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Structural Analyses of Helicobacter Pylori Folc Conducting Glutamation in Folate Metabolism

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Structural Analyses of Helicobacter Pylori Folc Conducting Glutamation in Folate Metabolism crystals Article Structural Analyses of Helicobacter Pylori FolC Conducting Glutamation in Folate Metabolism Joon Sung Park 1 , Hyoun Sook Kim 2, Sang Ho Park 1, Mi Seul Park 1, Sung-Min Kang 1 , Hyun-Jung Kim 3 and Byung Woo Han 1,* 1 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Korea 2 Division of Precision Medicine, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do 10408, Korea 3 College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Korea * Correspondence: [email protected]; Tel.: +82-2-880-7898 Received: 26 July 2019; Accepted: 17 August 2019; Published: 19 August 2019 Abstract: FolC plays important roles in the folate metabolism of cells by attaching l-Glu to dihydropteroate (DHP) and folate, which are known activities of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS), respectively. Here, we determined the crystal structure of Helicobacter pylori FolC (HpFolC) at 1.95 Å resolution using the single-wavelength anomalous diffraction method. HpFolC has globular N- and C-terminal domains connected by a single loop, and a binding site for ATP is located between the two domains. Apo-HpFolC was crystallized in the presence of citrate in a crystallization solution, which was held in the ATP-binding site. Structural motifs such as the P-loop and W-loop of HpFolC for binding of ATP and two magnesium ions are well conserved in spite of the low overall sequence similarity to other FolC/FPGSs. The W-loop would also recognize a folate molecule, and the DHP-binding loop of HpFolC is expected to exhibit a unique recognition mode on DHP, compared with other FolCs. Because human FolC is known to only exhibit FPGS activity, the DHFS activity of bacterial FolC is an attractive target for the eradication of pathogenic bacteria. Consequently, our structural analyses of HpFolC provide a valuable foundation for a universal antibacterial strategy against H. pylori as well as other pathogenic bacteria. Keywords: bifunctional FolC; dihydrofolate synthetase (DHFS); folate metabolism; folylpolyglutamate synthetase (FPGS); Helicobacter pylori 1. Introduction Helicobacter pylori is a pathogenic Gram-negative bacterium, which has been classified as a class 1 carcinogen that contributes to chronic inflammation within the human gastric niche and can lead to stomach cancer [1–3]. Eradication of H. pylori usually starts with triple therapy, which is a combination of a proton pump inhibitor, amoxicillin, and clarithromycin. Failure of this therapy as a result of antibiotic resistance to clarithromycin has been combated with newly developed approaches, including bismuth and tetracycline, but long-term plans still require novel antibacterial targets for H. pylori [4,5]. In cells, folate plays a central role in the single-carbon transfer system involved in DNA (thymidine and purines) and amino acid (Gly, Ser, and Met) synthesis; hence, it is essential for cell growth and replication [6]. Folate and its derivatives in cells are reported to exist mainly in polyglutamated forms, which were initially speculated to be a reservoir of cellular glutamate [6] and, further, to be substrate forms with elevated affinities to several folate-processing enzymes [7–9]. Several folate-derived drugs targeting folate metabolism also exhibit increased efficiencies by polyglutamation [10]. Folate polyglutamation is achieved through folylpolyglutamate synthetase (FPGS, EC 6.3.2.17), which attaches Crystals 2019, 9, 429; doi:10.3390/cryst9080429 www.mdpi.com/journal/crystals Crystals 2019, 9, 429 2 of 11 Crystals 2019, 9, x FOR PEER 2 of 11 polyglutamationl-Glu to acid moieties [10]. Folate of folate polyglutamation and its polyglutamated is achieved derivatives through fo inlylpolyglutamate an ATP-dependent synthetase manner (FPGS,(Figure 1EC). 6.3.2.17), which attaches L-Glu to acid moieties of folate and its polyglutamated derivatives in an ATP-dependent manner (Figure 1). Figure 1. ActivitiesActivities of of dihydrofolate dihydrofolate synthetase synthetase ( (DHFS)DHFS) and and folylpolyglutamate folylpolyglutamate synt synthetasehetase ( (FPGS)FPGS) in folatefolate metabolism. metabolism. FPGS isis knownknown as as a ubiquitousa ubiquitous enzyme enzyme found found in all livingin all cells.living Interestingly, cells. Interestingly, in some organisms in some organismssuch as Corynebacterium such as Corynebacteriumspecies, Escherichia species, coli, Escherichia Neisseria gonorrhoeaecoli, Neisseria, Mycobacterium gonorrhoeae, tuberculosis Mycobacterium, and tuberculosisPlasmodium, falciparumand Plasmodium, FPGS falciparum has been, reported FPGS has to been additionally reported perform to additionally the activity perform of dihydrofolate the activity ofsynthetase dihydrofolate (DHFS, synthetase EC 6.3.2.12) (DHFS, as aEC bifunctional 6.3.2.12) as enzymea bifunctional (referred enzyme to as (referred FolC in this to as paper) FolC in and this is paper)involved and in is the involved de novo in synthesis the de ofnovo folate synthesis [11–15] of (Figure folate1 ).