemistry, University of Michigan, Ann Arbor, MI, USA *Corresponding authors: Institute of Biological Chemistry, Academia Sinica, No. 128 Academia Road Section 2, Nan-Kang, Taipei 11529, Taiwan. Tel.: +886 2 2788 1981; Fax: +886 2 2788 2043; E-mail: [email protected] or Tel.: +886 2 2789 0110; Fax: +886 2 4705; E-mail: [email protected] The EMBO Journal (2006)25:5970-5982https://doi.org/10.1038/sj.emboj.7601440 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis—amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines. Introduction Parasitic diseases, such as Chagas' disease, African sleeping sickness and several widespread illnesses known collectively as leishmaniasis, cause millions of human deaths each year worldwide. Lack of suitable drugs or vaccines is a major concern. In contrast to bacterial or viral infections, development of effective antiparasitic chemotherapy has been hindered by the close similarities between parasite and host metabolisms. Many of the existing drugs suffer from poor efficacy, host toxicity or/and drug resistance. To aid the development of new drugs, a special emphasis should be placed on a metabolic pathway in parasites that differs from or does not exist in the host (Fairlamb and Cerami, 1992; Krauth-Siegel et al, 2003; Müller et al, 2003). Spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and glutathione (GSH, γGlu-Cys-Gly) are present at high concentrations (0.1−10 mM) in most cells. Spermidine is a polycationic molecule that interacts with proteins, phospholipids and nucleic acids (Marton and Pegg, 1995), affecting primarily cell proliferation and differentiation (Tabor and Tabor, 1984; Pegg, 1986; Wang, 1995). GSH, a primary antioxidant, is important in maintaining the redox balance as well as in reductively scavenging reactive oxygen species (Meister and Anderson, 1983). Notably, the enzyme glutathione reductase keeps GSH in a reduced/activated form. In contrast, protozoal parasites of the genera Trypanosoma and Leishmania lack GSH reductase and GSH peroxidase activities (Boveris et al, 1980; Fairlamb and Cerami, 1985; Penketh et al, 1987). These pathogenic parasites instead employ trypanothione (bis(glutathionyl)spermidine) to defend against oxidative stress (Shames et al, 1986). The analogous enzymes trypanothione reductase and trypanothione peroxidase exist exclusively in the Kinetoplastida (Fairlamb and Cerami, 1992). Thus, trypanothione-related metabolism appears to be an attractive target for therapeutic intervention. There are two biosynthetic steps to produce trypanothione from GSH and spermidine; the initial reaction requires glutathionylspermidine synthetase (GspS) to catalyze the coupling of GSH and spermidine to form glutathionylspermidine (Gsp) (Henderson et al, 1990; Smith et al, 1992). Gsp is then conjugated with another GSH to produce trypanothione by trypanothione synthetase (TryS) (Oza et al, 2002a, 2002b, 2003; Comini et al, 2003). Each step involves an amide bond formation that requires prior phosphorylation of the carboxy (C) terminus of GSH by ATP. Escherichia coli produces only the metabolic intermediate Gsp, but not trypanothione. The corresponding enzyme, GspS, was identified more than four decades ago (Dubin, 1959; Tabor and Tabor, 1975). Although the biological function of the E. coli GspS remains obscure, previous work indicates that the enzyme has a second activity to hydrolyze Gsp back to GSH and spermidine (Bollinger et al, 1995). The two activity domains are separate in this bifunctional protein: the amidase domain is located at the N terminus and the synthetase domain is at the C terminus (Kwon et al, 1997). Interdomain communication negatively regulates the amidase activity in the E. coli enzyme (Lin et al, 1997b). Some parasites, such as Trypanosoma cruzi and Trypanosoma brucei, use one enzyme to synthesize Gsp and trypanothione, but others (e.g., Crithidia fasciculata) utilize two separate enzymes for this purpose (Oza et al, 2002a, 2002b, 2003; Comini et al, 2003). The majority of these proteins are bifunctional, having both amidase and synthetase activities, which suggests the importance of regulating the physiological concentrations of substrate and/or product. The scarcity of homologous sequences and the lack of any structural information have impeded our understanding of the Gsp- or trypanothione-related enzymes. Herein we report the determination of five X-ray crystal structures of E. coli GspS, including the protein/substrate, protein/product and protein/inhibitor complexes. In particular, during crystallization, the nanomolar phosphinate inhibitor became phosphorylated to generate the phosphinophosphate intermediate at the active site despite its limited