Structural and functional studies of a novel Botulinum neurotoxin and of MTH1 Linda Henriksson Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Wednesday 10 October 2018 at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B.

Abstract X-ray crystallography visualizes the three dimensional molecular structures of proteins at atomic resolution. Seeing the molecular structure of a biomedically interesting protein enables a higher understanding of its function. The process of producing pure protein from genetic material to generate crystals and determine the molecular structure can be a long and challenging process. My thesis involves structural and functional studies of two different proteins, which are both biomedically interesting and important to learn about. X-ray crystallography is the method which has been used to determine the majority of the protein structures that we know of today and is also the method used in the results presented in my thesis. Today there are no cancer therapies defeating all types of cancers and they do not come without side effects. Battling cancer diseases often include long and painful treatments. Finding an anti-cancer drug targeting phenotypes characteristic of cancer cells is a compelling thought. MutT homolog-1 (MTH1) is an enzyme present in all proliferating cells. The enzyme seems to be crucial for cancer cell survival but not for the viability of normal cells. MTH1 cleans out oxidized and thereby damaged nucleotides from the free nucleotide pool and stops them from being used in DNA synthesis. This process is very important in fast proliferating cancer cells. The hypothesis is to inhibit MTH1 and thereby allow a limitless amount of DNA damage in the cancer cells. This action will eventually kill cancer cells while not affecting normal cells. The molecular structure of MTH1 with (PDB ID: 3ZR0) and without a product bound (PDB ID: 3ZR1) was determined and is presented in my thesis. These two structures aided in the synthesis of inhibitors. Botulinum neurotoxins (BoNTs) are the most potent toxins known. As little as one gram of pure toxin could potentially kill one million people. Due to its potency BoNT is a potential bioterrorism threat. The toxin is also a very potent drug used clinically to relieve the symptoms of an array of neuromuscular disorders. Most people know this neurotoxin by one of its commercial names: Botox™. Additionally BoNTs are the cause of botulism. BoNTs are neuro-specific enzymes that target neuromuscular signaling, inducing flaccid paralysis and potentially death. It is of importance to learn more about these toxins to enable the development of new countermeasures, vaccines or more efficient neuroparalytic drugs. BoNTs consist of three domains with different functions, all crucial for intoxication. The toxins are fragile and can easily be destroyed by harsh surroundings if not protected by non-toxic non-hemagglutinin (NTNH) proteins. The complex of some BoNT serotypes and their protective NTNH have proven to be pH-dependent. Parts of the intoxication process are not yet clear and their mechanisms are still puzzling researchers. Until recently seven BoNT serotypes were identified. We have now identified and characterized a novel serotype called BoNT/X. The molecular structure of the active domain is presented here (PDB ID: 6F47). The pH-dependent mechanism forming a complex as seen in other serotypes, is confirmed to be present in BoNT/X as well.

Keywords: Botulinum neurotoxin, botulism, cancer, gene cluster, molecular structures, MTH1, NTNH, oxidised nucleotides, progenitor complex, protein expression, protein purification, X-ray crystallography.

Stockholm 2018 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-159295

ISBN 978-91-7797-384-3 ISBN 978-91-7797-385-0

Department of Biochemistry and Biophysics

Stockholm University, 106 91 Stockholm

STRUCTURAL AND FUNCTIONAL STUDIES OF A NOVEL BOTULINUM NEUROTOXIN AND OF MTH1

Linda Henriksson

Structural and functional studies of a novel Botulinum neurotoxin and of MTH1

Linda Henriksson ©Linda Henriksson, Stockholm University 2018

ISBN print 978-91-7797-384-3 ISBN PDF 978-91-7797-385-0

Cover picture: "Olivkvisten står för utveckling, ny kunskap och framtidshopp. Den symboliserar växande och demokrati." Källa: https://www.su.se/medarbetare/kommunikation/grafisk-manual/grafiska-element-1.361974

Printed in Sweden by Universitetsservice US-AB, Stockholm 2018 Distributor: Department of Biochemistry and Biophysics, Stockholm University

Till Tea och Ida Till Magnus

Populärvetenskaplig sammanfattning

Min avhandling rör två ämnesområden, nämligen MutT homolog-1 (MTH1) och Botulinum neurotoxin (BoNT). De är i många aspekter fullständigt olika varandra men båda är proteiner och så kallade enzymer, vilka möjliggör biokemiska processer i våra celler genom att underlätta för reaktioner att ske. De tar isär och sätter ihop sina substrat till produkter. Det finns många olika enzymer, alla med en särskild uppgift. MTH1 och BoNT är avgörande i två dödliga sjukdomar och är därför intressanta och viktiga att studera. Syftet med mina studier har varit att få experimentella resultat som är värdefulla i utvecklingen av nya läkemedel. En molekyl består av atomer och en molekylstruktur är en tredimensionell (3D) bild som visar var i rymden atomerna är positionerade i förhållande till varandra. Processen att bestämma en molekylstruktur kan vara lång och krånglig. Man startar med genetiskt material, som är recept på proteiner och placerar det i proteinproducerande värdar. Bakterier är mycket funktionella protein-producenter. Därefter utvinns proteinet från bakterierna och renas stegvis. Under rätt förhållanden kan rent protein bilda kristaller. Proteinkristaller utgörs av många proteinmolekyler som är tätt packade i en viss ordning. De kan se ut som saltkristaller men är oftast mycket mindre. Den metod jag har använt mig av för att ta reda på hur strukturen ser ut kallas röntgenkristallografi. Det innebär att man skjuter en röntgenstråle på en proteinkristall. Ett speciellt mönster bildas utifrån spridningen från atomerna i kristallen. Det så kallade diffraktionsmönstret används för ta reda på var i rymden varje atom i molekylen är positionerad. Den informationen tillsammans med passande mjukvara möjliggör omvandling av data till en 3D bild. En sådan bild gör att man kan snurra på molekylen, se alla sidor av den, zooma in för att se detaljer och zooma ut för att få helheten. Det ger en bättre förståelse för hur enzymet fungerar. MTH1 verkar vara ett viktigt enzym för livsdugligheten hos cancer celler och är en gemensam nämnare mellan olika typer av cancer. MTH1 verkar dock inte ha så stor betydelse i normala celler. Om en cancerbehandling baserades på hämning av MTH1 så skulle bieffekterna troligtvis vara små eller frånvarande. Röntgenkristallografi-strukturen av MTH1 presenteras i min avhandling samt strukturen av MTH1 när det binder en av sina produkter. BoNT är ett av de mest potenta gift man känner till. Det har en toxicitet som är 1 miljon gånger högre än kobragift. BoNT är motsägelsefullt nog ett ömtåligt protein och kan lätt brytas ned i lågt pH eller i ogästvänlig miljö. Non-toxic non-hemagglutinin (NTNH) är också ett protein och det fungerar som BoNTs livvakt. Det kramar om BoNT när miljön är ofördelaktig och släpper när omgivningen återigen är duglig. Den här krama- och släpp-mekanismen är beroende av pH-värdet i omgivningen. BoNT-familjen består av sju olika nervgifter och de orsakar botulism. En av serotyperna, BoNT/A, är känt som Botoxä och används vid kosmetiska behandlingar men även som medicinsk behandling av många neuromuskulära åkommor. På grund av sin toxicitet är BoNT ett möjligt bioterroristhot. Det finns åtskilliga skäl till att lära oss mer om BoNT. Efter noggranna utredningar har vi upptäckt en ny serotyp och det var ca 50 år sedan det senast hände. Den kallas än så länge BoNT/X. Molekylstrukturen av den del av BoNT/X, där substratet omvandlas till produkt, presenteras i min avhandling. Avslutningsvis bekräftar ännu inte publicerad data att pH-beroendet i krama- och släpp-mekanismen också är tillämpbar på BoNT/X.

Popular science summary

My thesis concerns two topics, namely MutT homolog-1 (MTH1) and Botulinum neurotoxin (BoNT). These proteins are in many aspects completely different from each other, but both are proteins and are so called enzymes that enable biochemical processes in our cells by facilitating reactions. This involves taking apart or putting together their substrates to form products. There are many different enzymes, all of which perform specific tasks. MTH1 and BoNT are crucial players in two deadly diseases and are therefore interesting and important targets for further studies. The aim of my studies has been to get experimental data valuable for the development of new drugs. A molecule is built out of atoms and a molecular structure is a three-dimensional (3D) picture displaying where in space the atoms are positioned in relation to one other. The process to get to a molecular structure can be long and tricky. One starts with the genetic material, which is the recipes of the relevant protein and inserts it into a protein producing host. Bacteria are very useful and common hosts, which produce the proteins of interest. Proteins are then extracted from the bacteria and are purified in a stepwise manner. If the conditions are optimal the pure protein can potentially form crystals. Protein crystals are generated from many protein molecules densely packed in a certain order. They can look like salt crystals but are often much smaller. I have used a method called X-ray crystallography to determine 3D protein structures. X-ray crystallography involves shooting a protein crystal with a beam of X- rays. A specific pattern based on the scattering from the atoms in the crystal is generated. The so-called diffraction pattern can be used to determine the position of each atom in the molecule. With this information and the appropriate software it is possible to convert the data into a 3D picture. One can then turn the molecule around to see all sides of it, zoom in to see fine details and zoom out to see the entire protein. The molecular structure gives a better understanding of the enzymes function. MTH1 seems to be very important for the survival of cancer cells and is a common feature between different types of cancers. MTH1 does not seem to be very important in normal cells. Therefore the side-effects of a cancer therapy based on MTH1 inhibition would potentially be absent or small. The X-ray crystallographic structure of MTH1 is presented in my thesis, as well as the structure of MTH1 bound with one of its products. BoNT is one of the most potent toxins known and has a toxicity that is 1 million times higher than cobra venom. Interestingly, BoNT is actually a very fragile protein and can easily be broken down in low pH or in harsh surroundings. Non-toxic non-hemagglutinin (NTNH) is a protein and that functions as the bodyguard of BoNT. It hugs BoNT in unfavorable environments until conditions are favorable again, in a pH-dependent manner. The BoNT family consists of seven different neurotoxins and are the causative agents of botulism. One of the serotypes, BoNT/A, is known as Botoxä and is used for cosmetic treatments as well as for the medical treatment of many neuromuscular disorders. Because of its toxicity BoNT is a potential bioterrorism threat. The motives to find out more about BoNT are numerous. After thorough investigations we have found a new BoNT serotype. About 50 years have passed since that happened last time. It is currently called BoNT/X. The molecular structure of the part of BoNT/X where the activity occurs, is also presented in my thesis. Finally, unpublished data can confirm that the pH-dependency in the hug and release mechanism is applicable to the new serotype BoNT/X.

List of Publications

The following publications and manuscript are included in my doctoral thesis and are referred to by their roman numerals.

Reprints of the publications were made with permission from the publishers. * These authors contributed equally to this work.

I Crystal structure of human MTH1 and the 8-oxo-dGMP product complex. Svensson L.*, Jemth A.-S.*, Desroses M., Loseva O., Helleday T., Högbom M. and Stenmark P. FEBS Letters. 2011 Aug 19;585(16):2617-21. DOI: 10.1016/j.febslet.2011.07.017

II Identification and characterization of a novel botulinum neurotoxin. Zhang S., Masuyer G., Zhang J., Shen Y., Lundin D., Henriksson L., Miyashita S.-I., Martinez M., Dong M. and Stenmark P. Nature Communications. 2017 Aug 3;8:14130 DOI: 10.1038/ncomms14130

III Structural characterisation of the catalytic domain of botulinum neurotoxin X – high activity and unique substrate specificity. Masuyer G., Zhang S., Barkho S., Shen Y., Henriksson L., Košenina S., Dong M. and Stenmark P. Scientific Reports. 2018 Mar 14;8(1):4518 DOI: 10.1038/s41598-018-22842-4

IV pH dependence of the minimal progenitor complex of Botulinum neurotoxin X. Henriksson L. M. and Stenmark P. Manuscript. 2018

Additional Publications

Following publications are not included in my doctoral thesis.

V Crystal structures of botulinum neurotoxin DC in complex with its protein receptors synaptotagmin I and II. Berntsson R., Peng L., Svensson L. M., Dong M. and Stenmark P. Structure. 2013 Sep 3;21(9):1602-11. DOI: 10.1016/j.str.2013.06.026

VI Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts. Kmiec B., Teixeira P. F., Berntsson R. P.-A., Murcha M. W., Branca R. M. M., Radomiljac J. D., Regberg J., Svensson L. M., Bakali A., Langel Ü., Lehtiö J., Whelan J., Stenmark P. and Glaser E. Proc Natl Acad Sci. 2013 Oct 1;110(40):E3761-9. DOI: 10.1073/pnas.1307637110

VII Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2. Berntsson R., Odegrip R., Sehlén W., Skaar K., Svensson L., Massad T., Högbom M., Haggård- Ljungquist E. and Stenmark P. Nucleic Acid Research. 2014 Feb;42(4):2725-35. DOI: 10.1093/nar/gkt1119

VIII 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., Schultz N., Lundbäck T., Einarsdottir B. O., Saleh A., Göktürk C., Baranczewski P., Svensson R., Berntsson R. P., Gustafsson R., Strömberg K., Sanjiv K., Jacques-Cordonnier M. C., Desroses M., Gustavsson A. L., Olofsson R., Johansson F., Homan E. J., Loseva O., Bräutigam L., Johansson L., Höglund A., Hagenkort A., Pham T., Altun M., Gaugaz F. Z., Vikingsson S., Evers B., Henriksson M., Vallin K. S., Wallner O. A., Hammarström L. G., Wiita E., Almlöf I., Kalderén C., Axelsson H., Djureinovic T., Puigvert J. C., Häggblad M., Jeppsson F., Martens U., Lundin C., Lundgren B., Granelli I., Jensen A. J., Artursson P., Nilsson J. A., Stenmark P., Scobie M., Berglund U. W. and Helleday T. Nature. 2014 Apr 10; 508(7495):215-221 DOI: 10.1038/nature13181

IX Glycans confer specificity to the recognition of ganglioside receptors by botulinum neurotoxin. Hamark C., Berntsson R. P.-A., Masuyer G., Henriksson L. M., Gustafsson R., Stenmark P. and Widmalm G. Journal of the American Chemical Society. 2017 Jan 11;139(1):218-230. DOI: 10.1021/jacs.6b09534

X MutT homologue 1 (MTH1) catalyses the hydrolysis of the mutagenic O6-methyl-dGTP. Jemth A.-S., Gustafsson R., Bräutigam L., Henriksson L., Vallin K. S. A., Sarno A., Almlöf I., Homan E., Rasti A., Warpman Berglund U., Stenmark P. and Helleday T. Submitted to Nucleic Acid Research. 2018

List of Abbreviations

A Adenine ALS Amyotrophic lateral sclerosis, a neurodegenerative disease BabyBIG Botulism immune globulin intravenous BAT Heptavalent botulism antitoxin BoNT/A Botulinum neurotoxin serotype A, the naming model is valid to all BoNTs C Cytidine C. botulinum Clostridium botulinum CMPK1 Cytidine monophosphate kinase 1 Cryo-EM Cryo-electron microscopy C. tetanus Clostridium tetanus DNA Deoxyribonucleic acid dATP Deoxyadenosine triphosphate dNMP Deoxynucleoside monophosphate dNTP Deoxynucleoside triphosphate E. coli Escherichia coli E. faecium Enterococcus faecium EM Electron microscopy En Enterococcus ESRF European synchrotron radiation facility FDA US food and drug administration G Guanine GST Glutathione S-transferase HA Hemagglutinin HC Heavy chain HCC C-terminal of the heavy chain HCN N-terminal of the heavy chain HC/X Heavy chain of BoNT/X, the naming model is valid to all BoNTs His6-tag A polyhistidine tag, which contains six histidines Hsp90 Heat shock protein 90 LC Light chain LC/X Light chain of BoNT/X, the naming model is valid to all BoNTs LL-PTC Larger progenitor toxin complex L-PTC Large progenitor toxin complex MDR Multi-drug-resistant M-PTC Minimal progenitor toxin complex mRNA Messenger ribonucleic acid MTH1 MutT homolog-1 N1, 2, 3 Nitrogen 1, 2 or 3 NAP Neurotoxin associated protein Ni-NTA Nickel nitrilotriacetic acid NMJ Neuromuscular junction NMR Nuclear magnetic resonance NTNH Non-toxic non-hemagglutinin NUDIX Nucleoside diphosphates linked moiety-X O6-methyl-dGMP O6-methyl-deoxyguanosine monophosphate O6-methyl-dGTP O6-methyl-deoxyguanosine triphosphate OrfX Open-reading-frame-X PDB Protein data bank PPi Pyrophosphate PTC Progenitor toxin complex RAS Small guanosine nucleotide binding protein, named after rat sarcoma RMSD Root mean square deviations RNA Ribonucleic acid ROS Reactive oxygen species RT Room temperature SAM S-Adenosyl methionine SeMet Selenomethionine SNAP25 Synaptosomal-associated protein of 25 kDa SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein receptor SNP Single-nucleotide polymorphism SV2 Synaptic vesicle 2 Syt I/II Synaptotagmin I/II T Thymine Te, TeNT Tetanus Trx-TrxR Thioredoxin-thioredoxin reductase U Uridine VAMP Vesicle-associated membrane protein

2-OH-A 2-hydroxy-adenine 2-OH-dA 2-hydroxy-deoxyadenine 2-OH-dAMP 2-hydroxy-deoxyadenosine monophosphate 2-OH-dATP 2-hydroxy-deoxyadenosine triphosphate 2-OH-dGTP 2-hydroxy-deoxyguanosine triphosphate 3D Three-dimensional 8-oxo-dATP 8-oxo-deoxyadenosine triphosphate 8-oxo-dG 8-oxo-deoxyguanine 8-oxo-dGDP 8-oxo-deoxyguanosine diphosphate 8-oxo-dGMP 8-oxo-deoxyguanosine monophosphate 8-oxo-dGTP 8-oxo-deoxyguanosine triphosphate 8-oxo-G 8-oxo-guanine 8-oxo-GTP 8-oxo-guanosine triphosphate

Table of Contents INTRODUCTION ...... 1 PROTEINS ...... 2

STRUCTURES AND FUNCTIONS ARE RELATED ...... 3 ENZYMES...... 3 RECOMBINANT PRODUCTION OF SOLUBLE PROTEINS...... 4

ESCHERICHIA COLI – A PROTEIN PRODUCTION FACILITY...... 4 PURIFICATION METHODS ...... 4 STRUCTURAL STUDIES WITH X-RAY CRYSTALLOGRAPHY ...... 5

CRYSTALLIZATION ...... 6 SYNCHROTRONS AND X-RAYS...... 7 THE PHASE PROBLEM...... 8 OTHER METHODS – NMR AND CRYO-EM ...... 9 MTH1 ...... 11 CANCER ...... 11 NUDIX HYDROLASE FAMILY ...... 13 MTH1 ACTIVITY AND MECHANISMS ...... 14 ANTICANCER HYPOTHESIS ...... 14 PAPER I – CRYSTAL STRUCTURE OF HUMAN MTH1 AND THE 8-OXO-DGMP PRODUCT COMPLEX...... 16 Comparison between the NMR and the X-ray structure...... 16 The catalytic pocket ...... 16 The substrate 8-oxo-dGTP ...... 17 Coordination of the 8-oxo-dGMP product ...... 17 Distinction between oxidized and non-oxidized nucleotides ...... 18 NEWLY GAINED INSIGHTS AND YET UNPUBLISHED RESULTS ...... 19 CRITICS ...... 20 BOTULINUM NEUROTOXINS ...... 21 ONE OF THE MOST POTENT TOXINS ALSO HEALS ...... 21 Botulism ...... 22 THE BONT FAMILY ...... 22 Serotypes, subtypes and mosaic toxins ...... 24 Gene clusters ...... 25 NTNH AND OTHER NON-TOXIC PROTEINS ...... 25 pH dependent PTC assembly...... 26 Roles of the PTC proteins ...... 27 BONT ACTIVITY AND MECHANISMS ...... 27 Neuronal internalization ...... 28 Translocation and activation ...... 29 BoNT substrates ...... 30 PAPER II – IDENTIFICATION AND CHARACTERIZATION OF A NOVEL BOTULINUM NEUROTOXIN ...... 31 PAPER III – STRUCTURAL CHARACTERISATION OF THE CATALYTIC DOMAIN OF BOTULINUM NEUROTOXIN X – HIGH ACTIVITY AND UNIQUE SUBSTRATE SPECIFICITY ...... 34 PAPER IV – PH DEPENDENCE OF THE MINIMAL PROGENITOR COMPLEX OF BOTULINUM NEUROTOXIN X ...... 36 BIBLIOGRAPHY ...... 39

Introduction

I would like to think of evolution as fine-tuning the flow of matter coming to its best use, while balance is maintained. Biochemistry is the chemistry of life and is all about balance, feedback, communications and actions when needed. Biochemistry involves all the processes, reactions and metabolisms that are executed inside and between all cells, in all living organisms, from trees and viruses to humans. It is intriguing to reflect on the fact that all organisms are built up from essentially a few different classes of biomolecules: nucleotides, lipids, carbohydrates and proteins. These biomolecules are composed of atoms and particles that arose 14 billion years ago and eventually evolved in to living organisms. Microorganisms were the first inhabitants on Earth and were the beginning of life, 4 billion years ago (10). What initially caught my interest in natural science was a need to understand the flow of matter in organisms. The area of biomedicine introduced me to the human body and how the matter flows within us in sickness and in health; inhibition or stimulation of processes, compensating events, feedback loops, cascade reactions and long signaling pathways. This affects how everything is held together and makes up a functional unit. Structural biochemistry has been a prolongation of that understanding, utilizing tools to gain understanding of life and enabling development of potential drugs. Structural biochemistry is about finding out and learning from the three-dimensional molecular structures of substances of interest. When the structure is known it is possible to understand and characterize the molecule. Investigation of a molecular structure offers a better understanding of its function. Generating structures of protein molecules important in diseases is extremely useful, as it can give indications of how to fight them.

1

Proteins

One of the most important, abundant and variable types of molecules in our bodies are proteins. Proteins perform a variety of different tasks, from being signaling molecules communicating with the cell surroundings to being receptors and channels that decide what should be let in and out of the cells. Proteins can also function as structural building blocks like the fibrous proteins in muscles or as parts building up skin or hair. They can be antibodies defending the organism against intruders or function in storing and transporting oxygen. Proteins can also be hormones regulating our bodily functions or enzymes enabling reactions. Proteins are involved in basically all biochemical processes in all living organisms and are crucial for our survival. Deoxyribonucleic acid (DNA) contains the genetic code and is responsible for its transmission. DNA molecules are located in every cell and they are passed on from organisms to their progenies. DNA is built out of four nucleotides: thymine (T), cytidine (C), guanine (G) and adenine (A) in different combinations. Ribonucleic acid (RNA) is the second nucleic acid, which differs from DNA in that it has a fifth nucleotide, uridine (U) instead of T. These two nucleic acids, DNA and RNA, are responsible for the transcription of genes and their translation of them into amino acids. Amino acids are the building blocks of proteins. Even though there are only 20 amino acids, proteins can be very different from each other. They all have different molecular structures, properties and sizes. The foundation of all amino acids is the same, each containing a carboxyl group and an amino group bound to the same carbon atom. It is their functional group that gives each of them their unique features. Amino acids can be either hydrophobic or hydrophilic, acidic or basic, positive, negative or uncharged, with or without ring structures and be large or small. The endless combinations of amino acids make up protein molecules with unique and specific features, structures and functions (10). One can think of amino acids (also called residues) as different pearls on a string, as an explanation of the primary structure of a protein. All residues have names and abbreviations; Leucine/Leu/L is one example. The primary structure describes the order of the amino acids, connected to each other via peptide bonds, making up a polypeptide chain (Figure 1A).

(A) (B) (C)

Figure 1. Structural arrangements of MTH1 (PDB ID: 3ZR1). (A) LYTLVLV residues displayed as part of the MTH1 polypeptide chain to show the primary structure, each residue with a specific color. (B) The same residues in corresponding colors, making up a b-strand presenting the secondary structure. (C) The b-strand displayed as part of a b-sheet and the whole MTH1 tertiary structure.

2

The order of the residues together with their properties affect how the primary structure will fold and if it will form a-helices, b-strands or loops, which all are examples of common secondary structure elements of a protein (Figure 1B). The tertiary structure is the arrangement of these secondary structures in space, including the interactions between them (Figure 1C). The tertiary structure of a protein represents the overall fold showing how the secondary structures are held together by weak interactions. A quaternary structure is formed when more than one polypeptide chain makes up a functional protein, and it displays how these polypeptides interact with each other (10). Soluble proteins can be used in biochemical assays, in the production of antibodies or when determining the molecular structures with either X-ray crystallography, cryo-EM or NMR. To be able to produce and reproduce experiments, robust expression and purification protocols are needed. For new and uncharacterized proteins, there are no existing protocols. Earlier scientists have established the foundations found in literature, which together with experience and trial and error are the requirements needed for success.

Structures and functions are related

The 3D structures of proteins are fully related to their functions. Enzymes for example, have crevices or pockets available where the catalytic processes take place. These cavities need to be lined with suitable residues, necessary to generate affinity towards the substrate specific to the enzyme. To enable enzyme activity the active site needs to be spacious enough to avoid steric hindrance and to be able to fit the substrate and additional molecules in. At the same time, it needs to be restricted to avoid unwanted molecules. To elucidate the function of an enzyme it is of the utmost importance to know its 3D structure. Only when the molecular structure is known, is it possible to get an understanding of where the catalytic pocket is situated, how it looks, what residues are found in certain areas or domains and how the substrates are recognized and selected. Knowledge of the enzymes selectivity can provide indications for how potential drugs could be designed. Proteins often contain so called motifs, which describe specific combinations of certain residues, conserved at specific locations between species. Motifs in newly discovered proteins give an understanding of evolutionary relationships to other proteins, in the same or in other species.

Enzymes

Enzymes are catalysts that make biochemical processes more efficient and speed up the velocity of the reaction. The reaction where substrates are converted to products is made thermodynamically favorable by enzymes, which lower the activation energy needed for the process. One enzyme can act on many substrates, either by assembling or disassembling them. By regulating the enzyme activity the number of converted substrates is altered. Different enzymatic activities occur all the time in each cell, depending on internal and external biological signals. These characteristics make enzymes very appealing drug targets. Enzymes are divided into different classes depending on what reactions they perform: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Hydrolases use

3 water in their reactions to cleave chemical bonds (10). Both MutT homolog-1 (MTH1) and Botulinum neurotoxins (BoNTs) belong to the class of hydrolases. MTH1 cleaves off phosphate groups from its substrates and BoNTs cleave their substrates by proteolysis, meaning the hydrolysis of peptide bonds (7, 11).

Recombinant production of soluble proteins

The human body contains between 50-75 % water, depending on the age of a person. Without water we can only live for a few days. It is essential for the homeostasis in our cells and is needed where chemical reactions take place (12). Water stabilizes the native conformations of proteins via hydrophobic interactions and hydrogen bonds, as well as enabling their enzymatic activities (13). Most of the proteins crucial for our bodily functions are soluble and need water to work properly. To enable drug development and understand how to influence proteins of biomedical importance, it is necessary to evaluate the protein in structural studies and in biochemical assays. Prior to those studies, milligram amounts of acceptably pure protein is needed. Soluble proteins are commonly overexpressed recombinantly in hosts such as bacteria, insect cells, mammalian cells or in yeast. A common host used for recombinant expression of soluble proteins, is the prokaryotic bacteria Escherichia coli (E. coli) (14).

Escherichia coli – a protein production facility

Our intestines contain E. coli strains important for the uptake of nutrients. Other E. coli strains produce toxins, are infectious and can cause stomach pain if ingested (14, 15). The genome of E. coli is small and simple and consists of only ~4.400 genes (16). E. coli is successfully used in transformations because the bacteria easily takes up foreign DNA (15). In 1997 the E. coli strain K-12 was the first genome to be completely categorized (16). E. coli is haploid, which means that the genes are organized in one chromosome. This conveniently ensures the viability of mutations during genetic engineering. Not many expression systems beat E. coli when it comes to growth rate. In only 20 minutes E. coli produces a new generation. It is practical to be able to express several mg of protein during a time frame of less than 24 h. Additionally they are easy to grow both aerobically and anaerobically (15). E. coli have been the expression organism delivering proteins for structural determinations in ~104.000 entries out of ~143.000 in the Protein Data Bank (PDB, 25 Jul 2018)(17). One of the most common E. coli expression strains is BL21(DE3) (18). E. coli was first identified in 1885 and is today very commonly used in recombinant protein production (19). In the future, the bottle neck with upscaling will likely concern problems like the large amount of proteins expressed, their export out of the cell and feedback inhibition (20).

