Structural analysis of a shrimp thymidylate synthase reveals species-specific interactions with dUMP and raltitrexed
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.
Keywordsthymidylate synthase (TS) closed conformation deoxyuridine monophosphate (dUMP) thymidine monophosphate (TMP) raltitrexed Litopenaeus vannamei
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We thank the staffs from the BL17U1 and BL19U1 beamline stations at SSRF for assistance during data collection.
- 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.CrossRefGoogle Scholar
- 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.Google Scholar
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar
- 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.Google Scholar
- 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.CrossRefGoogle Scholar
- Food and Agriculture Organization of the United Nations Globefish. 2018. Market Reports, http://www.fao.org/in-action/globefish.
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar