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STRUCTURAL AND FUNCTIONAL STUDIES OF PROTEINS OF MEDICAL RELEVANCE Robert Gustafsson Structural and functional studies of proteins of medical relevance Protein-ligand complexes in cancer and novel structural folds in bacteria Robert Gustafsson ©Robert Gustafsson, Stockholm University 2018 ISBN print 978-91-7797-097-2 ISBN PDF 978-91-7797-098-9 Cover picture by Robert Gustafsson and Elisabeth Westergren Printed in Sweden by Universitetsservice US-AB, Stockholm 2018 Distributor: Department of Biochemistry and Biophysics, Stockholm University To my family, and Elisabeth, with all my love List of Publications The following publications and manuscripts are included in this thesis and are referred to by their roman numerals in the text. Paper I: MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool Gad, H.*, Koolmeister, T.*, Jemth, A.-S.*, Eshtad, S.*, Jacques, S. A.*, Ström, C. E.*, Svensson, L. M., Schultz, N., Lundbäck, T., Einarsdottir, B. O. , Saleh, A., Göktürk, C., Baranczewski, P., Svensson, R., Bernts- son, R. P.-A., Gustafsson, R., Strömberg, K., Sanjiv, K., Jacques- Cordonnier, M.-C., Desroses, M., Gustavsson, A.-L., Olofsson, R., Jo- hansson, F., Homan, E. J., Loseva, O., Bräutigam, L., Johansson, L., Höglund, A., Hagenkort, A., Pham, T., Altun, M., Gaugaz, F. Z., Vi- kingsson, S., Evers, B., Henriksson, M., Vallin, K. S. A., Wallner, O. A., Hammarström, L. G. J., Wiita, E., Almlöf, I., Kalderén, C., Axelsson, H., Djureinovic, T., Carreras Puigvert, J., Häggblad, M., Jeppsson, F., Mar- tens, U., Lundin, C., Lundgren, B., Granelli, I., Jenmalm Jensen, A., Ar- tursson, P., Nilsson, J. A., Stenmark, P., Scobie, M., Warpman Berglund, U., Helleday, T. Nature 508(7495), 215-221 (2014). DOI: 10.1038/nature13181 Paper II: Hypoxic signaling and the cellular redox tumor environment deter- mine sensitivity to MTH1 inhibition Bräutigam, L., Pudelko, L., Jemth, A.-S., Gad, H., Narwal, M., Gus- tafsson, R., Karsten, S., Carreras Puigvert, J., Homan, E. J., Berndt, C., Warpman Berglund, U., Stenmark, P., Helleday, T. Cancer research 76(8), 2366-2375 (2016). DOI: 10.1158/0008-5472.CAN-15-2380 Paper III: Crystal structures and inhibitor interactions of mouse and dog MTH1 reveal species-specific differences in affinity Narwal, M.*, Jemth, A.-S.*, Gustafsson, R., Almlöf, I., Warpman Ber- glund, U., Helleday, T., Stenmark, P. Biochemistry, in press (2018). DOI: 10.1021/acs.biochem.7b01163 Paper IV: Fragment-based discovery and optimization of enzyme inhibitors by docking of commercial chemical space Rudling, A.*, Gustafsson, R.*, Almlöf, I., Homan, E. J., Scobie, M., Warpman Berglund, U., Helleday, T., Stenmark, P., Carlsson, J. Journal of Medicinal Chemistry 60(19), 8160-8169 (2017). DOI: 10.1021/acs.jmedchem.7b01006 Paper V: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP Jemth, A.-S., Gustafsson, R., Bräutigam, L., Henriksson, L. M., Carreras Puigvert, J., Homan, E. J., Warpman Berglund, U., Stenmark, P., Helleday, T. Manuscript Paper VI: Crystal structure of the emerging cancer target MTHFD2 in complex with a substrate-based inhibitor Gustafsson, R., Jemth, A.-S., Gustafsson, N. M. S., Färnegårdh, K., Lo- seva, O., Wiita, E., Bonagas, N. Dahllund, L. Llona-Minguez, S., Hägg- blad, M., Henriksson, M., Andersson, Y., Homan, E. J., Helleday, T., Stenmark, P. Cancer Research 77(4), 937-948 (2017). DOI: 10.1158/0008-5472.CAN-16-1476 Paper VII: Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster Gustafsson, R.*, Berntsson, R. P.-A.*, Martínez-Carranza, M., El Tekle, G., Odegrip, R., Johnson, E. A., Stenmark, P. FEBS Letters 591(22), 3781-3792 (2017). DOI: 10.1002/1873-3468.12889 Reprints of the published articles were made with permission from the pub- lishers. * These authors contributed equally to this work. Additional Publications Crystal structure, biochemical and cellular activities demonstrate sepa- rate functions of MTH1 and MTH2 Carter, M., Jemth, A.-S., Hagenkort, A., Page, B. D. G., Gustafsson, R., Griese, J. J., Gad, H., Valerie, N. C. K., Desroses, M., Boström, J., Warp- man Berglund, U., Helleday, T., Stenmark, P. Nature Communications 6, 7871 (2015). DOI: 10.1038/ncomms8871 Glycans confer specificity to the recognition of ganglioside receptors by botulinum neurotoxin A Hamark, C., Berntsson, R. P.-A., Masuyer, G., Henriksson, L. M., Gus- tafsson, R., Stenmark, P., Widmalm, G. Journal of the American Chemical Society 139(1), 218-230 (2017). DOI: 10.1021/jacs.6b09534 Contents Introduction .................................................................................................... 1 Cancer ........................................................................................................................... 2 Clostridium botulinum neurotoxin .................................................................................. 3 Cancer and the cancer cell ............................................................................ 5 Somatic mutation theory ................................................................................................ 6 Critiques of the somatic mutation theory .................................................................. 7 Genetic instability and general cancer therapy .............................................................. 7 Synthetic lethality ........................................................................................................... 9 Redox regulation and reactive oxygen species ........................................................... 10 Generation of reactive oxygen species .................................................................. 11 Reactive oxygen species in health and disease .................................................... 11 Hypoxia and reactive oxygen species .................................................................... 12 Oxidised nucleotides ................................................................................................... 13 Oxidative damage to nuclear and mitochondrial DNA leads to cell death ............. 14 Methylated nucleotides ................................................................................................ 15 MutT homologue 1 (MTH1) .......................................................................... 17 The MutT protein from E. coli degrades 8-oxo-dGTP .................................................. 17 MTH1 can degrade several oxidised nucleotides ........................................................ 17 Structures of MTH1 show a versatile binding site ....................................................... 18 mth1 gene and isoforms .............................................................................................. 19 MTH1 is part of the NUDIX hydrolase protein family ................................................... 20 MTH1 prevents damage caused by ROS .................................................................... 21 Cancer phenotypic lethality: MTH1 as a drug target for cancer .................................. 22 MTH1 controversy ....................................................................................................... 24 New MTH1 function: degradation of O6-methyl-dGTP ................................................ 24 De novo nucleotide synthesis and one-carbon metabolism ......................... 25 One-carbon metabolism .............................................................................................. 26 Methylene-tetrahydrofolate dehydrogenase family ...................................................... 27 Outcomes from the one-carbon metabolism ............................................................... 29 Drugs for one-carbon metabolism: antifolates and antimetabolites ............................. 30 Importance of transport and polyglutamation of THF and drugs ................................. 31 MTHFD2 is upregulated in cancers and is a potential target for cancer phenotypic lethality ........................................................................................................................ 32 Structural information on the MTHFD family ............................................................... 33 Clostridium botulinum .................................................................................. 35 Botulism ....................................................................................................................... 36 Botox and medical use of botulinum neurotoxins ........................................................ 36 Botulinum neurotoxin and mechanism of action .........................................................