Abstract
Thymidylate synthase (TS) is a key enzyme in the de novo biosynthesis of thymidine monophosphate, serving as a well-known drug target in chemotherapy against cancers and infectious diseases. Additional to its clinical value, TS is supposed to be a promising drug target in aquatic-disease control. To facilitate designing pathogen-specific TS inhibitors for shrimp-disease control, we report the crystal structures of TS from Litopenaeus vannamei (LvTS) in the apo form, LvTS-dUMP complex and LvTS-dUMP-raltitrexed complex at 2.27 Å, 1.54 Å, and 1.56 Å resolution, respectively. LvTS shares a similar fold with known TSs, existing as a dimer in the crystal. The apo LvTS and LvTS-dUMP take an open conformation, and raltitrexed binding induces structural changes into a closed conformation in LvTS-dUMP-raltitrexed. Compared to those in other known TS-dUMP-raltitrexed complexes with the closed conformation, the C-terminal loop in LvTS-dUMP-raltitrexed shifts its position away from the bound raltitrexed; the distance between C6 of dUMP and Sγ of the catalytic cysteine is obviously longer than that in the known TS structures with closed conformations, resembling that in the TS structures with open conformations. Other species-specific interactions with dUMP and raltitrexed are also observed. Therefore, LvTS-dUMP-raltitrexed adopts a loosely closed conformation with structural features intermediate between the closed and the open conformations that were reported in other TSs. Our study provides the first crustcean TS structure, and reveals species-specific interactions between TSs and the ligands, which would facilitate designing pathogen-specific TS inhibitors for shrimp-disease control.
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Afonine P V, Grosse-Kunstleve R W, Echols N, Headd J J, Moriarty N W, Mustyakimov M, Terwilliger T C, Urzhumtsev A, Zwart P H, Adams P D. 2012. Towards automated crystallographic structure refinement with Phenix. refine. Acta Crystallographica Section D: Structural Biology, 68(4): 352–367, https://doi.org/10.1107/S0907444912001308.
Arvizu-Flores A A, Aispuro-Hernandez E, Garcia-Orozco K D, Varela-Romero A, Valenzuela-Soto E, Velazquez-Contreras E F, Rojo-Domínguez A, Yepiz-Plascencia G, Maley F, Sotelo-Mundo R R. 2009. Functional identity of the active sites of crustacean and viral thymidylate synthases. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 150(3): 406–413, https://doi.org/10.1016/j.cbpc.2009.06.008.
Aslanidis C, de Jong P J. 1990. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Research, 18(20): 6 069–6 074, https://doi.org/10.1093/nar/18.20.6069.
Cardinale D, Guaitoli G, Tondi D, Luciani R, Henrich S, Salo-Ahen O M H, Ferrari S, Marverti G, Guerrieri D, Ligabue A, Frassineti C, Pozzi C, Mangani S, Fessas D, Guerrini R, Ponterini G, Wade R C, Costi M P. 2011. Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase. Proceedings of the National Academy of Sciences of the United States of America, 108(34): E542–E549, https://doi.org/10.1073/pnas.1104829108.
Carreras C W, Santi D V 1995. The catalytic mechanism and structure of thymidylate synthase. Annual Review of Biochemistry, 64: 721–762, https://doi.org/10.1146/annurev.bi.64.070195.003445.
Chen D, Jansson A, Sim D, Larsson A, Nordlund P. 2017. Structural analyses of human thymidylate synthase reveal a site that may control conformational switching between active and inactive states. The Journal of Biological Chemistry, 292(32): 13 449–13 458, https://doi.org/10.1074/jbc.M117.787267.
Choi Y M, Yeo H K, Park Y W, Lee J Y. 2016. Structural analysis of thymidylate synthase from Kaposi’s sarcoma-associated herpesvirus with the anticancer drug raltitrexed. PLoS One, 11(12): e0168019, https://doi.org/10.1371/journal.pone.0168019.
Davis I W, Leaver-Fay A, Chen V B, Block J N, Kapral G J, Wang X Y, Murray L W, Arendall III W B, Snoeyink J, Richardson J S, Richardson D C. 2007. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Research, 35(S2): W375–W383, https://doi.org/10.1093/nar/gkm216.
de Clercq E, Balzarini J, Descamps J, Bigge C F, Chang C T C, Kalaritis P, Mertes M P. 1981. Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2′-deoxyuridine and their 5′-phosphates. Biochemical Pharmacology, 30(5): 495–502, https://doi.org/10.1016/0006-2952(81)90635-3.
Deschamps P, Réty S, Bareille J, Leulliot N. 2017. Crystal structure of the active form of native human thymidylate synthase in the absence of bound substrates. Acta Crystallographica Section F: Structural Biology Communications, 73(6): 336–341, https://doi.org/10.1107/S2053230X17007233.
Dowiercial A, Wilk P, Rypniewski W, Rode W, Jarmula A. 2014. Crystal structure of mouse thymidylate synthase in tertiary complex with dUMP and raltitrexed reveals N-terminus architecture and two different active site conformations. Biomed Research International, 2014: 945803, https://doi.org/10.1155/2014/945803.
Emsley P, Lohkamp B, Scott W G, Cowtan K. 2010. Features and development of Coot. Acta Crystallographica Section D: Structural Biology, 66(4): 486–501, https://doi.org/10.1107/S0907444910007493.