Purification methods

After protein expression the E. coli cells need to be lysed to make the proteins inside them available. Common methods to break cells involve liquid homogenization or high frequency sound waves (21). The lysate is a mixture of growth media, cellular parts and

4 different proteins. The recombinantly over-expressed proteins are produced along with host endogenous proteins. The protein of interest needs to be extracted from the lysate and this is usually done in a stepwise manner, using suitable purification methods. The method of choice and outcome depends on the protein and how it was genetically engineered, the purity needed and also what equipment the laboratory facility offers. Affinity chromatography is a good start in the purification process and involves six histidines (His6-tag) added to either the N- or the C-terminus of the protein sequence. To get a His6-tag fused to the protein of interest, six histidines need to be added next to the gene of interest in the vector during genetic engineering. Fusing a His6-tag to the sequence is thus done prior to expression. A His6-tag binds with high affinity to nickel- nitrilotriacetic acid (Ni-NTA) resin, while non-interacting proteins in the lysate are washed away (22). To avoid non-specific binding of endogenous E. coli proteins, it is common to use a low concentration of imidazole in the wash. Elution can be done either by lowering the pH, which protonate and release the histidines from the resin or by using a high concentration of imidazole. Histidine has an imidazole side-chain, therefore imidazole and histidine will compete for the binding to the metal (22, 23). Chromatography purification methods are basically columns packed with resins, which are solid phases with specific features. Metal chelating affinity chromatography involve interactions between metal ions in the solid phase and ions in the liquid phase (23). Size- exclusion chromatography is based on a column withholding a resin consisting of porous beads. The molecules are sorted depending on their size and the largest are eluted first (24). Ion-exchange chromatography involves a resin that binds charged molecules. There is cation and anion-exchange chromatography. The pH of the applied buffers change the charges and enables elution of the desired proteins (25). Metal chelating affinity chromatography (already described) is a method based on interactions. Enzymes interacting with their substrates, receptors with their ligands or antibodies binding to their antigens are all examples of what natural interactions that can be used in separation experiments (26).

Structural studies with X-ray crystallography

The method of crystallizing proteins was discovered more than 150 years ago and was at that time used to demonstrate the purity of proteins (27, 28). X-ray crystallography was discovered in the late 1930s and is the main method to determine 3D structures of proteins, even though other methods exist (29-34). X-ray crystallography has become an important method in structural biochemistry because of the knowledge gained from crystallization and the importance of available high quality 3D structures in drug development. X-ray crystallography allows visualization of the exact atomic positions in macromolecules. The quality of the data is a direct reflection of the quality of the crystal (6, 35). The genetic revolution in the 1980´s to the 1990´s, when many interesting genes encoding proteins in biological systems suddenly became available, has also been very important in this field. The evolution of X-ray crystallography has happened over several decades and technical solutions, screens and robots have been developed to make the most time consuming work about crystal production significantly easier (36, 37). The statistics in the PDB for proteins and nucleic acids shows that an overwhelmingly majority of these structures were determined using X-ray crystallography (17, 38). The number of structures released annually is increasing each year, reflecting the gains from 5 improved techniques and experience. Approximately 127.000 out of about 143.000 molecular structures are determined using the X-ray crystallography technique. In contrast Nuclear magnetic resonance (NMR) has so far generated ~12.000 structures and 3D Electron microscopy (EM) has produced ~2.200 structures (PDB, 27 July 2018).

Crystallization

The first protein crystal that was published was hemoglobin by Hünefeld in 1840 (27). A single protein molecule will not generate a measurable signal in X-ray crystallography. To magnify the signal and generate a diffraction pattern, an ordered 3D arrangement of protein molecules is needed, which is the exact nature of a protein crystal (39). Hünefeld suggested that crystals could be produced from a protein solution by slow and controlled evaporation (27). The process of generating crystals is the bottleneck of X-ray crystallography (6, 35, 37, 40). A protein crystal is a structured and densely packed array of many protein molecules, forming a certain lattice. The lattice describes how repeating particles are arranged in unit cells, which are the repeating units that form the crystal. A unit cell is made up of a set of asymmetric units, which are defined by the space group (35, 41). The signals from many ordered protein molecules produces a stronger diffraction signal, which can be detected. Hence protein crystals are needed for this method. To succeed in protein crystallization specific conditions are needed and those are unique to each batch of produced protein. Factors affecting the crystallization process are protein concentration, temperature, the type and concentration of precipitant, protein purity and solubility, additional molecules, pH and chemicals needed for crystallization to occur (5, 31, 41). Protein molecules that contain too many flexible regions are significantly more difficult to crystallize. Thermodynamics must be favorable for crystal formation to happen, the free energy, DG, must be negative and the kinetics must be advantageous (42). There are several different crystallization methods and their common factor is to get the protein supersaturated. Methods mentioned in the literature are dialysis, vapor diffusion and liquid-liquid diffusion (35, 43). Vapor diffusion was the method of choice when MTH1 and the light chain of BoNT/X (LC-X) were crystallized (7, 9). A certain volume of protein solution is mixed with a specific volume of mother liquor in a droplet, commonly in the µl range. The mother liquor is also found in excess in the bottom of the Figure 2. General vapor diffusion set up of a well. The mixed droplet is incubated separate hanging drop experiment. Modified picture (5, 6). from the mother liquor but is enclosed in the

same chamber with a grease-sealed lid (Figure 2)(40, 42). A sealed vapor diffusion experiment mimics a cell, which is a closed compartment. The droplet on the other hand is an open system. The open system tries to reach equilibrium with the mother liquor and the content in the drop will therefore change over time. The slow change of contents in the drop will hopefully result in a favorable crystallization condition (42).

6

A phase diagram describes were it is most likely to produce protein crystals, indicating how protein and precipitant concentrations can be adjusted in relation to each other (Figure 3). There is an equilibrium between the solid phase, where protein molecules are organized into a 3D lattice forming a crystal and the liquid phase, where protein molecules are free in solution (6, 42). Crystals can only grow from a supersaturated solution and this is achieved through addition of Figure 3. A phase diagram where protein concentration and the concentration of precipitating agents are varied precipitating agents like polymers or to reach supersaturation where crystals can grow. salts, and by changing other Modified picture (6). parameters such as pH or temperature. Supersaturation is a state of non-equilibrium and in order to recover equilibrium in solution, protein molecules congregate into a crystal (42). In the process of crystallization there are many factors crucial for the formation of a crystal such as metal ions, cofactors, inhibitors or other small molecules that need to be present to form a stable structure. There are billions of protein molecules in a crystal and if they are well-ordered they will diffract to high resolution and generate good data (6). Crystallizing a protein usually includes a lot of trial and error, starting with screening, using plates with hundreds of different conditions. Small hits like seeds or tiny crystal needles that diffract to weakly for good data collection need to be improved (6, 37). After identifying favorable crystallization conditions one needs to optimize them to get high quality crystals that are big enough. Optimization is therefore a narrowing down process (6). Optimizations are often done manually and using this technique requires larger drops because of the limitations in manual pipetting compared to crystallization robots. Some years ago cryo-protection of protein crystals was not routine. Nowadays crystals are mainly cryoprotected and flash frozen (vitrified) prior to X-ray data collection. The vitrifying of crystals is done manually. The cryo-protection method has been developed and improved over the years and is now the most common way of taking care of and storing protein crystals. Cryoprotected and frozen crystals have been shown to have a longer lifetime and are less damaged by radiation, which in turn increases the possibility of collecting complete datasets from one crystal. These crystals generally generates better data and to higher resolutions. Another advantage is of course the chance of saving them for another round of data collection (44, 45).

Synchrotrons and X-rays

The history of X-ray crystallography started in the beginning of 1900 when Wilhelm Conrad Röntgen discovered X-ray radiation. He was awarded the first Nobel Prize in physics in 1901 for this groundbreaking discovery. Other discoveries, which eventually lead to today’s profound knowledge of X-ray crystallography methods are numerous. M. von Laue discovered that crystals diffracted X-rays and W. H. Bragg together with W. L. Bragg found out how to analyze and describe diffraction patterns in order to determine structures. C. J. Davisson and G. P. Thomson understood the diffraction of electrons and L.

7

Pauling, the importance of chemical bonds in structural determination. These scientists are just a handful, which all received the Nobel Prize during the last century in the research area of X-ray crystallography (46, 47). After producing and vitrifying well-ordered protein crystals, high quality data can be collected from them at synchrotrons. The resulting data is then analyzed for the purpose of determining the molecular structure of the protein. A synchrotron is a circular building and their circumferences vary significantly. The European synchrotron radiation facility (ESRF) has a circumference of 844 m. The purpose of having a circular building is to accelerate particles until they almost reach the speed of light. The particles travel in vacuum and their travel around the circular pipe is coordinated by a strong electromagnetic field. Accelerated electrons emit energy as X-ray light when changing direction. All X-ray waves produced in this way are focused into a beam (48, 49). The beam is channeled from the circular pipe and is targeted towards a protein crystal, which is mounted on a goniometer (Figure 4A-B). The goniometer rotates the crystal in the X-ray beam and ensures images of the diffraction pattern in different orientations (Figure 4B) (40). The detectors located behind the crystal measure the intensity of the scattered X-rays that hit them (Figure 4C). A diffraction pattern is displayed when the beam of X-ray hits the protein crystal. Incoming X-ray will interact with the electrons surrounding the nuclei in atoms and X-rays Figure 4. A general X-ray crystallography set up at a synchrotron. (A) will be reflected in a Incoming X-rays. (B) X-rays interact with the crystal mounted on a certain diffraction pattern rotating goniometer. (C) A detector records the reflected X-rays. (D) A (40, 48, 49) diffraction pattern is generated. (Figure 4D) .

The phase problem

The intensity of the spots in the diffraction pattern depends on the amplitude of the wavelength from the emitted photons. Because each electron will be hit by several X-ray waves, it is necessary for these waves to be in phase and not cancel each other out (destructive interference). If the waves are in phase the amplitude will be reinforced (Figure 5A-B) Destructive interference occur if the waves are completely out of phase (Figure 5C-D). If they are a little out of phase they will still affect each other, either amplifying or attenuating the signal. If destructive interference occurs, no spots are generated and a diffraction pattern is not formed (40). Using X-ray crystallography to determine the molecular structure of a protein requires a solution to the phase problem. The data obtained in X-ray crystallography provides information on the amplitudes of the waves not their phases. This is referred to as the “phase problem” (50). There are several different methods that can be used to determine the phases of the scattered waves. The

8

Figure 5. (A) Two waves in phase generates (B) a reinforcement of the amplitudes, while (C) two waves 180° out of phase, generates (D) depletion of the amplitudes. Modified pictures (8). most common method when solving structures using X-ray crystallography is Molecular replacement, which was also the method used solving the structures of MTH1 (PDB ID: 3ZR1) and the light chain of BoNT/X (PDB ID: 6F47) (7, 9). In Molecular replacement a homology model is used to determine the phases and the sequence identity of the model should be > 25 % (50). When a completely new structure is determined and no homology models are available, the phases from special atoms introduced into the structure can be used. These atoms generate anomalous signals and can be metals, sulfur or phosphorus. These atoms can already be present in native structures or they can be introduced into the protein by genetic engineering methods or by soaking existing crystals (51). Introduction of selenomethionine (SeMet) into the protein is a common way to generate an anomalous signal (51, 52). Another way of gaining the phase information is to use Isomorphous replacement. The crystals used have the same space group and unit cell dimensions and their structures look the same. The only difference between them is that at least one heavy atom is introduced into one of the crystals. Comparing those structures enables the scientist to get around the phase problem (40, 53).

Other methods – NMR and cryo-EM

Determining molecular structures can be done using methods other than X-ray crystallography. NMR was first described in 1938 and is not dependent on crystal formation (54). With NMR, signals from oscillating atom nuclei in solution are measured. At certain frequencies the nuclei in atoms produce electromagnetic signals while they are passing through a very strong magnetic field. At this frequency the nuclei absorb energy and this is characteristic for each nuclei. The signal reveals the chemical surroundings at a specific position in the structure. Protein dynamics can be assessed with this method

9

(55). A prerequisite of this technique is the use of ionic liquids, which is also a drawback of this method. Ionic liquids make the reactions happen very fast and solubilize the reactants efficiently, which makes their extraction difficult (56). Determining molecular structures with NMR is limited to smaller molecules. The samples should be very pure and the method requires fairly large amounts of protein (57). NMR measures the reactions immediately, which is especially advantageous when the time frames of the reactions are small. With NMR it is possible to control the temperature accurately and the method does not destroy the sample. Quantifying the reactants while monitoring several reactants simultaneously is a convenient feature. Overlapping signals are common problems and there are several ways suggested of getting around this. Some of these solutions are expensive and others are difficult to manage (56). NMR is used in the clinic to generate pictures of tissues where it can be used to image cancer tumors (58). NMR can be used to determine molecular crystal structures and give information about dynamics and physics (55). The scientists who developed cryo-EM during the 1980´s were awarded the Nobel Prize in chemistry in 2017 (59). Disturbances between scattered electrons and non-scattered electrons generates an image (33). EM sample preparation previously required certain fixation or dehydration of the sample, which could generate defects (57). Vacuum is a pre- requisite in EM, which does not agree well with hydrated specimens or living cells (33). Drawbacks with this method are the destruction of the sample by either vacuum or radiation (57). The cryo-EM technique visualizes 3D molecular structures of proteins at almost atomic resolution (33). By vitrifying the sample of interest in liquid ethane the vapor pressure is reduced. The result is a sample in a hydrated state in thin layer of non- crystalline and clear ice (33, 57). Direct electron detectors have been developed that now count the numbers of electron events instead of the energy from them (which could vary). Different energies in electrons do not affect the new detectors or interfere with the output signal (57). The electron beam causes the specimen to move, which is another problem that has been addressed. Problems regarding complications with samples that are conformational or compositional heterogenous have also been dealt with. If different molecules in a heterogenous sample are generating a 3D structure, the result will be unclear. The radiation induced movements in the specimen were compensated for by realigning the images. With the new cryo-EM technique it is possible to solve structures at resolutions where even the side-chains of a protein are visualized. There is no phase problem associated with this method, which is an advantage over X-ray crystallography. The limitations of this technique are constantly diminishing and small molecules are now possible to visualize. However, to solve smaller complexes (< 300 kDa) further developments are required (57).

10

MTH1

The MTH1 project involved determining the molecular structure of this potential anti- cancer target in humans. The enzyme was discovered in humans and rodents due to its homology with the E. coli enzyme MutT, thus giving it the name MutT homolog-1 (MTH1)(60, 61). MTH1 is present in all proliferating cells and in post-mitotic neurons, having a housekeeping function of clearing damaged nucleotides from the free nucleotide pool. The 18 kDa enzyme is located in both the nucleus and in the mitochondria, where it performs its nucleotide hydrolase activity (62). In MTH1-deficient mice, the enzyme has shown to have an important role in minimizing the accumulation of 8-oxo-guanine (8-oxo-G) in DNA, in both cultured cells and in mice brains (62). In MTH1 deficient-mice carcinogenesis has been shown to increase (63). Significant increases in the accumulation of oxidized nucleotides in certain neurons have been discovered in Alzheimer´s, Parkinson´s and Amyotrophic lateral sclerosis (ALS) diseases (64-68). MTH1 is produced from the genetic information of five exons in the mth1 gene. Due to alternative splicing seven messenger RNA (mRNA) transcripts are produced (69). The transcripts formed have single nucleotide polymorphisms (SNPs) and yield four different MTH1 isoforms, namely MTH1a (p26), MTH1b (p22), MTH1c (p21) and MTH1d (p18) (70). Other studies have shown that alternative splicing produces SNPs and therefore several MTH1 isomers exist. The MTH1d isomer has a SNP on exon 4 (71). Increased prevalence of MTH1d is found in patients with gastric and hepatocellular cancer (70, 72, 73). All four known MTH1 isoforms have been proven to have activity towards 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 2-hydroxy-deoxyguanosine triphosphate (2-OH-dGTP) (74). The role of MTH1 in cells is becoming clearer and studies show that its hydrolase activity is important in cleaning out of oxidatively damaged nucleotides from the free nucleotide pool.

Cancer

In 2016, 60.000 people in Sweden were diagnosed with malignant tumors. Another 38.000 were reported as having developed basal-cell carcinoma, a less critical type of skin cancer. The incidence of cancer diseases have increased since 1970 but the mortality is decreasing (75). Cancer is an array of diseases including around 200 different types. There are approximately 200 different cells in the human body and almost all of them can proliferate into cancer cells (76). The definition of cancer is a population of cells that are abnormal and have a limitless growth. Cancer cells can metastasize, spreading from the site of origin to distant and completely different tissues (77). Cancer is caused by mutations in genes that may confer growth benefits (78). The need for effective cancer treatments is serious, preferably with small or no side-effects. One of the largest challenges to overcome within the area of cancer treatment is the resistance against chemotherapy developed in cancer cells (79, 80). Cancer cells developed from normal cells that are unable to form organized and well- functioning structures due to mutations. The reasons for tumorigenesis are numerous and include radiation damage, somatic mutations, infections or hazardous chemicals. All of these factors render mutations possible (81). A mutation is an alteration in the genome, and if mutations are accumulated the risk of developing cancer cells increases. If modified

11 nucleotides are incorporated into DNA via mismatches in the base pairing, it can result in a constant alteration in the genetic code (Figure 6) (82). DNA damage can allow the cell- cycle to proceed into the S phase where new DNA is replicated and cause double strand breaks. These are DNA lesions that are most toxic as both strands in the DNA molecule are broken (83-86). Mutations are frequently occurring in our cells and it is very seldom that they generate cancer cells. Most of the time the mutations are taken care of by proof- reading enzymes, regulated check points in the cell cycle and DNA repair mechanisms. Rats display a level of ~106 oxidative deviations in each cell and new oxidative abnormalities are produced every day at a rate of ~105 events. These are levels common in normal proliferating cells that are due to metabolic events (87).

Figure 6. Mutations are manifested after two rounds of replications. In normal base pairing, C pairs with G. (A) G is oxidatively damaged by ROS generating GO (B) During the 1st round of replication, GO mismatches with A, which normally pairs with T. (C) After the 2nd round of replication A base pair with its normal partner T. A mutation has arisen. Modified picture (63).

A variety of cancer treatments are available today, but there is no single cancer treatment that act on all different types of tumors. Existing treatments can be exhausting for the patient and complete recovery cannot be promised. Research to find genotypic and personalized treatments are ongoing, but extensive investigations are most likely necessary prior to therapy (88, 89). Finding a cancer phenotype to target, which is common between different cancer cells, would be advantageous. Human MTH1 and E. coli MutT hydrolyze 8-oxo-dGTP to its monophosphate form, 8-oxo- deoxyguanosine monophosphate (8-oxo-dGMP). Hydrolysis of oxidized nucleotides inhibits their incorporation into the DNA, thus reducing mutations (62). As cancer cells are fast proliferating they are dependent on copious amounts of nucleotides. 8-oxo-dGTP is a serious contributor to mutations and increases the risk of cancer development (90). Earlier studies have shown that there is significant accumulation of 8-oxo-G and an elevated incidence of carcinogenesis in mth1 knockout mice compared to wild-type mice (91-93). Cytidine monophosphate kinase 1 (CMPK1) prevents oxidized cytidine species from being incorporated into DNA by converting them to uridine (94, 95). Together MTH1 and CMPK1 act in the defense against oxidized nucleotides in cancer cells and are potential cancer therapy agents. MTH1 is a potential anti-cancer drug target, but is far from the only one. Reactive oxygen species (ROS) are a group of ions, radicals or molecules derived from oxygen, which have an unpaired valence electron and readily participate in bond (96, 97) . - . formation . Examples of ROS are superoxide ( O2 ), hydroxyl radicals (OH) and (10) hydrogen peroxide (H2O2) . Oxygen radicals can oxidize nucleotides with guanine being the nucleotide most prone to oxidation (98). Different oxidized nucleotide species are present in DNA and of those, 8-oxo-dGMP and 2-hydroxy deoxyadenosine

12

Figure 7. Oxidatively damaged nucleotides. (A) 2-hydroxy-deoxyadenosine monophosphate (2-OH-dAMP) (B) 8-oxo-deoxyguanosine monophosphate (8-oxo-dGMP).

monophosphate (2-OH-dAMP) are able to base pair with A and G (Figure 7) (63). ROS can arise due to radiation, air pollutants, tobacco and drugs (99). In general, cancer cells have increased levels of ROS, which are involved in tumor growth (96). ROS are produced in all cells as byproducts of normal metabolic events. Oxidative phosphorylation, peroxisome activity and the immune system all generate ROS (100-103). ROS can be responsible for generating mutations in both nuclear and mitochondrial DNA (104). Nucleic acids can be oxidatively damaged by either incorporation of oxidized nucleotides or by directly being oxidized (63, 105, 106). If oxidations are introduced the DNA lesions can lead to cancer or neurodegenerative diseases, which are related to increased age (107). An accumulation of 8-oxo-G in genomes has been shown to be age-related (107, 108). The same accumulation is also present in patients with cancer tumor diseases (97).

Nudix hydrolase family

Nucleoside diphosphates linked moiety-X (NUDIX) enzymes are present in all species. They make up a large family of hydrolases and not much is known about their functions (109). Although there are indications of the enzymes having roles in the cell-cycle regulations (110, 111). There are more than 130 members in the NUDIX family and they have very different substrate affinities with not much in common regarding molecular structures (109, 112). The substrates are nucleotides and their derivatives. The substrates usually contain two phosphates connected to each other with an ester bond. MTH1 is a NUDIX family member and has the typical fold of a b-sheet between two a- helices and contain the characteristic NUDIX motif (7, 113). The NUDIX motif is a conserved sequence of 23 residues (7, 104, 114). NUDIX enzymes hydrolyze their substrates with assistance from a water, which makes a nucleophilic attack towards the b-phosphate on the substrate and hence inorganic pyrophosphate (PPi) is cleaved off. The result is a deoxynucleoside monophosphate (dNMP) that cannot be used in DNA synthesis (104, 115). MutT is also a NUDIX family member but has low sequence similarity with MTH1, except in the typical motif (104). Earlier studies have proven that both E. coli MutT and human MTH1 have the same activity, hydrolyzing 8-oxo-dGTP and 8-oxo-guanosine triphosphate (8-oxo-GTP) (72, 116, 117). MTH1 has shown an even broader substrate specificity compared

13 to MutT and hydrolyses 2-hydroxy-deoxyadenosine triphosphate (2-OH-dATP), 8-oxo- deoxyadenosine triphosphate (8-oxo-dATP) and other nucleotide analogues (72, 116, 118, 119). In vivo mutagenesis of 8-oxo-dGTP and 2-OH-dATP have been demonstrated (63).

MTH1 activity and mechanisms

The structure of MTH1 in solution was solved with multidimensional heteronuclear NMR spectroscopy in 2004. In the same study the MTH1 binding pocket was identified as well as the mechanism of the hydrolysis (104). MTH1 hydrolyzes nucleotides that are oxidatively damaged and thus supplying a pool of non-oxidized nucleotides available in the production of new DNA molecules (Figure 8A-B) (7, 104, 109). A free nucleotide pool with undamaged nucleotides is a prerequisite for building new and proper DNA molecules required during cell division. Free existing nucleotides are more susceptible to damage than nucleotides already incorporated into DNA. There they are shielded from the surroundings inside DNA helices, densely packed into chromosomes (120).

(A) (B) (C)

Figure 8. (A) A normal cell with low amounts of ROS generates limited amounts of oxidized nucleotides and only require low concentrations of MTH1. (B) A cancer cell with high concentrations of ROS generates high levels of oxidized nucleotides, which require high concentrations of MTH1. (C) Inhibition of MTH1 in cancer cells lead to incorporation of oxidatively damaged nucleotides into DNA. Interpretations and illustrations by L. Henriksson (63, 90, 121).

A normal proliferating cell does not have high amounts of ROS present and thereby the amounts of oxidized nucleotides are limited. The need for MTH1 activity is thus low (Figure 8A). In cancer cells the ROS levels are high and the amounts of damaged nucleotides are therefore high as well. The amount of MTH1 activity in cancer cells is clearly increased compared with in normal cells (Figure 8B). In cancer cells with high amounts of ROS and accordingly high amounts of oxidatively damaged nucleotides, inhibition of MTH1 would allow introduction of those nucleotides into DNA and consequently the death of the cancer cells (Figure 8C) (63, 90, 121).

Anticancer hypothesis

Normal proliferating cells have controlled cell divisions and controlled cell cycles, which makes the proliferation highly regulated. The risk of oxidized and in other ways damaged nucleotides being introduced into DNA are low. The development and the division of cancer cells are not as controlled. They need to rely on a few control systems to avoid utilization of damaged nucleotides in synthesizing DNA. One of those systems is MTH1 (63, 90).

14

There are many different types of oxidative damage in DNA. Two examples are 8-oxo-G and 2-hydroxy-adenine (2-OH-A) and they can base pair with adenine and guanine. These bases are not their common partners and this will eventually lead to point mutations (Figure 6) (63). 8-oxo-deoxyguanine (8-oxo-dG) is mostly formed when DNA is oxidized, while 2-hydroxy-deoxyadenine (2-OH-dA) is formed when deoxyadenosine triphosphate (dATP), in the free nucleotide pool, is oxidized (105). Without MTH1 activity damaged nucleotides could potentially be part of DNA molecules and increase the risk of mutagenesis and cancer development (63, 104). Increased numbers of deoxy-nucleoside triphosphates (dNTPs) in DNA, more double strand break lesions in DNA and faster development of senescence can be caused by suppressed MTH1 (122). MTH1 has shown to inhibit RAS-induced oxidative damages in earlier studies (123). By inhibiting MTH1 the introduction of damaged nucleotides into DNA during translation is increased. Eventually the amount of damaged nucleotides in newly synthesized DNA molecules, will reach a limit where it is not possible for the cancer cell to survive. Therefore it is of great interest in the battle against cancer and other neurodegenerative diseases to develop MTH1 inhibitors (86, 121).

15

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The substrate 8-oxo-dGTP Co-crystallization of MTH1 with 8-oxo-dGTP, revealed that 8-oxo-dGMP product was bound in the active site, confirming that a hydrolyzation of the substrate had taken place. This result gave insight into the binding of the product and how it was coordinated in the catalytic pocket (Figure 11). MTH1 binds the base of 8-oxo-dGMP in an anti-conformation while MutT binds the same product base in a syn-conformation (Paper I, Figure 2B) (127). 8-oxo-GTP is an inferior substrate to 8-oxo-dGTP for MTH1, which is confirmed by the difference in activity between these two substrates (119). The 3-OH group of the deoxyribose sugar in the substrate forms a hydrogen bond with Thr8. A hydrogen bond between Thr8 and the 2-OH group of a ribose sugar, is not advantageous or even possible due to the hydrophobic and thereby unfavorable surroundings of that hydroxyl group. The positioning of the phosphate group is slightly shifted and this could be an explanation of the increased activity towards 8-oxo-dGMP. Intriguingly MutT has the same activity towards both 8-oxo-GTP and 8-oxo-dGTP (128). 8-oxo-dGTP is altered and is naturally present in two different tautomers, the 6, 8- diketo tautomer (Figure 10A) and the 6-enol-8-keto tautomer (Figure 10B). By taking away the 6-keto group from 8-oxo- dGTP a higher affinity towards Figure 10. Tautomers of 8-oxo-G. (A) The 6, 8 di-keto tautomer and MTH1 is generated. The (B) the 6-enol-8-keto tautomer. Modified picture (7). conclusion is drawn that this group is not crucial for the interaction between 8-oxo-dGTP and MTH1 (7, 129). Instead, the substrate shows an increased affinity towards MTH1. Nitrogen 1 (N1) becomes a better hydrogen acceptor by losing the hydrogen when the 6-keto group is lost and thereby strengthening the hydrogen bond with Asp120 (Figure 11). Other nucleotides lacking a hydrogen at the N1 position show good quality as MTH1 substrates (129). The 6-OH group in the 6-enol-8-keto tautomer forms a hydrogen bond with a short distance (2.4 Å) towards Asp119 (Figure 11). This indicates the formation of a strong hydrogen bond, which in turn suggests that this is the superior tautomer (Figure 10B).

Coordination of the 8-oxo-dGMP product 8-oxo-dGMP is located in a pocket between the b-sheet and a-helice one. In close vicinity of the base there are three charged residues, Asn33, Asp120 and Asp119. All positioned in such a way that formation of hydrogen bonds with the product are possible (Figure 11). Two of the residues seem to be especially important for the specificity of the binding and they are Asn33 and Asp119. N3 in 8-oxo-dGMP is hydrogen bonding (2.8 Å) with the nitrogen in Asn33. The oxygen of the same residue is hydrogen bonding (2.8 Å) with the 2-amino group. Asn33 is crucial for MTH1 to discriminate between the oxidized and the unoxidized form of purines. Asn33 has proven to be very important enabling the binding of a substrate as well as being substrate specific. The bulkier residue in an Asn33Glu mutation inhibits activity by introducing steric hindrance into the active site (104).

17



     



 

 

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of 8-oxo-dGTP and a hydrogen bond donating group in the N2 position (127). The most important positions regarding specificity are possibly position 1 and 6. In those positions it is possible to either donate or accept hydrogens, which agrees well with the 8-oxo-dG 6-enol-8-keto tautomer (Figure 10B). This speaks in favor of this tautomer having a higher natural occurrence in solution (131). The 8-oxo group does not interact with MTH1 directly but is probably contributing to the stability of the 6-enol-8-keto tautomer by changing the electrostatic potential of the base. The electrons in the ring structure will be affected by the 8-oxo-group and thereby create stronger interactions with the base surrounding residues. An unoxidized nucleotide will thereby have a changed electrostatic potential and this could be how oxidized and unoxidized nucleotides are discriminated between. The X-ray structures of MTH1 alone and together with 8-oxo-dGMP have enabled further research about and synthesizing of inhibitors (121, 132). At the moment clinical trials are ongoing and only future can tell what will come out of this project. To be part of this project has been very rewarding for me both personally and as a scientist.

Newly gained insights and yet unpublished results

Nucleotides can also be damaged by methylations. S-adenosyl-methionines (SAM) are naturally occurring in our cells and are involved in non-enzymatic transfers of methyl groups (133). There are methylating agents present in our surroundings and in chemotherapeutics. A methylated nucleotide can potentially introduce mutations and DNA lesions (134). The free nucleotide pool is approximately 102 – 105 times more sensitive to damage caused by methylations, than nucleotides already incorporated in DNA (135). In yet unpublished results O6-methyl-deoxyguanosine triphosphate (O6-methyl-dGTP) is revealed to be another MTH1 substrate and is hydrolyzed to its monophosphate form, O6- methyl-deoxyguanosine monophosphate (O6-methyl-dGMP). Structural studies show that the same residues in the active site are involved in binding O6-methyl-dGMP. Compared to the binding of 8-oxo-dGMP there are very small changes in the hydrogen binding pattern. Both products are positioned in almost the same way even though the bases are modified differently. O6-methyl-dGMP is slightly shifted towards a hydrophobic pocket. Comparison between human MTH1 and zebra fish MTH1 upon binding O6- methyl-dGMP confirms the hydrogen binding pattern, involving the same residues forming hydrogen bonds to the base of 8-oxo-dGMP. When MTH1 binds 8-oxo-dGMP and O6-methyl-dGMP the phosphate groups are positioned in exactly the same spot. When human MTH1 and zebrafish MTH1 bind to the same products, O6-methyl-dGMP, the same results appears: the phosphate groups are positioned in exactly the same spot. In comparison with the unpublished structure it is clear that this part of the active site is conserved. The phosphate group on the product is primarily surrounded by the negatively charged residues: Glu52, Glu100 and Glu56, and the positively charged Lys23. The same residues are involved in the binding of O6-methyl- dGMP in human MTH1 as in zebrafish MTH1 as well as in the binding of 8-oxo-dGMP in human MTH1 (7).

19

Critics

The link between induced cancer cell death by MTH1 inhibition have been questioned and the importance to evaluate MTH1 as a potential anticancer target, emphasized. A potent and selective MTH1 inhibitor with superior pharmacokinetics, BAY-707, is presented and did not show any potential as an anticancer drug in vitro or in vivo (136). Chemically and structurally different potential MTH1 inhibitors were developed and evaluated. No cancer cell death was observed. The anticancer effect of developed MTH1 inhibitors is suggested to be due to off-target cytotoxic effects and not related to the inhibition of MTH1 (137). Another MTH1 inhibitor, IACS-4759, was identified and evaluated. It was revealed as a good candidate with great metabolic stability being a sub-nanomolar inhibitor. IACS-4759 can be a valuable tool in further investigations of MTH1 as an anti-cancer target (138). Tumors cells can adapt to increased ROS levels and generate damage repair mechanisms and to use these pathways as anticancer targets can be convenient because it is not likely that normal cells are affected (139). MTH1 as a potential anticancer target is appealing, which is underlined by all groups doing research about the same. Additional investigations were done to address the critics that have arisen (132, 137, 138, 140). Evaluation of the inhibitors that have shown to fail as anti- cancer treatments, visualized by immunofluorescence, reveal that 8-oxo-dGMP never are introduced into DNA. Accordingly no 8-oxo-G DNA lesions appeared, which are pre- requisites for MTH1 inhibition to work. TH588 raises the levels of 8-oxo-dG DNA lesions. This could be due to off-target activity, where 8-oxo-dGTPase is inhibited while MTH1 is not. Other off-targets creating synergy effects with MTH1 could also be an explanation. Several isoforms of MTH1 exist and there are different post-translational modifications. Different responses to inhibition can be expected (132). Kettle et al. reported that siRNA did not affect the survival of cancer cells (137). A contradiction is reported where the same siRNA is used and resulted in increased cancer cell death (132). Cancer cells depleted of MTH1 can possibly survive if using siRNA, which does not function in all types of cells (121). Kawamura et al. reported that TH588 and TH287 restrain beta-tubulin and display similar proteomics features as known anti- microtubule agents, in vitro (140). A proteomics study was performed and comparisons between TH588 and anti-microtubule agents were done. TH588 revealed to have a unique mode of action, which also was completely different compared to the anti-microtubule agents tested by Kawamura et al. (132). 8-oxo-dGTP and 2-OH-dATP are only toxic if MTH1 inhibitor TH588 is present. This has been shown by microinjections into zebrafish embryos (141). TH588 greatly inhibit cell viability in a resazurin assay (132). MTH1 deficient mice (-/-) have shown to stay alive and grow old, which indicates that MTH1 is less dependent in normal proliferating cells (121, 142). An optimized version of inhibitor TH588, TH1579, has proven to have excellent anti-cancer properties in vivo as well as pharmacokinetics and good bioavailability (132). Overexpression of MTH1, MutT and an antioxidant (NAC) prevented 8-oxo-G from being introduced into DNA. This proves that MTH1 treated with and inhibited by, TH588 and the optimized inhibitor TH1579, lead to DNA lesions (132). MTH1 is a hot topic and the involvement of many scientists and the sharing of knowledge about this enzyme, is of great importance for cancer research.

20

Botulinum neurotoxins

In 1919, the first BoNTs were discovered, namely BoNT/A and BoNT/B (143). During the following half decade five other BoNT serotypes were discovered and the last one, BoNT/G was found in 1969 (144). In 2017, almost 50 years after the previous BoNT was found, a new serotype was discovered. It is called BoNT/X (2). The part of my thesis that will include the BoNT project will be all about this recently found serotype, which has not been categorized until now. BoNTs are the most potent toxins found in nature (145). They are the causative agents of botulism, which is a potentially deadly disease (146-148). BoNTs are produced by several different strains of Clostridium botulinum (C. botulinum) (149). Most strains of Clostridium bacteria express toxins and have pathogenic features but only C. botulinum and Clostridium tetanus (C. tetanus) are specifically acting on neurons (150, 151). C. botulinum are gram positive, anaerobic and spore forming bacteria. Spores produced by C. botulinum are found in soil and are also present, but sparsely, in feed crops and in animal intestines (152). The sequence homology between serotypes is approximately 30 % even though the molecular structural fold and the domain organization are conserved (2, 150, 153-155). The light chain (LC) where the catalytic site is located is a Zn2+ dependent endopeptidase (154). BoNTs are so called AB protein toxins, containing an active part (A) and a binding part (B) (156). Today there are eight different serotypes known based on their serological specificity. They are named A-G together with the most recent discovered serotype, called BoNT/X (2, 146, 150, 157).

One of the most potent toxins also heals

BoNTs are amongst the most potent toxic agents known today (145). There is an estimation of it being 106 times more potent than the venom from cobra (158). At the same time, BoNT/A is used in extremely low doses to relief symptoms in an increasing number of neuropathological diseases. Most people are familiar with the commercial name Botoxä and its usage in cosmetic corrections. An extremely diluted form of BoNT/A is marketed as BotoxÔ (Allergen, Inc, US) or as DysportÔ (Speywood, UK) and is used as treatment for many neuromuscular disorders. In 1989 BoNT/A was approved as treatment for strabismus, hemifacial spasm and blepharospasm by the US Food and Drug Administration (FDA) (159-161). The number of disorders that can be treated with Botoxä is growing rapidly (159, 162-165). The neurospecificity and the potency make BoNTs excellent treatments (145, 166). One unit of BotoxÔ corresponds to 50 x 10-12 g of BoNT/A neurotoxin protein (161). The symptoms in diseases where neurons are overactive can be eased with local injections and thereby increase the wellbeing and the quality of life for many people suffering (162, 166). Extensive sweating, spasms, squint or an overactive bladder are just a few of the disorders that also can be regulated with Botoxä (165). Large amounts of BoNTs are easily produced and together with their potency and the possibility of being aerosolized, they belong to the category A of biothreat agents (Centers (145, 166, 167) for Disease Control, Atlanta, GA) . If orally ingested the human LD50 is approximated to 1 µg/kg body weight. If inhaled an even lower amount is needed with an (168, 169) approximated human LD50 of 10-13 ng/kg body weight . It is not only a lethal potential bioterrorism weapon that kills but intoxication result in people needing medical attention for a very long time (162). Available countermeasures or treatments are antidotes

21 against serotype A-G (170). To get an efficient treatment it is crucial to administrate antitoxin as soon as possible after intoxication (171). There are so far no approved vaccines against botulism (172). Cheap, available and easily administrated antidotes and vaccines are of concern and need to be developed as well as antidotes against the novel BoNT/X.

Botulism BoNT is causing the disease botulism. The first description of the symptoms of the sausage poisoning, which later came to be known as food-borne botulism, was carried out by Justinus Kerner in 1817-1822 in Germany (173). The serotypes involved in human botulism are A and B and very seldom E and F (155, 174). In nature, decomposing animal cadavers can contain spores from C. botulinum. These spores are often found in the intestines of live animals. Spores turns into germinating bacteria if the conditions are favorable: anaerobic, nutrient rich, humid and with a slightly raised pH. The presence of spores contaminates maggots feeding from the carcass. The maggots stay unaffected but are spreading the spores when they are feeding birds and other animals. In this way spores are transferred to favorable environments where they can germinate and become sporulating bacteria, which express and release toxin. BoNT intoxicates the host and induce flaccid paralysis and possibly death and thereby the production of toxins is amplified (155). Cattle develops botulism mainly from serotype C and D. Animal botulism outbreaks can be due to silage contaminated with BoNT or C. botulinum. BoNT/G do not cause botulism (152). There are five different types of botulism and the most common are food-borne botulism arising from ingestion of toxin-contaminated food-stuffs and infant botulism evolving from spore-contaminated food. A third, very rare type of botulism arising from ingestion of food contaminated with spores and is seen in adults with a deteriorated intestinal bacteria flora. The fourth, very rare variant, is wound botulism in which C. botulinum grows in wounds. The final type is iatrogenic botulism, which arises in persons overdosing BoNT as an agent of treatments of different kinds (155). Blurred vision, swallowing impairments and muscle weakness are typical symptoms of botulism. Thereafter flaccid paralysis arises (175). Food-borne botulism symptoms are stomach pain, diarrhea and vomiting followed by flaccid paralysis. The onset is fast and the outcome is dose dependent (176). It is crucial to be treated with antitoxins as soon as possible if intoxicated. Severe cases of botulism causes paralysis for several months and requires ventilatory support (145). During months of paralysis motor neurons are rebuilding, creating new nerve paths and eventually recover the motility in the muscles (176). There are different treatments available for infant botulism and non-infant botulism. The first is attended with botulism immune globulin intravenous (BabyBIG), which are human derived antibodies (177). The latter is treated with heptavalent botulism antitoxin (BAT). BAT is a combination of antitoxins towards all the seven serotypes (BoNT/A-G) known before 2017 (170). Addition of an antitoxin towards the eighth serotype requires thorough characterization of the potency of BoNT/X (2).

The BoNT family

Georgina Burke discovered two BoNTs in 1919, which were named BoNT/A and BoNT/B and thereby she established the still used naming convention of the BoNT family members (143). The categorization of BoNTs into different serotypes are based on loss of cross- neutralization from existing antitoxins. There are eight BoNT serotypes known today,

22 several subtypes and mosaic toxins as well as the closely related Tetanus neurotoxin (TeNT) that are included in the clostridial neurotoxin family (2, 150, 153, 155, 178). They are so far named according to the established system from 1919 except from BoNT/X, the most recent serotype discovered (2). Thanks to computational technologies, databases and possibilities of advanced searches, more BoNT family members will most likely be discovered in the future. The overall domain arrangement is homologous between serotypes (179). BoNTs have a MW of 150 kDa and are expressed as a polypeptide chain and consists of the light chain (~50 kDa) and the heavy chain (HC, ~100 kDa) (Figure 12) (155). Each domain has a specific function: a binding domain, which is the C-terminal of the heavy chain (HCC), a translocation domain, which corresponds to the N-terminal of the heavy chain (HCN) and the LC, which carries the catalytic site of this Zn2+ endopeptidase (155). There is a linker region present surrounding the LC like a belt and connecting it to the HC. Two conserved Cys residues form a disulfide bond, which needs to be proteolytically cleaved to activate the toxin (154, 155, 180).

Figure 12. The overall domain arrangement of BoNT/A valid for all BoNTs. From left the LC (blue and turquoise), wrapped with a belt (green) holding it close to HCN (light green and yellow), which is connected with HCC (orange and red)(PDB ID: 3V0B). Modified picture (181).

BoNTs are also divided into four groups based on their phenotypic features and what type of botulism that is generated (152). Group I consists of serotype A, B and F causing human botulism and with a growth temperature of 12-37°C. Group II is made up from serotype B, E, and F, which also is the cause of human botulism but is having a lower growth

23 temperature, between 3-30°C. Group III causes animal botulism and the serotypes are C and D, growing in the temperature range 15-40°C. Finally there is a group IV, which do not cause botulism and the only serotype in this group is BoNT/G, having a maximal temperature enabling growth, set to 37°C (152).

Serotypes, subtypes and mosaic toxins BoNT serotypes are divided into seven groups that varies serologically (143, 146, 182). Serotypes are further categorized and divided into subtypes and mosaic toxins. The relationship between them is yet complicated by involving different gene carriers, more than one gene present in each gene carrier, silent bont genes as well as the phenomenon of horizontal gene transfer. Subtypes are inactivated by the same antisera as its parental serotype and have at least 2,6 % variations in the sequence. The sequence alterations do affect the affinity of the BoNT/A monoclonal antibodies when binding to subtype A1 and A2 (179). Subtypes are designated BoNT/A1 where A indicates the parental serotype and 1 identifies the subtype (182, 183). Studies have additionally shown that functional variants in the phenotype of subtypes exist (184-186). Mosaic toxins have domain sequence homology towards different serotypes (187). Mosaic BoNTs are neutralized by antibodies toward either of the parental BoNTs (188). BoNT/DC is a mosaic toxin and share binding domain with BoNT/C and catalytic domain with BoNT/D (189). Intriguingly this mosaic toxin does not share the affinity for the same protein receptor as the parental BoNT/C. Instead BoNT/DC bind to the same protein receptor as BoNT/B (189). Different bont genes can be located on various types of gene carriers, which could be either a chromosome, a plasmid or a phage. This phenomenon is explained by genes being motile. Horizontal gene transfer enables gene carriers to have more than one bont gene present (190-192). The motility of toxic genes into non-toxic strains convert them into toxic strains. This movement contributes to the development of new strains (153). Two or three bont genes can be found in the same strain and these serotypes are designated as BoNT/Af, where A identifies the serotype expressed at higher levels and f indicates the serotype expressed at lower levels (188, 193, 194). When more than one bont gene is present in a gene carrier and only one of them is expressed the others are identified as silent (195, 196). The silent gene is designated within brackets, like B in BoNT/A(B) (197). A new Botulinum neurotoxin was isolated from a BoNT strain IBCA10-7060, found in an infant with botulism in 2013. The toxin was not neutralized by antitoxins against other serotypes and it cleaved common BoNT substrates, namely vesicle-associated membrane protein 1 (VAMP1), VAMP2 and VAMP3. It had the typical motifs in the binding domain and in the catalytic domain. It was characterized as a new toxin and was named BoNT/H (198, 199). Sequential alignment revealed a high similarity, more than 80 %, towards the catalytic domain in BoNT/F5 and towards the binding domain in BoNT/A1. The new serotype H was suggested actually being a BoNT/F5A1 hybrid (188). Because existing antisera neutralized the toxin it was suggested another name: BoNT/FA (188). This strain was further investigated and BoNT/A specific monoclonal antibodies neutralized the toxin. A third name, BoNT/HA, was suggested (200). If the nomenclature established by Georgina Burke is to be followed, the procedure of how to characterize new BoNT serotypes must be set and agreed upon (143). Serotypes A-G

24 were already defined when the supposed BoNT/H was discovered and which later on needed further investigations (199). Therefore the last serotype is called BoNT/X (2). A gene encoding a Botulinum neurotoxin like protein (BoNT/En) was discovered in an Enterococcus faecium (E. faecium) strain found in cow faeces. The gene product was characterized and was found cleaving the common BoNT substrates VAMP2 and synaptosomal-associated protein of 25 kDa (SNAP-25) and was located in an open- reading-frame-x (orfx) gene cluster. Sequential alignment revealed BoNT/X as the closest related botulinum neurotoxin (178). E. faecium is the major reason for multi-drug-resistant (MDR) infections (201, 202). The GI-tract of animals and humans are colonized by E. faecium and have therefore been exposed to antibiotics for many years (202). The bontEn gene is located on a conjugative plasmid (178). Conjugative plasmids offer one of the most competent ways of horizontal gene transfers. That makes it highly involved in the increasing number of bacteria that are resistant to multiple antibiotics. This is a development that agrees well with the evolution and the increased use of antibiotics (203). The classifications of BoNTs are eagerly debated and the future will show how BoNT continually will be categorized.

Gene clusters Bont genes are always located in gene clusters, which can be of two different kinds: the orfx or the hemagglutinin (HA) gene cluster (Figure 13) (156). Orfx and HA are referred to as non-toxic neurotoxin associated proteins (NAPs). BoNT gene clusters withhold bont, ntnh and ha or orfx genes along with genes expressing regulatory proteins (156, 204). The BoNT gene clusters closest neighbours are helicases, transposases and insertion sequences, which indicate that horizontal gene transfer may occur (153). Combinations of non-toxic genes along with ntnh are expressed together with the bont gene. BoNT/A1, B, C, D and G are expressed together with HA proteins, while BoNT/A2, E, F and X are expressed with OrfX proteins. BoNT/A is the only serotype expressed from both types of gene clusters (1, 156).

Figure 13. Gene clusters of BoNT/X, A1 and A2. Modified picture (1-4).

NTNH and other non-toxic proteins

The expressed gene products are non-covalently bound and are forming progenitor toxin complexes (PTCs) (Figure 14A-B and 20A) (156, 169). BoNT and NTNH is making up the minimal PTC (M-PTC) with a MW of ~300 kDa (Figure 20A) while the largest is made up from four NAPs, NTNH and BoNT with a MW of about 900 kDa (205). A combination of the

25 three HA proteins (HA17, HA33 and HA70), NTNH and BoNT/A has a MW of ~760 kDa (156, 206-208). The combination of HA33, HA17 and HA70 was structurally determined for serotype C and is an example of how a PTC can look like (Figure 14A-B) (209).

Figure 14. The HA70 PTC of BoNT/C (PDB ID: 4EN6). (A) View from the top. (B) View from the side. Modified pictures (209, 210).

The ntnh gene is likely to have arisen from a duplicate of the bont gene. The gene product NTNH seems to have developed into possessing a protective role of BoNT (153, 181, 211). Keeping in mind that C. botulinum exists and thrives in decaying cadavers, which offer degradation and denaturation of proteins, protection of BoNT is needed (174). BoNT and NTNH have the same overall fold with a similar domain arrangement (Figure 20B). The functions of the domains are different between BoNT and NTNH and the sequence similarity only reaches 20 % (181). C. botulinum releases expressed gene products from bont gene clusters as aggregates. These proteins are forming different kinds of oligomeric PTCs in various sizes by non-covalent interactions (156, 212-214). BoNT and NTNH are together making up the M-PTC and addition of other non-toxic proteins generate the large PTC (L-PTC) or the larger PTC (LL-PTC) (212, 214). Activity studies of BoNT/D revealed that M-PTC contain NTNH and BoNT with no HA activity, while the L-PTC and the LL-PTC both contain HA activity (214). pH dependent PTC assembly The purpose of the formation of M-PTC is to protect BoNT from the harsh surroundings (174, 181). The heterodimeric M-PTC is formed via induced fit at acidic pH in BoNT/A and E (215, 216). Low pH in the environment protonates pH sensitive residues, which induce assembly of the complex. The M-PTC is disassembled in neutral or high pH because the same residues are deprotonated and this enables disruption of the M-PTC complex (181, 215). It was revealed that BoNT/A has positive electrostatic potential close to its pH sensitive residues, while corresponding NTNH has negative electrostatic potential around its pH sensitive residues. Changing the charges of the pH sensitive residues will impoverish the interaction between BoNT/A and NTNH and a disassembly of the M-PTC is favorable (181). There are conserved carboxylates found in all three domains of BoNT and they contribute with high local pKa (155, 217). These residues are possibly pH-sensors

26 and when protonated they increase the net positive charge and enable interactions with the negatively charged membrane (217-220).

Roles of the PTC proteins The progenitor clusters have protective roles shielding BoNTs from being broken down in harsh surroundings (181). The size of the complexes seem to have effect on the toxicity because the larger a PTC is, the more potent it is (221). Free BoNT is orally less potent compared to the PTC of the same serotype (207). The evidence of their roles in the intoxication process, were the monolayer of epithelial cells need to be crossed, are convincing. Different mechanisms have been proposed. A mechanism where the monolayer of epithelial cells are broken and BoNT/A can pass in between cells have been suggested (208). Results show that BoNT/A is more easily internalized when it is in its PTC, compared with being a free BoNT/A (169). BoNT/A seem to be transcytosed after binding to a non-specific receptor displayed on the human bronchial epithelial cells (169, 222). Studies reveal that transcytosis is the mechanism by which BoNT is transferred from gut to the general blood stream. Other studies suggest that NAPs are endocytosed into epithelial cells, from where they assist the BoNTs to pass into circulation (222-224). The L- PTC of serotype A is suggested to consist of two heterodimers, one contain BoNT and NTNH at 290 kDa while the other at 470 kDa contain NAPs. The smaller heterodimer is protecting BoNT, while the larger complex enables absorption across intestinal epithelial cells, by binding to carbohydrate receptors displayed on the cell surface (207). Studies report that HA proteins adhere to glycoproteins abundant on epithelial cells and thereafter BoNT/A is transported via the epithelial cells, to the basolateral side (207, 225). Expressed HA33 proteins are involved in the binding and the transfer of BoNT/A across the epithelial cell layer. HA33 has been investigated as part of the PTC serotype A and proved to increase the movement over epithelial cells lining the gut lumen. By doing so, HA33 demonstrated its importance in the role of intoxication. Oligosaccharides presented on mammalian erythrocytes, functions as non-specific binding receptors to HA33 proteins (169). Additionally HA33 protects BoNT from proteolytic degradation (214). HA have shown to bind to E-cadherins displayed on epithelial cells and break up the cell-to- cell adhesion (208). NAPs seem to have additional functions and have proven to add thermostability to the molecular structure of BoNT/A. The complex of BoNT/E is also more functionally resistant to temperatures (226). Mice were intralumenally administrated with fluorescently marked HC/A, to elucidate the transfer across the epithelial monolayer. Intestinal crypt cells were shown to be their targets (227). Further research is needed to fully understand the roles of different NAPs and what specific combinations result in and if synergy effects are reached. The roles of the OrfX proteins is an interesting area of research and some structures have already been determined (213).

BoNT activity and mechanisms

The BoNTs aim the neuromuscular junctions (NMJ) and the presynaptic neurons, where they bind with a dual binding mechanism (Figure 15B). After binding, BoNT is internalized into a presynaptic neuron (Figure 15C). The low pH in the vesicle induces conformational changes, enabling the LC to be externalized into the neuronal cytosol (Figure 15D). The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are BoNT substrates and are localized in the neuronal cytosol. SNARE

27 proteins are involved in vesicle fusion events (Figure 15A)(228). This is where the BoNT enzymatic activity proteolytically cleaves SNARE proteins, stopping them from fusing acetylcholine-containing vesicles with the neuronal membrane. The neuromuscular signaling is inhibited and flaccid paralysis is induced (155, 156).

Figure 15. Illustration of BoNT action in the presynaptic NMJ. (A) Normal acetylcholine release with assistance from SNARE proteins. (B) BoNT present in NMJ binds to gangliosides abundant on neuronal membranes and to a protein receptor displayed from the emptied synaptic vesicle. (C) BoNT is internalized when the synaptic vesicle is re-formed. (D) Lowering of pH inside the vesicle drives the translocation of LC into the neuronal cytosol. (E) The LC is hydrolyzing SNARE proteins. (F) Acetylcholine release is not possible.

Neuronal internalization Depending on the route of administration, BoNT travels over endothelial cells lining bronchi or intestines to reach the general blood stream. Prior to being transferred over the endothelial barrier, BoNT travels with the company of NAPs. Via blood circulation, BoNT reaches the NMJs of peripheral motor neurons (Figure 15B) (156). The C-terminal of the heavy chain (HCC) first binds to gangliosides abundant on the axon terminal of the presynaptic neuron (229). Most BoNT serotypes bind the same type of ganglioside receptor, namely GT1b (230-232). Although other gangliosides do function as BoNT initial receptors (233, 234). Gangliosides are composed of a sialic acid part and a ceramide part. The ceramide is composed of a fatty acid and a sphingosine. A majority of all sialic acid moieties in the neuronal membranes are present in the composition of gangliosides (235). SNARE proteins are responsible for fusing acetylcholine vesicles in presynaptic neurons with the neuronal membrane. When the vesicle fuses with the membrane its lumen is exposed and BoNT protein receptors are displayed to the NMJ (Figure 15A) (236, 237). BoNT is thereby able to connect to a second anchoring point, a serotype specific protein receptor. (155, 189, 234) After binding to its receptors BoNT is internalized when the vesicle is once again formed (Figure 15B) (189, 238). The second step of the binding mechanism contributes with specificity and affinity (189, 239-243). The dual binding mechanism amongst BoNTs was proposed in the 1980´s and involves initial binding to a ganglioside following binding to a protein receptor (238). The binding affinities of some serotypes have been characterized but far from all are known (189, 234). Serotypes B, DC and G are using synaptotagmin (Syt) I and II as protein receptors, while serotypes A, D, E and F bind to

28 synaptic vesicle 2 (SV2) (156). Accordingly BoNT is internalized into an empty presynaptic acetylcholine vesicle, forming from the neuronal membrane where BoNT is anchored (Figure 15C) (244). Recent mutational studies of BoNT/B, DC and G have revealed a third binding mechanism where a hydrophobic loop between the ganglioside binding site and the peptide binding site, has a crucial role. The loop is present in above mentioned serotypes, which all have Syt I and II affinities (245). The structures and the activity between a truncated and a full-length version of LC/A have been compared. The full- length variant shows a greater flexibility and an increased enzyme activity. This might be due to a more rapid binding of the substrate SNAP-25 and a change in conformation compared to the truncated variant, which is staying in a compact conformation (246). Studies have shown that BoNT/DC is being neatly internalized without binding to any gangliosides. Instead the toxin binds to other molecules displayed on the neuronal surface, which contain sialic acids. Another interaction is reported in the same study and occurs between a flexible loop located on the HCC and lipid membranes (247).

