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Cell Cycle Regulation and Novel Structural Features of Thymidine Kinase, an Essential Enzyme in Trypanosoma Brucei

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Cell Cycle Regulation and Novel Structural Features of Thymidine Kinase, an Essential Enzyme in Trypanosoma Brucei This is a repository copy of Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/108063/ Version: Accepted Version Article: Valente, Maria, Timm, Jennifer, Castillo-Acosta, Víctor M et al. (7 more authors) (2016) Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei. Molecular Microbiology. pp. 365-385. ISSN 0950-382X https://doi.org/10.1111/mmi.13467 Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei Maria Valente1§, Jennifer Timm2§, Vctor M. Castillo Acosta1, Luis M. Ruiz P%rez1, Tom Balzarini1, Joanne E. Nettleship3, Louise E Bird3, Heather Rada3, Keith S. .ilson2* / 0olores 1onz2lez Pacano3ska1* 1 Instituto de Parasitología y Biomedicina "López-Neyra", Consejo Superior de Investigaciones Cientficas, ,ranada, Spain 2 .or/ Structural Biology La0oratory, 1epartment of Chemistry, 3niversity of .or/, .or/ .O10 511, 36 3 The O8ford Protein Production 9acility, Research Comple8 at Harwell, Rutherford Appleton La0oratory, R92 Harwell, 1idcot, O8fordshire OX11 09A, 36 *Correspondence to: 1olores ,onz@lez-Pacanows/aA dgonzalezBip0.csic.es and 6eith S. DilsonA /eith.wilsonByor/.ac.u/ Running title: Cell cycle regulation and structure of TbT6 6eywords: Trypanosoma brucei, thymidine /inase, cell cycle, protein structure, pyrimidines This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/mmi.13467 This article is protected by copyright. All rights reserved. Molecular Microbiology Page 2 of 65 Abstract Thymidine /inase ET6F is a /ey enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the G-phosphate of ATP to 2H-deo8ythymidine EdThdF forming thymidine monophosphate EdTIPF. 3nli/e other type II T6s, the Trypanosoma brucei enzyme ETbT6F is a tandem protein with two T6 homolog domains of which only the C-terminal one is active. In this study, we esta0lish that TbT6 is essential for parasite via0ility and cell cycle progression, independently of e8tracellular pyrimidine concentrations. De show that expression of TbT6 is cell cycle regulated and that depletion of TbT6 leads to strongly diminished dTTP pools and 1NA damage indicating intracellular dThd to 0e an essential intermediate meta0olite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbT6 in comple8 with dThd and dTIP at resolutions up to 2.2 J. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleo0ase moiety compared to the human enzyme. Our findings strongly support TbT6 as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could 0e e8ploited in the process of rational drug design. 2 This article is protected by copyright. All rights reserved. Page 3 of 65 Molecular Microbiology Introduction Trypanosoma brucei is a unicellular parasite of the class 6inetoplastida and the causative agent of Human African trypanosomiasis EHAT, EDorld Health Organisation, 2014FF. In the last decade Trypanosoma brucei has 0een intensely studied in order to find new drugs and new drug targets that could improve parasitic chemotherapy and overcome the deficiencies of e8isting treatments. The identification of major differences in the 0iochemical pathways 0etween the parasites and their human hosts would help reduce to8icity of novel drug candidates. 1eo8ynucleotide meta0olism provides drug targets for many diseases as a 0alanced and well-controlled pool of deo8yri0onucleotides EdNTPsF is essential for 1NA replication and repair. In most species, two meta0olic routes contri0ute to the intracellular dNTP pool, the de novo and the salvage pathway EReichard, 1988F. Dhile deo8ythymidine triphosphate EdTTPF 0iosynthesis via deo8yuridine monophosphate Ed3IPF formation has 0een proven to 0e necessary for parasite survival in vitro ESien/iewicz et al., 2008F, very little is /nown a0out the relevance of dTTP formation via thymidine salvage. Thymidine /inases ET6s, EC 2.7.1.21F are /ey enzymes in the pyrimidine salvage pathway, catalyzing the magnesium-dependent transfer of the G-phosphate of ATP to thymidine EdThdF, forming thymidine