[11H–15]. pylori (Figisure also 1). predictedH. pylori tois havealso predictedbifunctional to FPGShave bifunctional exhibiting DHFS FPGS activity exhibiting [16], DHFS which activity implies [16] that, awhich selective implies inhibitor that againsta selective the inhibitorDHFS activity against of Hthe. pylori DHFSFolC activity (HpFolC) of H could. pylori be FolC an attractive (HpFolC) candidate could be for an an attractive antibacterial candidate drug [17for]. an antibacterialSo far, structures drug [17] of FPGSs. from five different bacterial species have been determined: E. coli FolC (EcFolC,So far, PDB structures codes: 1W78 of FPGSs and 1W7K) from five [18], differentLactobacillus bacterial casei FPGS species (Lc haveFPGS, been PDB determined: codes: 1FGS, E 1JBV,. coli FolC1JBW, (Ec 2GC5,FolC, 2GC6, PDB codes: 2GCA, 1W78 and 2GCB)and 1W7K) [19–21 [18]], M, Lactoba. tuberculosiscillus caseiFolC FPGS (MtFolC, (LcFPGS, PDB PDB codes: codes: 2VOR 1FGS, and 1JBV,2VOS) 1JBW, [22], Thermotoga 2GC5, 2GC6, maritima 2GCA,FolC and (Tm 2GCB)FolC, [19 PDB–21] code:, M. 1O5Z),tuberculosis and YersiniaFolC (Mt pestisFolC,FolC PDB (Yp codes:FolC, 2VORPDB codes: and 2VOS) 3N2A, [22] 3NRS,, Thermotoga 3PYZ, and maritima 3QCZ). FolC Here, (Tm weFolC, determined PDB code: the 1O5Z), crystal and structure Yersinia of pestisHpFolC FolC at (1.95YpFolC, Å resolution PDB codes: using 3N2A, the single-wavelength3NRS, 3PYZ, and 3QCZ). anomalous Here, di weffraction determined (SAD) the method. crystal We structure analyzed of HptheFolC structure at 1.95 of ÅHp resolutionFolC in comparison using the single with- thewavelength other FolC anomalous/FPGS structures, diffraction which (SAD should) method. enable We a analyzeddeeper understanding the structure of of Hp folateFolC polyglutamation in comparison with in H the. pylori otherand FolC/FPGS provide structures, a structural which basis should for the enabledevelopment a deeper of newunderstanding antibiotics of against folateH polyglutamation. pylori. in H. pylori and provide a structural basis for the development of new antibiotics against H. pylori. Crystals 2019, 9, 429 3 of 11 2. Materials and Methods 2.1. Cloning, Protein Expression, and Purification The HpFolC gene (hp_1545, residues 1–394) was cloned into the pET-21a(+) vector (Novagen, Darmstadt, Germany) with a C-terminal hexahistidine tag. The recombinant vector was transformed into Rosetta 2(DE3), an E. coli strain. The transformed cells were selected and cultured with Luria-Bertani media containing 50 µg/mL ampicillin. Cells were shake-incubated at 37 ◦C until OD600 reached 0.5, and 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added for induction. After induction, the cells were incubated for an additional 16 h at 20 C. Cultured cells were harvested at 6,000 g and ◦ × lysed by sonication in a buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 35 mM imidazole, and 1 mM phenylmethanesulfonyl fluoride. The cell lysate was centrifuged at 35,000 g for 1 h and the × supernatant was filtered with 0.45 µm syringe filter (Sartorius, Göttingen, Germany). The filtrate was loaded onto a HiTrap chelating HP column (GE Healthcare, Chicago, IL, USA) and eluted by adding an increasing gradient of a buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 1 M imidazole. The eluted fractions containing HpFolC protein were desalted using a HiPrep 26/10 desalting column (GE Healthcare, Chicago, IL, USA) with a buffer containing 20 mM Tris-HCl (pH 9.0) and 50 mM NaCl. The desalted sample was loaded onto a HiTrap 5 ml Q HP column (GE Healthcare, Chicago, IL, USA) and eluted by adding an increasing gradient of a buffer containing 20 mM Tris-HCl (pH 9.0) and 1 M NaCl. The eluted protein was loaded onto a HiLoad 16/600 Superdex 200 pg column (GE Healthcare, Chicago, IL, USA) equilibrated with a buffer containing 10 mM HEPES-NaOH (pH 7.0) and 100 mM NaCl. The eluted protein was concentrated for crystallization. Selenomethionine (SeMet)-derived HpFolC was expressed with B834(DE3), a methionine auxotrophic E. coli strain. The transformed cells were cultured in the media containing M9 minimal salts (Sigma-Aldrich, Darmstadt, Germany) and an amino acid mix with l-selenomethionine. Cells were shake-incubated at 37 ◦C until OD600 reached 0.5, and 0.5 mM IPTG was added for induction. After induction, the cells were incubated for an additional 16 h at 20 ◦C. The subsequent purification procedure was the same as for native HpFolC. 2.2. Crystallography Initial crystal of HpFolC was obtained using the sitting-drop vapor diffusion method from a commercial crystallization-screening reagent containing 20% (w/v) polyethylene glycol 3350 and 0.2 M ammonium citrate tribasic (pH 7.0) (The Classic II suite; QIAGEN, Hilden, Germany). HpFolC crystals were further optimized using the hanging-drop vapor diffusion method for X-ray diffraction experiments. Before exposure to X-ray beams, crystals were soaked in the crystallization solution supplemented with 25% glycerol for cryoprotection and flash-cooled in a 100 K nitrogen gas stream. Diffraction data were indexed and scaled with HKL2000 [23].
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