Translocation and activation To prepare the lumen of an empty presynaptic neuronal vesicle for acetylcholine refill, ATPase lower the pH inside by pumping in H+. Vesicle internalized BoNT, already present, will be affected by the decreasing pH, which enables the translocation mechanism of the LC (248). The translocation domain HCN, transports the LC out of the vesicle and into the neuronal cytoplasm where the BoNT substrates are located (Figure 15D-E) (249). The mechanism of how the LC is transferred over the vesicle membrane, out of the lumen and into the neuronal cytosol is not completely established but some mechanisms have been reported (154). The translocation mechanism is pH driven (250, 251). As response to low pH the translocation domain forms a transmembrane pore in the synaptic vesicle bilipid layer (252-254). To be activated, a short linker needs to be proteolytically cleaved between LC and HC. This enables a conformational change and render translocation possible (255, 256). The pore allows LC to translocate into the cytosol of the neuron. Studies have shown that LCs from BoNT/A1 and E1 are transferred via the transmembrane pore. The width is only about 15-20 Å and fit the size of an unfolded LC (257, 258). An unfolding event must take place to allow LC entry into the cytosol (259). It has been reported that HCN in BoNT/A can form a channel through the vesicle membrane and has an additional role as a chaperone assisting in refolding LC to its native conformation (260). The unfolding and the passing through the pore must occur without disrupting the disulfide bond. The disulfide bond needs to be reduced in the neuronal cytosol when the LC has crossed the synaptic vesicle membrane. If the disulfide bond is reduced before or during the translocation, a pore will not be formed and render translocation impossible (217, 259). Studies show that keeping the disulfide bond intact is crucial for BoNT activity (217, 259, 261, 262). Reduction of the disulfide bond releases the LC from the pore-forming translocation domain and is necessary for catalytic activity (259). The thioredoxin- thioredoxin reductase (Trx-TrxR) system reduces the interchain disulfide bond in the clostridial family member Tetanus (TeNT) (261). Trx-TrxR inhibitors were used in cultured neurons to show the systems involvement in the entry process. The inhibitors also proved to stop BoNT activity (263, 264). The heat shock protein 90 (Hsp90) is a chaperone aiding other proteins to fold correctly. Hsp90 does not have specific substrate interactions, instead it recognizes secondary structures not properly folded (265). Refolding of the LC is required for catalytic activity.

29

Studies have shown that a Hsp90 inhibitor stops the neurotoxicity of BoNT/A1, D and TeNT (266). Hsp90 is suggested having a role in assisting the LC refolding process (265). Activation of BoNT takes place when the linker between LC and HC is proteolytically cleaved by host or bacterial proteases and an active di-chain is formed (154, 180). The durability of the action is determined by the LC in subtypes of serotype A. The lasting effect was shown to vary between subtypes. Recombinant hybrid toxins maintained antitoxin sensitivity and were still catalytically active. This could be useful in the clinic. (267). Proteolytic cleavage of SNARE proteins is performed by the refolded and activated LC (154). Cleaved SNARE proteins are unable to fuse vesicles with the neuronal membrane (Figure 15E). The result is no release of acetylcholine into the NMJ (Figure 15F). Without neuronal muscular signaling it is not possible to activate the post-synaptic motor neurons and the muscles are left unresponsive and flaccid paralysis is induced (171, 228). Recently a study revealed that BoNTs do not only act locally but is also travelling along the neuronal axon and exert distal effects. It was also reported that BoNT enters the neuron via other, not yet known, pathways and into organelles that are not acidic (268).

BoNT substrates The varying substrates for BoNTs are found in the cytoplasm of the presynaptic neuron. BoNT/A, C and E hydrolyze SNAP-25, while BoNT/B, D, F, G and X all act on VAMPs. Additionally BoNT/C also acts on Syntaxin and BoNT/X on Ykt6 (2, 269, 270). Some serotypes share substrates but each serotype hydrolyses a specific peptide bond (271). SNAP-25, VAMP and Syntaxin are all SNARE proteins. SNAREs contain a conserved and characteristic motif, which involves 60-70 residues in certain repeats. There are several members of the SNARE family in different organisms and all of them are crucial in an array of membrane fusion events (228, 272). BoNTs act on SNAREs crucial for acetylcholine vesicle fusion with the neuronal membrane (155). Structures of BoNT/F in complex with inhibitors mimicking VAMP increase the understanding of substrate recognition (PDB ID: 3FIE and 3FII). Three exosites are presented and specific secondary structure elements in LC/F bind to the substrate. These exosites are located away from the catalytic pocket and is settling the substrate specificity when coordinating the substrate during proteolysis (271). The crystal structure of BoNT/A binding its substrate SNAP-25, reveals several exosites as well as structural alterations in proximity of the catalytic pocket. Comparisons between structures of BoNT/A, B and E reveal almost identical catalytic pockets and this indicates that substrate specificity is determined away from the catalytic site (11).

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Paper II – Identification and characterization of a novel Botulinum neurotoxin

C. botulinum strain 111 was categorized as the cause of one case of infant botulism in Japan in 1996. It was recognized as BoNT/B and later on characterized as BoNT/B2 sitting on a plasmid (273, 274). In about 8 % of all reported cases of infant botulism BoNT/A(B) strains are identified as the cause of the disease, in where serotype B is silent (275). In an attempt to get an overview of the BoNT family members, using Uniprot sequence database, we found a new bont gene and it was located on the chromosome in strain 111. A new BoNT serotype was discovered and it is called BoNT/X. In this article BoNT/X is identified and characterized. No structure of full-length BoNT/X has yet been determined even though crystallization trials have been initiated. The sequence similarities are approximately 30 % compared with all other serotypes (179). The similarities and differences in the sequences are not concentrated to one domain but are evenly distributed, which speaks in favor of BoNT/X not being a mosaic toxin. The sequence confirms the presence of conserved motifs in both the binding site (SXWY) and the active site (HEXXH), two crucial motifs for BoNT action (9, 276, 277). The bontX gene is positioned down-stream of ntnh in an orfx gene cluster in the same way BoNT/A2, E and F are (156). The new toxin has some unique features not found in already known BoNTs. In the bontX gene cluster, orfx1, orfx2 and orfx3 have their reading frames in an opposite direction compared to in other orfx gene clusters (Figure 13). Surprisingly an additional gene is present in the cluster and it is called orfx2b, which is found down-stream of bontX. The function of OrfX2b needs to be investigated in the future.

Figure 16. Alignment between VAMP1, VAMP2 and VAMP3. The two SNARE motifs are shadowed (grey). The cleavage sites of BoNT/F, D, X, B and G are colored (orange) and are appointed with arrows. Modified picture (2).

The catalytic activity was investigated. A His6-tagged LC/X was produced in E. coli and proved its activity by cleaving the common BoNT substrates VAMP1, VAMP2 and VAMP3 (Figure 16). Surprisingly they were cleaved in a novel site, between Arg and Ala, and is located in a conserved SNARE motif. BoNT/X also showed activity towards three new BoNT substrates, namely VAMP4, VAMP5 and Ykt6 (278). The cleavage sites in VAMP4 and VAMP5 are homologous to the corresponding site in VAMP2 cleaved by BoNT/X.

31

A recombinant X-LC-HCN was produced for investigation. In the linker region present in all BoNT serotypes, two conserved Cys are located. These cysteines, Cys423 in LC and Cys467 in HCN, are forming a disulfide bond between the chains. In BoNT/X a third Cys, Cys461 in HCN, is found (Figure 17). Mutational studies revealed that Cys423 could bind to either of the two cysteines located in the HCN. This rearrangement of disulfide bonds is referred to as disulfide bond shuffling and is possible between adjacent cysteines. Mutants were treated with an endoproteinase, Lys-C, and were thereafter analyzed. Cys423Ser does not hold the LC and HCN together, while the other two mutants have disulfide bonds between the chains. It is confirmed in X-LC-HCN that mutation Figure 17. Illustration of disulfide binding in BoNT/X wild type and three of Cys423Ser is not active mutants. Modified picture (2). while Cys461Ser and Cys467Ser are. A fully active toxin was needed to elucidate the activity of BoNT/X. Due to its expected high toxicity, the amount produced was needed to be kept at a minimum. There are no antidotes towards BoNT/X and it belongs to an extremely toxic family, therefore the precautions. Sortase-mediated ligation was the chosen method, enabling the production of small amounts of the toxin under strict control by joining together two non-toxic fragments (Figure 18) (279, 280). The sortase recognizes certain motifs in LC-HCN and HCC respectively. Initially the HCC is prepared by addition of a glycine, a thrombin cleavage site and a glutathione S-transferase (GST) tag at the N-terminal. Thrombin cleaves off the tag and leaves an N-terminal with a glycine. Thereafter the added His6-tag is cleaved off from the C-terminal of LC-HCN by sortase, which is a transpeptidase. The cleavage of a peptide bond in the LC-HCN motif make the fragment susceptible to new peptide bond formation with N-terminals having a glycine. The two non- toxic fragments can be ligated together with the sortase. By sortase- mediated ligation, a fully active BoNT/X was Figure 18. Full length, inactive BoNT/X is produced via sortase mediated generated in restricted ligation. Modified picture (2). and low amounts.

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The full-length and active BoNT/X penetrated rat cortical neurons and cleaved VAMP2 and thereby demonstrated its capacity. BoNT/X was tested against all known BoNT antisera in a dot blot assay to find out if it was recognized by antisera targeting existing serotypes. It was not, which underlines the discovery of a new serotype. Testing the activity in vivo is of interest and the toxin was therefore injected into one of the hind limbs of mice. The toxin induced flaccid paralysis, which was confirmed by the inability of spreading toes upon stimulation. Inactive full-length BoNT/X is generated by mutating two conserved residues located in the active site. The mutations are Arg360Ala and Tyr363Phe. The inactive toxin was expressed to be used in production of neutralizing antibodies and in crystallization trials. The cDNA was produced in house (by Professor Min Dong, Harvard Medical School, USA), containing the genetic information needed together with a C-terminal His6-tag. The cDNA was cloned into a pET22b vector. The E. coli, strain BL21(DE3) was taking up the vector during transformation and thereafter expression could take place. A large-scale protein production protocol was carefully elaborated, enabling and ensuring the supply of material for further explorations. Purified full-length and inactive BoNT/X could be very useful in crystallization trials to elucidate the molecular structure of the holotoxin, in biochemistry assays to further characterize the new serotype, in production of neutralizing antibodies and possibly production of antidotes enabling investigations of active BoNT/X. A robust purification protocol was produced, with a proper yield of pure, inactive full- length BoNT/X. Crystallization trials were performed with the prospect to determine the structure of BoNT/X and have so far resulted in low diffraction initial hits. This remains a challenge to improve.

33

Paper III – Structural characterisation of the catalytic domain of Botulinum neurotoxin X – high activity and unique substrate specificity

The LC of the recently discovered BoNT/X is here characterized and structurally studied (2). The structure of LC/X, the catalytic domain containing the active site, is presented and hereby all eight BoNT LCs are structurally known (232, 281-286). There are three different SNARE proteins being proteolytically cleaved by the seven other known serotypes. All serotypes vary in cleavage sites, even if they share substrate (272). A common motif is present and is shared between the SNARE proteins and consists of nine residues (272). The motif is found in duplicates in VAMP and Syntaxin, while SNAP-25 displays four identical motifs (272). If the substrate contains the proper cleavage site for a certain substrate but is too short, the LC fails to cleave it (270, 272). The substrate contains more than one identical peptide bonds and intriguingly the LC only hydrolyses one of them (270). This indicates there are sequences distant to the cleavage site, possibly important for recognizing and binding the substrate (272). LC/X containing a N-terminal His6-tag was recombinantly expressed in E. coli and thereafter purified. A protein concentration of 13,6 mg/ml was used in crystallization trials. Crystal screening with commercially available screens were performed and crystals appeared at room temperature (RT). Two structures of the LC/X are presented in this paper. One was determined to 1.35 Å, displaying residues 1-413 but lack electron density for N- and C-terminals (Figure 19). This structure had space group P212121. The other 2+ structure was solved to 2.4 Å, had space group P22121 and did not contain Zn in the active site. With molecular replacement and the use of the earlier structure of LC/B (PDB ID: 1EPW), the structure of LC/X was determined. LC/X was structurally compared to other LCs with affinity for the same substrates, namely VAMPs.

Figure 19. The light chain of BoNT/X at 1.35 Å resolution containing Zn2+ (ball) in the active site (PDB ID: 6F47). Modified picture (9).

The overall fold and secondary structures are as expected similar to other LCs, cleaving the same substrates. Comparing the structural folds of LC/X with other LCs reveal low root mean square deviations (rmsd) values between 2.3 to 2.9 Å. The flexible areas are observed in loop regions, representing parts of the surface. Structure elements

34 surrounding the active site show higher homology in the fold and therefore seem conserved. A loop present on the surface, consisting of residues 195-217, diverges between serotypes and this might be to enable interactions with the rest of the protein. This type of interaction has been seen in other serotypes (154). The molecular structure of LC/X (1.35 Å) shares the conserved binding motif, HEXXH and the overall organization of the catalytic site as well as being a Zn2+ dependent endopeptidase, with other serotypes. Three charged residues located in the catalytic pocket, His227, Glu228 and His231, are together coordinating the Zn2+ (Figure 19). In the 2.4 Å LC/X structure no Zn2+ is present in the active site, which rule out proteolytic activity. The overall arrangement of the catalytic site is maintained despite lacking the Zn2+ crucial for enzymatic activity. Comparing the active site of LC/X with the overall arrangement of other serotypes, reveal a more restricted access to the catalytic pocket. Inside the catalytic pocket residues 235- 242 in LC/X, are aligned to bind to certain element on the substrate and together they are constituting a so called S subpocket. The residues binding to the N-terminal of the substrate is denoted with S, while residues binding to the C-terminal of the substrate is marked S´ (287). S subpockets are present in other serotypes and seem to aid in recognizing and binding the substrate, enabling proteolysis at specific peptide bonds (288). The S subpocket and the S´ subpocket are coordinating the substrate by interacting with each of the two ends of the substrate. The S subpocket in LC/X is not conserved while the S´ subpocket displays conserved residues between serotypes. Arg360 and Tyr363 are two prominent and well conserved residues being a part of the S´ subpocket. Mutational studies of BoNT/A have shown that these residues are important for the activity and based on that knowledge, they are most likely crucial for the activity of BoNT/X as well (289). One can hypothesize that the conserved S´ subpocket is more robust in substrate recognition, while S subpocket is evolving to eventually accept changes in the substrate. This might be a part of an evolutionary development towards recognizing different substrates. Except from the common BoNT substrates, VAMP1, 2 and 3, LC/X additionally and surprisingly cleaves VAMP4, 5 and Ykt6. Those are completely new substrates for the BoNT family (278). VAMP1, 2 and 3 in LC/X share cleavage sites and a peptide bond between Arg and Ala is hydrolyzed. VAMP4 and Ykt6 share cleavage sites with each other and are hydrolyzed between Lys and Ser, while VAMP5 is cleaved between Arg and Ala. When a BoNT/F bound inhibitor, structurally based on VAMP (PDB ID: 3FIE), is superimposed with LC/X (PDB ID: 6F47), one can clearly see how the substrate is lining the surface like a belt around the LC/X, with a starting point in the active site (Paper III, Figure 5A) (271). Three new substrates are presented as unique to BoNT/X. In addition, the cleavage sites in VAMP1, 2 and 3 are completely new (2). The efficiency of the LC/X enzymatic activity compared to LC/B and LC/Tetanus (LC/Te) is significantly higher when hydrolyzing VAMP1 (290). The less accessible active site in LC/X does not conflict with its activity and LC/X shows a higher turnover number than both LC/B and LC/Te.