monophosphate EdTIPF. SubseLuently, dTIP is phosphorylated further to dTTP, a substrate for 1NA synthesis and therefore essential for replication EAl-Iadhoun et al., 2004A Birringer et al., 2005A Birringer et al., 2006F. There are two types of T6s, I and II, with Luite different 31 3 This article is protected by copyright. All rights reserved. Molecular Microbiology Page 4 of 65 folds and substrate specificities. Type I T6s include the herpes simple8 virus T6 EHSM- T6F and human mitochondrial T62, and their 0road su0strate specificity is exploited in antiviral therapy. Type II T6s ET6IIsF are present in many organisms, for e8ample protozoan parasites li/e Trypanosoma brucei and Leishmania spp., plants, mammals EcytosolicF, 0acteria and certain viruses ERanj0arian et al., 2012A Timm et al., 2014A Lee N Cheng, 1976F. The crystal structures of many T6IIs have 0een solved, including those from human EHsT61, EDelin et al., 2004A Birringer et al., 2005FF, Ureaplasma urealyticum EUuT6, E6osins/a et al., 2005FF, Bacillus anthracis and B. cereus EBaT6 and BcT6, E6osins/a et al., 2007FF, Thermotoga maritima ETmT6, ESegura-Pena et al., 2007aA Segura-Pena et al., 2007bFF, Clostridium acetobutylicum and L. major ELmT6, ETimm et al., 2014FF. All these structures have the enzyme pac/ed as a tetramer in the crystal with similar Luaternary interactions: most forming a OclosedP tetramer with no ligand in the phosphate donor site, and a smaller number with an OopenP tetramer with the site occupied 0y a nucleobase. The majority of T6IIs are reported to 0e either dimers or tetramers in solution, or an eLuili0rium 0etween 0oth EEri/sson et al., 2002A Iutahir et al., 2013F. Iost are highly specific, phosphorylating only dThd and deo8yuridine Ed3rdF, and tolerate only minor changes in the 5-position of the pyrimidine 0ase and/or in the 3H-position of the 2H-deo8yri0ose sugar ERohansson N Eri/sson, 1996F. 3nli/e other T6IIs, TbT6 deviates from the usual homodimer/homotetramer form. Instead, it is a tandem protein with two homologous HsT61-li/e domains in a single chain 0oth with the conserved T6II fold, assumed to have evolved as a result of gene duplication and subseLuent fusion. The full length TbT6, has 0een reported to 0e a pseudo-dimer, with its two domains corresponding to two adjacent subunits of a typical T6II ERanj0arian et al., 2012F. The two domains have 67S seLuence identity on the 4 This article is protected by copyright. All rights reserved. Page 5 of 65 Molecular Microbiology amino acid level. Ranj0arian et al furthermore showed that only the C-terminal domain 2 is active, while in the N-terminal domain the catalytically important 1286 is replaced 0y an asparagine and two residues proposed to 0e involved in substrate 0inding E1245F and transition state sta0ilization ER247F are also different. In addition, TbT6 was shown to have 0roader substrate specificity compared to HsT61, which provides the potential for identifying inhi0itors that specifically target the parasitic enzyme. The essential character of TbT6 has not 0een fully analyzed to date, yet the e8istence of the de novo 0iosynthesis would preclude the reLuirement of thymidine salvage for parasite survival in T. brucei. However, in the closely related species L. major, LmT6 was shown to 0e important for infectivity of the parasites EThiel et al., 2008F. On the other hand, cellular production of dTTP is tightly modulated throughout the cell cycle EReichard, 1988F. In human cells, cell cycle-dependent regulation of enzymes involved in dTTP formation has 0een e8tensively descri0ed ENavalgund et al., 1980A Bradshaw, 1983A Sherley & 6elly, 1988F and HsT61 has 0een shown to have an important role in this process. 1uring mitosis, the enzyme is phosphorylated on Ser13 EChang et al., 1998F, which interferes with active tetramerization and gives rise to a reduction of catalytic efficiency ELi et al., 2004F. HsT61 function is practically undetecta0le at the entry to the ,1 phase due to anaphase promoting comple8/cyclosome EAPC/CF- dependent degradation at the end of mitosis E6e N Chang, 2004F. Here we assess the importance of TbT6 for parasite growth and correct cell cycle progression of T. brucei
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