stability (t1/2=25 min) (Chen et al, 1997; Lin et al, 1997a). These results clarify the mechanistic details of the synthetase reaction and will contribute to our understanding of structural and functional differences within the TryS enzyme family. Results Overall structure We obtained five crystal structures: apo_GspS and the GspS_AMPPNP, GspS_GSH_ADP, GspS_inhibitor and GspS_ADP complexes. All these structures contain a dimeric GspS in each asymmetric unit (Figure 1). Each of the final refined structures includes 571–603 total residues per GspS monomer, with some disordered regions at the N terminus (1–10) and in some surface loop regions (536–542, 547–563 in apo_GspS, and 30–40, 455–457 in the GspS complexes). The overall structure description is based on the information from the GspS_GSH_ADP structure that has the highest resolution (2.2 Å) and is more intact in the refined model. Figure 1.Overall structure of E. coli glutathionylspermidine synthetase/amidase. A ribbon diagram of the overall structure of E. coli GspS, showing two monomers in the asymmetric unit, and a pseudo-two-fold axis between the two monomers. The amidase domain (N-terminal 1–195), synthetase domain (C-terminal residues 206–619) and linker region (Glu196 to Ala205) are labeled. Active sites of the synthetase domain are revealed by the substrates represented as sticks (ADP and GSH) and spheres (Mg2+). Side chains of catalytic residues Cys59 and His131 in the amidase domain are designated in the same way. The dash represents a portion of the undefined region (residue 30–40) in the solved structure. The ribbon figures were drawn using PyMOL. Download figure Download PowerPoint The globular structure reveals a mixed α/β fold with a size of 30 × 35 × 40 Å3 in the N-terminal amidase domain and an equilateral triangle shape in the C-terminal synthetase domain with the sides of the triangle of ∼60 Å and thickness of 30 Å (Figure 2A). Residues 196–205 between the two domains are defined as the linker region. The N-terminal amidase domain has an open-sandwich topology comprising two central α-helices (α2 and α3) surrounded by four (β1, β2, β3 and β4) and eight (β5, β6, β7, β8, β9, β10, β11 and β12) antiparallel twisted strands, as shown in Figure 2B. As we will demonstrate, the C-terminal synthetase domain belongs to the ATP-grasp superfamily (Murzin, 1996) and is structurally similar to that of human glutathione synthetase (PDB code: 2HGS) (Polekhina et al, 1999), despite no obvious sequence homology. The synthetase domain is composed of three main structural units, including (1) an antiparallel β-sheet (strands β15, β16, β29, β30 and β31; green in Figure 2B), together with α6 (green), α7, α8 (gray), α14 and α15 (yellow) packing on one side of the sheet, α4, α5 (gray), α9, α10 (blue), β13, β14 and β32 (gray) packing on the other side; (2) a parallel β-sheet (β17, β18, β21 and β22; red in Figure 2B) together with α11, α13 (red), α12, β19 and β20 (gray) and (3) a lid domain (orange in Figure 2B) composed of an antiparallel sheet of β23, β24, β25, β26, β27 and α16. Figure 2.Structure analysis of the two activity domains in E. coli GspS. (A) Folding of the amidase domain (left, residues 1–195) and synthetase domain (right, residues 206–619). The amidase domain contains two central α-helices (red) that are surrounded by four and eight antiparallel twisted strands (yellow and blue, respectively). The synthetase domain mainly consists of antiparallel β-sheets (green), parallel β-sheets (red) and a lid domain (orange). Please see Results for the detailed description. (B) A topology diagram corresponding to each activity domain. The color codes for the secondary structural elements are identical to those in (A). (C) A stereo view of the catalytic region of the synthetase domain. The substrates ADP and GSH are shown as ball-and-stick structures and Mg-O as spheres (green–red). Download figure Download PowerPoint The active site of the synthetase domain, clearly demarcated by the bound ligands in the complex structures, is located at the central antiparallel β-sheet and is surrounded by five loops (Figure 2C for stereo view); that is, P-loop (residues 535–543, designated in orange), loop1 (441–444, yellow), loop2 (332–338, cyan green), loop3 (601–609, red) and the D–E loop (387–392, green). As a part of the lid domain, P-loop (536AGRCGS542) is disordered in the apo_GspS structure, but forms a closed conformation when bound with substrate, product or inhibitor. Figure 3A and 3B show the surface charge potential of the synthetase active site of the GspS_GSH_ADP and GspS_inhibitor complexes, respectively. As shown in Figure 4A, P-loop, loop2 and loop3 have different conformations due to the binding