35

Paper IV – pH dependence of the minimal progenitor complex of Botulinum neurotoxin X

All bont genes known are expressed in progenitor complexes (156, 205, 215). The M-PTC of serotype X contain BoNT/X and NTNH and are together ~300 kDa. Free BoNT is fragile and susceptible to low pH or peptidases. Formation of a complex with NTNH, protects BoNT from being destroyed in unfavorable environments. (181). In the M-PTC, the surfaces of the HC of both proteins are facing each other in something that looks like a hug (Figure 20A). It is easy to understand that residues displayed on those surfaces will affect each other. NTNH and BoNT are only 20 % sequence homologous and do not share functions. The overall arrangement of the domains are conserved between the two proteins (Figure 20B) (181).

(A) (B)

Figure 20. BoNT/A (deep purple) and NTNH (light pink) (PDB ID: 3V0B). (A) The M-PTC of BoNT/A and NTNH. (B) BoNT/A and NTNH in an overlay picture showing the overall domain similarities. Modified pictures (181).

BoNT M-PTCs of serotype A and E have shown to assemble and disassemble using a mechanism, dependent on pH. In low pH the M-PTCs are forming stable complexes. High or neutral pH enables the release of BoNT/A and E as free molecules, for example upon entering the blood stream. Many interactions are broken when free BoNT is leaving the GI-tract. Studies have shown that there are clusters of pH-dependent residues displayed on both BoNT/A and E and their corresponding NTNH surfaces, which are facing each other (181, 215). The new BoNT serotype is here investigated to elucidate if the M-PTC is staying together or not, in the same pH dependent manner as the M-PTCs of serotype A and E (181, 215). The pH-sensitive residues found in HC of BoNT/A are electrostatically interacting with pH-sensitive residues in HC of NTNH. The pH-sensitive residues in BoNT/A are Glu982, Asp1037, Asp1118 and Asp1171 and they are surrounded by positive electrostatic potential. Mutational studies reveal them as crucial in the formation of M-PTC of serotype A. The pH-sensitive residues in NTNH are surrounded by negative electrostatic potential. When the pH is neutral or high, the positive electrostatic net charge of BoNT/A is changed to negative. The pH-sensitive residues are most likely deprotonated and repel the

36 negatively charged residues on NTNH. By doing so, the M-PTC dissociates into free molecules of BoNT and NTNH (181). Upon a change in pH, the pH-sensitive residues change their charges and their protonation state and hence a conformational change is induced. The electrostatic interactions between BoNT/A and NTNH are deteriorated (215). The study of BoNT/A in complex with NTNH shows that the M-PTC is associated at pH 6.0 and disassociated at pH 7.5 (181). Similar results have been shown for BoNT/E (215). Other studies have shown that the pKa increases when BoNT/A and NTNH form a complex, which leads to protonation of the pH-sensitive residues Glu982 and Asp1037, displayed on BoNT/A (181, 291). To elucidate if the pH-dependent mechanism is applicable to BoNT/X and NTNH, both plasmids were co-transformed to be expressed. Two different vectors holding their DNA, were co-transformed into E. coli strain BL21(DE3). Both proteins expressed well and the following purifications were done in several steps. To encourage BoNT/X and NTNH to form and stay in complexes, the buffers in the first purification steps held a pH value of 5.5. Both BoNT/A and BoNT/E were in stable complexes at pH 6.0 (205, 215). In the first purification step the His6-tag of BoNT/X came to use. Ni-NTA agarose were prepared and equilibrated with a pH 5.5 buffer. The protein solution was incubated with Ni-NTA to enable the His6-tags to bind to the Ni2+ in the solid phase. The solid phase was cleansed from other proteins by washing with imidazole. Elution was done with an increasing concentration of imidazole. The purified protein batch was used in upcoming purification steps and was dialyzed over night to change buffer to gel filtration buffer, still at pH 5.5. The obtained protein batch was run on a Superdex-200 26/60 column, where the resin allows separation by molecular size under isocratic elution. The chromatogram reveals what peaks and which fractions contain the protein of interest. Those fractions were pooled together and used as starting material for the last step in this experiment. Superdex-200 16/60 columns were used for the analytical size exclusion liquid chromatography runs, exploring varying pH-values. Based on earlier pH-dependence studies of M-PTC of serotypes A and E, pH 5.5, pH 6.5 and pH 7.5 were chosen pH-values (205, 215).

Figure 21. Overlay chromatograms from BoNT/X and NTNH in pH 5.5 (grey), in pH 6.5 (blue) and pH 7.5 (orange).

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At pH 5.5 a clear peak is eluted where the M-PTC is expected to (Figure 21). SDS-PAGE confirms the presence of a complex. These results agree with the pH dependence in serotype BoNT/A and E (181, 215). Following the first peak in pH 5.5, a smaller peak is eluted containing monomers of NTNH. During expression an excess of NTNH was most likely produced and these remaining monomers, not busy forming a complex with BoNT/X, are probably the ones visible here. The peak is less obvious in pH 6.5. The same peak is broader and seems to include a second peak. The first and highest peak in pH 6.5 consist of the M-PTC. The second peak in pH 6.5 is higher compared to in pH 5.5, indicating a higher amount of monomers. SDS- PAGE confirms the presence of the M-PTC in the first peak and in the second peak mostly NTNH monomers, but also a small amount of BoNT/X monomers are confirmed. These results indicate that the complex is under disassociation in pH 6.5 and this does not conflict with similar studies of BoNT/A and E where the complex is stable in pH 6.0 and dissociated in pH 7.5 (181, 215). When eluting BoNT/X and NTNH in pH 7.5 a clear peak is eluted where the elution profile indicates the size of monomers. The presence of both NTNH and BoNT/X monomers are confirmed with SDS-PAGE and underline the results achieved for BoNT/A and E (205, 215). The pH-dependence in the formation of the M-PTCs in other BoNT serotypes, is hereby confirmed applicable to the novel BoNT/X. Studies have revealed certain residues with importance regarding the pH-sensitive complex formation in BoNT/A and E (181, 215). The appointed residues found on the HC of BoNT/A are Glu982, Asp1037, Asp1118 and Asp1171 and they interact electrostatically with Asp808, Glu810, Asp455 and Asp1110 on the HC of NTNH. Mutagenesis studies confirm their importance in the pH-dependence (181). To see if they are conserved between serotypes the sequences of BoNT/A, E and X were aligned (Paper IV, Supplementary Figure 1). Alignments between NTNHs from serotype A, E and X were also performed (Paper IV, Supplementary Figure 2). The results reveal that some of these residues are fully conserved between serotypes. BoNT display one fully conserved residue, while NTNH has two fully conserved residues. Residues with similar features are aligned in some of the sites. The results reveal that NTNHs are slightly more conserved between serotypes than BoNT/A, E and X are. Asp1171 on BoNT/A and Asp1110 on NTNH are interacting with each other. They are conserved and are localized in flexible loops, which increase the steric flexibility. To elucidate how BoNT/X and NTNH actually interact with each other, structural studies of the M-PTC is needed. Only when the 3D molecular structure is known it is possible to understand what residues that are interacting with each other.

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Acknowledgements

I wish to thank Pål Stenmark for not once, but twice, accepting the important role as my supervisor during my PhD studies. You are a great scientist and you have been a true inspiration to me. Your dedication and your involvement and the passion that sparkles in your eyes when you talk about science or see a crystal have been a great driving force to me. Thank you for being a role model showing that our families is the most important thing in our lives, even though science is close behind. Thank you for taking good care of me and calming me down when the heat was on. You never once allowed me to believe that I could not do this. I would also like to thank my co-supervisor Martin Högbom for all the good advice you have shared during my time at DBB and thank you for spreading so much joy around you. My mentor Peter Brzezinski, thank you for being such an inviting and friendly person. I have always felt welcome to you with potential issues and questions. Thank you Stefan Nordlund, Peter Brzezinski and Martin Ott for setting high standards and for failing me in the Oral Exam and thank you Pia Ädelroth, Lena Mäler and Stefan Nordlund for passing me. During that exhausting and demanding period I learnt a lot. Thanks to all of you I am a better biochemist. I have had the pleasure of working with fantastic scientists outside DBB and wish to thank Tomas Helleday, Min Dong, Göran Widmalm and their research groups for great collaborations. A special thanks to Ann-Sofie Jemth for the nice talks, both scientific and personal. I wish to thank the DBB Administration for their great work. Especially I would like to thank Maria and Alex for their admirable patience and for always being friendly and helpful. “My” first Post doc. Ronnie, thank you for always taking the time to help me and for never making me feel stupid when I asked questions. You are an excellent scientist and a very friendly and warm person. Now you have your own group and its great for you. I wish you and Josy the best. “My” second Post doc. Geoffrey, thank you for being such a kind and helpful person. You are generous with your smart scientific advice and with your time. Your patience is admirable. You are a very skilled scientist and I am convinced that you can reach the stars. I wish you and Sarah the best. StenHögers, there have been so many of us during the years I will not even try to remember all persons. The early StenHög crew, thank you for filling the lab with laughter, music, pranks and the color pink. I have so many memories together with you and you taught me that “sharing is caring”. I will forever cherish this time of my life. The present StenHög crew, I wish to thank each and every one of you for always being helpful, friendly, humble and generous with time, patience and scientific advice. We have a great lab filled with great people and we do great science. Keep it up and never stop having Friday fikas. I would especially like to thank my desk neighbour Robert, for managing the MTH1 project in such a fantastic way. You have made the difference. You are an inspiration, a fantastic scientist and a great friend. I wish you and Elisabeth the best. Dan, we first met in the end of the 90´s and then we luckily ended up sharing an office and a lab. Thank you for reminding me about my Hälsingland heritage, for always being caring and for the talks about life and our important families. Thank you Robert, Geoffrey and Emma for careful reading of my thesis and for accurate and humble suggestions to improvements. Thank you to my former biomedicine class mates from my time at KI, my past and present friends and colleagues outside and within the area of science from Söderhamn, Upplands Väsby, Stockholm, Eskilstuna, SGC, KI, DBB and Perrigo (Omega Pharma) for lightening up my life with inspiring discussions, dinners, laughter’s, fikas, great lab work, nice working places, wine of course and for making my life meaningful and filled with joy. Especially I would like to thank Perrigo (Omega Pharma) who generously gave me three years leave and the opportunity to finish my PhD. I am forever thankful. Susanne F., tack för att du kom in i mitt liv och blev min vän när jag verkligen behövde en. Vi kommer alltid att finnas för varandra. Tack, för att du inte doktorerade. Utan dig hade jag inte skrivit den här boken. Anki, din vänskap betyder mycket för mig. Tack, för att du gör mig lugn och glad med ditt smittande skratt och ditt värdiga sätt. Berit, i dig har jag mött en kvinna som ständigt visar mig hur jag vill leva. Tack, för att du inspirerar mig och tack, för att du aldrig slutade att tro på mig. Johanna och Josephine, jag värdesätter vår vänskap mycket. Tack, för alla gemensamma studietimmar och allt stöd genom åren. Att studera med er gjorde det så mycket roligare. Tack till mina närmsta och älskade Familjemedlemmar, för att ni skapar den inkluderande, varma och kärleksfulla atmosfär som finns i vår bullriga och kaotiska familj. Jag älskar att vara en del av Den. Tack Amanda och Matilda, för att ni med er energi och livslust är en sådan källa till inspiration. Tack Sofia, för att du är så generös med din kärlek och omtanke. Ni är fantastiska på det ni gör så fortsätt att vara envisa, målmedvetna, tuffa och grymma. Jag är stolt över att vara er storasyster. Tack Mamma, för att du fortfarande alltid har tid för mig och för att du ger mig kloka råd och uppmuntrande ord när jag behöver. Tack, för ditt härliga skratt, din förmåga att lyssna, din generositet och ditt pragmatiska sätt att lösa problem eller att se det positiva i något negativt. Det gör dig till en fantastisk person som har hjälpt mig och andra så många gånger. Kärlek och respekt. Tack Magnus, för att du så ihärdigt uppmuntrade mig att återuppta studierna och tack, för ditt outtröttliga stöd under min doktorandtid. Du har en fantastiska förmåga att omvandla ord till handling och det, tillsammans med dina kreativa, generösa och pragmatiska lösningar har hjälpt mig igenom det här. Du har funnits hos mig med te, uppmuntrande ord, data-support, förståelse när det har varit tufft och en varm famn när jag har behövt. Din omtänksamhet har givit mig energi och du har varit stabil när det har stormat. Jag kunde inte ha bett om mer. Vi är så jäkla bra tillsammans och jag älskar dig så mycket. Mina kära döttrar Tea och Ida, ni är de underbaraste och roligaste personer jag känner och ni får mig jämt att skratta och på bra humör. Tack, för att ni låter mig ta del av era liv, tankar och funderingar. Jag älskar er så otroligt mycket och det gör mig lycklig och stolt över att se vilka fina personer ni har blivit. I mer än halva ert liv har jag studerat och ni har aldrig varit annat än stöttande, förstående eller uttryckt er stolthet. Det har betytt allt för mig. Tack, för att ni lät mig uppfylla min dröm.

Pappa och Mormor, Ni är saknade.