Flegel T W. 2012. Historic emergence, impact and current status of shrimp pathogens in Asia. Journal of Invertebrate Pathology, 110(2): 166–173, https://doi.org/10.1016/j.jip.2012.03.004.
Food and Agriculture Organization of the United Nations Globefish. 2018. Market Reports, http://www.fao.org/in-action/globefish.
Gibson L M, Lovelace L L, Lebioda L. 2008. The R163K mutant of human thymidylate synthase is stabilized in an active conformation: structural asymmetry and reactivity of cysteine 195. Biochemistry, 47(16): 4 636–4 643, https://doi.org/10.1021/bi7019386.
Holm L, Rosenström P. 2010. Dali server: conservation mapping in 3D. Nucleic Acids Research, 38(S2): W545–W549, https://doi.org/10.1093/nar/gkq366.
Jackman A L. 1999. Antifolate Drugs in Cancer Therapy. Humana Press, Totowa NJ. 456p, https://doi.org/10.1007/978-1-59259-725-3.
Lightner D V, Redman R M, Pantoja C R, Tang K F J, Noble B L, Schofield P, Mohney L L, Nunan L M, Navarro S A. 2012. Historic emergence, impact and current status of shrimp pathogens in the Americas. Journal of Invertebrate Pathology, 110(2): 174–183, https://doi.org/10.1016/j.jip.2012.03.006.
McCoy A J, Grosse-Kunstleve R W, Adams P D, Winn M D, Storoni L C, Read R J. 2007. Phaser crystallographic software. Journal of Applied Crystallography, 40(4): 658–674, https://doi.org/10.1107/S0021889807021206.
Perry K M, Fauman E B, Finer-Moore J S, Montfort W R, Maley G F, Maley F, Stroud R M. 1990. Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases. Proteins, 8(4): 315–333, https://doi.org/10.1002/prot.340080406.
Phan J, Koli S, Minor W, Dunlap R B, Berger S H, Lebioda L. 2001. Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug. Biochemistry, 40(7): 1 897–1 902, https://doi.org/10.1021/bi002413i.
Robert X, Gouet P. 2014. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(W1): W320–W324, https://doi.org/10.1093/nar/gku316.
Rustum Y M, Harstrick A, Cao S, Vanhoefer U, Yin M B, Wilke H, Seeber S. 1997. Thymidylate synthase inhibitors in cancer therapy: direct and indirect inhibitors. Journal of Clinical Oncology, 15(1): 389–400, https://doi.org/10.1200/JCO.1997.15.1.389.
Satow Y, Cohen G H, Padlan E A, Davies D R. 1986. Phosphocholine binding immunoglobulin Fab McPC603: an X-ray diffraction study at 2.7 Å. Journal of Molecular Biology, 190(4): 593–604, https://doi.org/10.1016/0022-2836(86)90245-7.
Schiffer C A, Clifton I J, Davisson V J, Santi D V, Stroud R M. 1995. Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site. Biochemistry, 34(50): 16 279–16 287, https://doi.org/10.1021/bi00050a007.
Smart O S, Womack T O, Flensburg C, Keller P, Paciorek W, Sharff A, Vonrhein C, Bricogne G. 2012. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallographica Section D: Structural Biology, 68(4): 368–380, https://doi.org/10.1107/S0907444911056058.
Stout T J, Tondi D, Rinaldi M, Barlocco D, Pecorari P, Santi D V, Kuntz I D, Stroud R M, Shoichet B K, Costi M P. 1999. Structure-based design of inhibitors specific for bacterial thymidylate synthase. Biochemistry, 38(5): 1 607–1 617, https://doi.org/10.1021/bi9815896.
Stroud R M, Finer-Moore J S. 2003. Conformational dynamics along an enzymatic reaction pathway: thymidylate synthase, “the movie”. Biochemistry, 42(2): 239–247, https://doi.org/10.1021/bi020598i.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10): 2 731–2 739, https://doi.org/10.1093/molbev/msr121.
Vonrhein C, Flensburg C, Keller P, Sharff A, Smart O, Paciorek W, Womack T, Bricogne G. 2011. Data processing and analysis with the autoPROC toolbox. Acta Crystallographica Section D: Structural Biology, 67(4): 293–302, https://doi.org/10.1107/S0907444911007773.
Zaware N, Sharma H, Yang J, Devambatla R K V, Queener S F, Anderson K S, Gangjee A. 2013. Discovery of potent and selective inhibitors of Toxoplasma gondii thymidylate synthase for opportunistic infections. ACS Medicinal Chemistry Letters, 4(12): 1 148–1 151, https://doi.org/10.1021/ml400208v.
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We thank the staffs from the BL17U1 and BL19U1 beamline stations at SSRF for assistance during data collection.
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Supported by the National Natural Science Foundation of China (Nos. 31572660, 31872600), the “1000 Talents Program”, and the Qingdao Innovation Leadership Project (No. 18-1-2-12-zhc)
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Liu, C., Zang, K., Li, S. et al. Structural analysis of a shrimp thymidylate synthase reveals species-specific interactions with dUMP and raltitrexed. J. Ocean. Limnol. 38, 1891–1899 (2020). https://doi.org/10.1007/s00343-019-9184-8
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DOI: https://doi.org/10.1007/s00343-019-9184-8