of ATP and GSH. Figure 3.Substrate GSH (left) and inhibitor (right) binding in the Gsp synthetase domain and its electrostatic surface. Electrostatic surface representations of the GspS_GSH_ADP (A) and GspS_inhibitor (B) complexes. The colors red, white and blue indicate negative, neutral and positive charges, respectively. Download figure Download PowerPoint Figure 4.Details of the interactions of GspS with substrate/product or inhibitor in the synthetase domain. (A) Interaction of substrate with loops. Three loops are shown, including the P-loop (residues 536–542), loop2 (332–338) and loop3 (601–609). The loops are represented by ribbons, and side chains of the residues interacting with GSH are shown as thicker lines. GSH is shown as ball-and-stick structures. The figure shows the comparison of positions of the three loops and the corresponding side chains between the structures apo_GspS (olive-green) and GspS_GSH_ADP (magenta). Hydrogen bonds between GspS and GSH are depicted as black dotted lines. The disulfide bond between Cys338 and the Cys of GSH is shown in yellow. (B) ADP-binding site. ADP is represented as a ball-and-stick structure, and the interacting residues are shown by their side chains as thicker lines in cyan. The P-loop, including Arg538 and Gly540, is in red. Magnesium ions are shown as green spheres. (C) Magnesium-binding site. Mg2+ ions are shown as light green and water molecules as red. The side chains of the coordinating residues are presented as ball-and-stick structures. Coordination of Mg2+ ions is depicted by dashed lines, and the distances are listed. The 2Fo−Fc electron density map of Mg-ADP contoured at 1σ level is shown. (D) Details of the interactions between the phosphinate inhibitor and GspS. Five loops are presented here in different colors, including the P-loop in orange, loop1 in yellow, loop2 in forest green, loop3 in red and D–E loop in green. Residues making hydrogen bonds with the inhibitor are shown by thicker lines. Download figure Download PowerPoint The amidase domain is a member of the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) superfamily (Bateman and Rawlings, 2003). It is a cysteine protease with Cys59 and His131 as the catalytic dyad, and these two amino acids are invariant among all GspS and TryS enzymes. Dimerization GspS exists as a dimer in solution, as supported by analytical ultracentrifugation (see supporting information). The sedimentation velocity of E. coli GspS estimates the molecular mass to be 138 kDa. Because the GspS polypeptide has a mass of 70 kDa, this result suggests that GspS should exist as a dimer in solution. Thus, the dimeric GspS structure in the asymmetric unit is considered as a functional dimer. The intersubunit contacts have a total buried surface area of 3400 Å2. The intersubunit interactions are between the amidase domain from one monomer and the synthetase domain from another monomer (Figure 1). Hydrophobic interactions between the two monomers are Leu15 with Ala424, Pro20 with Ala461, Ala114 with Ala460 and Leu303 with Val94. A salt-bridge interaction exists between Arg307 in one monomer and Asp49 in another monomer with a distance of 2.85 Å. Additionally, hydrogen bonds are observed in the dimeric interface, such as Tyr18 with Arg481, and Gln160 with Thr466. ATP-binding site ADP was located at the antiparallel β-sheet of GspS in a manner analogous to that observed in other ATP-grasp proteins (Fan et al, 1994, 1995; Polekhina et al, 1999; Thoden et al, 2000, Figure 2C for stereo view). The adenine ring is buried in a hydrophobic pocket that is shaped by Tyr329, Ala531, Leu570, Leu603, Val604 and Leu515. The exocyclic 6-amino group of the adenine base is hydrogen bonded with the main-chain oxygen of Gln569 and the N1 with the amide hydrogen of Trp571 (Figure 4B). The O2′ atom of the ribose forms hydrogen bonds with the main-chain oxygen of Leu603 and amide of Ile605, and the O3′ atom with Nε2 of Gln582. The negative charges on the α- and β-phosphates are compensated by two conserved residues, Lys498 and Lys533. Both ε-amino groups of Lys residues form salt bridges with Oδ− of ADP. The position of the γ-phosphate of ATP is deduced from the GspS_AMPPNP structure where Nη of Arg316 is close to the γ-phosphate within the hydrogen-bonding distance. The main-chain nitrogen of Gly540 and Cys539 in the P-loop interact with the β- and γ-phosphates, respectively. Magnesium binding Figure 4C and 4D shows the location of two magnesium ions in the complex structures. They are both bound in an octahedral geometry. Mg1 (left green ball in Figure 4C) is ligated by an α-phosphate oxygen and a β-phosphate oxygen atom of ADP, Oδ1 of Asp318, a carboxylate oxygen of Glu330 and two water molecules. The metal-ligand distances vary from 1.97 to 2.15 Å. The γ-carboxylate group of Glu330 also interacts with Mg2 (right green ball in Figure 4C) using both oxygen atoms (Mg–O distances are 2.19 and 1.97 Å). The other four ligands of Mg2 include a β-phosphate oxygen atom of ADP, Oδ of Asn332 and two water molecules, with the distances ranging from 1.96 to 2.04 Å. Glu330, a highly conserved residue in all ATP-grasp proteins, seems to play a vital role in enzyme catalysis, because it bridges between the two metal sites. The transferred phosphate during phosphorylation of the inhibitor In our previous report (Chen et al 1997; Lin et al, 1997a), the phosphinate analog of Gsp was found to exhibit an ATP-dependent, slow-binding inhibition against E. coli Gsp synthetase. The mixture of GspS, ATP and the phosphinate inhibitor was co-crystallized for structural analysis. In the final refined structure, ATP was found to be hydrolyzed to ADP. In addition, an extra phosphate was attached to the phosphinate oxygen, indicating that phosphorylation of the inhibitor was driven by ATP hydrolysis to give the tetrahedral phosphinophosphate that is bound at the active site. The intermediate mimics the tetrahedral adduct formed by the nucleophilic addition of spermidine to the acylphosphate (see Supplementary data). The γ-phosphate in AMPPNP or transferred phosphate in phosphinophosphate interacts with both Mg2+ ions, the main-chain amide of Cys539 in the P-loop, and Nη of Arg316. Arg316 is an important residue that plays a role in the transfer of γ-phosphate from ATP and the stabilization of the anionic tetrahedral intermediate. Arg316 hydrogen bonds to the γ-phosphate of AMPPNP (Figure 5A) as well as the phosphinyl oxygens of the inhibitor (Figure 5B). The main-chain amide of Cys539 contacts the γ-phosphate in the GspS_AMPPNP structure and the transferred phosphate in the GspS_inhibitor structure. The interaction stabilizes the pentavalent phosphate intermediate in the phosphorylation step (Figure 5A and B). Bridging between the transferred phosphate and ADP, the two Mg2+ ions serve as Lewis acids to assist the phosphate transfer and compensate the resulting negative charges during catalysis. Figure 5.Two different binding sites of GSH indicated by comparing the complex structures. (A, B) A special emphasis is placed on the positions of the γ-phosphate and transferred phosphate. Ligands are drawn as ball-and-stick structures and Mg2+ as spheres. (A) The stereo view of the AMPPNP-binding site in the GspS_AMPPNP structure. The P-loop and the interacting residues are green. (B) The stereo view of the ADP and inhibitor-binding site in the GspS_inhibitor structure. The P-loop and the interacting residues are in magenta. (C, D) Comparison of the GspS_GSH_ADP and GspS_inhibitor structures with a special focus on the substrate-binding sites. Boxes show the substrate and the inhibitor-binding sites in the complex structures. Download figure Download PowerPoint Furthermore, the Gsps_inhibitor structure was found similar to the complex structure of E. coli GSH synthetase with a phosphinyl peptide (Hiratake et al 1994; Hiratake, 2005). The peptide was phosphorylated, and ADP and the resulting phosphorylated phosphinate were located at the enzyme active site, as shown by the X-ray crystal structure analysis. Despite no obvious homology between this enzyme and E. coli GspS, both enzymes utilized the same residues to interact with the phosphinate and phosphates, including Arg316, D318 and E330 of GspS (corresponding to R210, D273 and E 281 of GSH synthetase, respectively). Two GSH-binding sites The first GSH-binding site (S2) is observed in the structure of GspS_GSH_ADP (Figure 5C). Surprisingly, the substrate is bound in the active site by forming a disulfide bond between its own Sγ atom and the Cys338 Sγ atom. The Gly portion of GSH also forms an isopeptide bond with Nζ of Lys607 (see Supplementary data).The orientation of the GSH binding in this structure is considered to be opposite to what it should adopt in the catalysis because the C-terminal carboxylate of Gly, serving as a nucleophile during the phosphate transfer, is located far from the ADP-binding site (S1). The formation of this isopeptide bond may be an accidental trap for nonproductive reactions in the absence of spermidine. GSH forms many hydrogen bonds with Arg316, Ser335, Ser337, Arg538, Arg598 and water molecules. Formation of the mixed disulfide and the isopeptide, considered as a nonproductive mode, was observed only when the enzyme was incubated with ATP and GSH. In contrast, we never saw such formation in additional presence of spermidine (data not shown). The proteins were crystallized at pH 8.5 that is very different from the optimum pH (7.0). The basic condition gives 20% of the optimum activity at pH 7.0 and also favors the disulfide bonding formation. The second GSH-binding site (S3) is revealed by the GspS_inhibitor structure (Figure 5D). The phosphinate inhibitor contains a tripeptide moiety of γ-Glu-Ala-Gly that is analogous to GSH (Figure 6). The tripeptide moiety interacts with several amino acids from the enzyme, including Ser335, Asp387, Glu392, Ala443 and Thr446 (Figure 4D). Figure 6.Proposed reaction mechanism of GspS in comparison with phosphorylation of the phosphinate inhibitor. Download figure Download PowerPoint Spermidine binding The possible spermidine-binding site is illustrated in the GspS_inhibitor structure, because the inhibitor is a Gsp analog containing GSH and spermidine. The interactions include hydrogen bonding between the terminal NH3+ group of inhibitor and Oδ1 of Asp610, as well as bidentate H-bonding of Glu391 Oε with the middle nitrogen of the spermidine moiety. The middle nitrogen is also within hydrogen-bonding distance with the main-chain oxygen of Lys607 (Figure 4D). The D–E loop was found to have different conformations between the GspS_GSH_ADP (Figure 5C) and GspS_inhibitor structures (Figure 5D). The four negatively charged residues Asp387, Asp389, Glu391 and Glu392 are closer to the S2 and S3 sites in the GspS_inhibitor structure in comparison with the GspS_GSH_ADP structure. (see Supplementary data). Notably, the side chain of Glu391 is positioned very differently in the two structures, revealing its key role in spermidine binding. The hydroxyl group of Ser337 is at a distance of 3.2 Å from the first carbon of the spermidine moiety of the inhibitor, indicating that the residue may facilitate the nucleophilic attack during catalysis by interacting with spermidine. Ser337 and Glu391 Oε2 form a hydrogen bond and are involved in the deprotonation of spermidine (Figure 6). Discussion Comparison of GspS with other ATP-grasp enzymes Although GspS lacks any significant sequence identity or homology with other members of the ATP-grasp superfamily, our GspS crystal structures suggest that the enzyme indeed belongs to the superfamily. Based on published structures and structural comparisons among members, our consensus structure is related to human glutathione synthetase (hGS, Figure 7A and B), because the two structures can be superimposed to define the conserved structural units, including the parallel β-sheet, antiparallel β-sheet and lid domain (Figure 7C). Figure 7D shows structure-based alignment of E. coli GspS and hGS, revealing that a number of important residues and loops are conserved in the two proteins. The structure of E. coli GspS can be superimposed on hGS with an rms deviation of 320 Cα atoms of 3.8 Å. The most structurally similar units between the two structures are the parallel β-sheets (Figure 7C). hGS has two relatively long α-helices in the lid domain, in contrast to one short helix of E. coli GspS. The antiparallel β-sheet of hGS is more compact with extra and larger helices. As this domain facilitates substrate binding in both enzymes, the loose arrangement in GspS either accommodates larger substrates (or products) or undergoes significant conformational changes during catalysis. Figure 7.Comparison of hGspS and E. coli GspS domain. (A) Ribbon diagrams of hGS (PDB: 2HGS) and (B) E. coli GspS synthetase domain. (C) The comparisons of the lid domain, antiparallel β-sheet and parallel β-sheet units between the human glutathione synthetase and E. coli GspS synthetase domain. (D) Structure-based sequence alignment of E. coli GspS and hGS. The secondary structure and some residue numbering of E. coli GspS are shown above the alignment. All residues numbering of the two proteins are shown on both sides of each line. Catalytically related and conserved residues are shown in sky-blue; identical or similar residues are pink, and the P-loop defined in the E. coli GspS structure is indicated. The figure was produced using ALSCRIPT (Barton, 1993). Download figure Download PowerPoint Two rare non-proline, cis-peptide bonds in the helical connection were found in our structure, including K56−W57 and F487−E488. K56−W57 is located in the N-terminal amidase domain. Interestingly, E488 corresponds to P295 in hGS, N114 in E. coli glutathione synthetase, G104 in biotin carboxylase and G85 in D-Ala-D-Ala ligase. Moreover, there is a great extent of variation in the residues forming the ATP-binding pocket; only a few residues are strictly conserved, such as Arg316, Lys498 and Lys533, which interact with the phosphates, E330 with Mg2+, and D318 and N332. The contributions of these comparativ