Synthesis and investigation of bacterial effector molecules Michael Franz Albers Doctoral Thesis, Department of Chemistry Umeå University, 2016 Responsible publisher under swedish law: the Dean of the Faculty of Science and Technology This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-411-0 Electronic version available at http://umu.diva-portal.org/ Tryck/Printed by: VMC-KBC Umeå Umeå, Sweden, 2016 Table of Contents Table of Contents i Abstract iii List of Abbreviations iv List of Publications vii Author contributions viii Papers by the author, but not included in this thesis viii Enkel sammanfattning på svenska ix Introduction 1 Post-translational modifications 1 Nucleotidylylation and phosphocholination 2 Small GTPases 7 Pathogens modify host cells at a molecular level 9 Quorum sensing in Legionella pneumophila 12 Proteomics towards PTMs 13 Chapter 1: Towards the identification of adenylylated proteins and adenylylation-modifying enzymes (Paper I – III) 19 Previous work 19 Outline: From building blocks to antibodies 22 Synthesis of a tyrosine-AMP building block 23 Synthesis of a threonine- and serine-AMP building block 26 Synthesis of adenylylated Peptides 28 Generation of AMP specific antibodies 30 Mass fragmentation patterns of adenylylated peptides 37 Immunoprecipitation of adenylylated proteins 42 Non-hydrolysable mimics for the study of deadenylylating enzymes 47 Future work 52 Ongoing Work – Covalent trapping of substrates of adenylyl transferases 53 Conclusions 60 Chapter 2: Tools for the investigation of phosphocholination (Paper IV – V) 61 Outline 61 Synthesis of PC building blocks 61 Synthesis of phosphocholinated peptides 63 Site-directed chemoenzymatic labelling 64 Future work 65 Conclusions 66 Chapter 3: Small molecule signalling of Legionella pneumophila (Paper VI – VII) 67 Introduction 67 Synthesis of LAI-1 and Am-LAI-1 67 i LAI-1 regulates Lqs-dependent signalling 69 Inter-kingdom signalling 72 Future work 74 Conclusions 76 Acknowledgements 77 References 79 Appendix 93 Chapter 1: General procedure for immunoprecipitation experiments 93 Chapter 1: Synthesis of non-hydrolysable mimics of adenylylated motifs 97 Chapter 1: Organic synthesis of ATP and NAD+ derivatives 101 Chapter 3: Organic synthesis of LAI-1 derivatives 109 ii Abstract During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. ATP-derivatives containing an electrophilic trap were used in conjunction with modified adenylyl transferases, to isolate and analyse the covalent complex between enzyme and protein substrate. Furthermore, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino- derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42. iii iv List of Abbreviations A adenine aa amino acid ab antibody ABC ammonium bicarbonate ADP adenosine diphosphate AMP adenosine monophosphate ARTD diphtheria toxin-like ADP-ribosyl transferase Atase adenylyl transferase ATP adenosine triphosphate BCA bicinchoninic acid BSA bovine serum albumin BTT 5-benzylthio-1H-tetrazole Bz benzoyl CAI-1 Cholerae autoinducer-1 cAMP cyclic adenosine monophosphate CDP cytidine diphosphate CID collision induced dissociation CMP cytidine monophosphate CoA Coenzyme A DAPI 4’,6-diamidino-2-phenylindole DCM dichloromethane DEAE diethylethanolamine DIAD diisopropyl azodicarboxylate DMAD dimethyl azodicarboxylate DMF dimethyl formamide DNA deoxyribonucleic acid DIPEA diisopropyl ethyl amine DTT dithiothreitol EDTA ethylene diamine tetraacetate eq equivalent ER endoplasmic reticulum ETD electron transfer dissociation FACE filter aided antibody capturing and elution FASP filter assisted sample preparation Fic filamentation induced by cAMP GAP GTPase activating protein GDI GTP dissociation inhibitor GDP guanosine diphosphate GEF guanosine nucleotide exchange factor GFP green fluorescent protein v GlcNAc N-acetylglucosamine GPI glycosylphosphatidylinositol GS glutamine synthetase GTP guanosine triphosphate HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HCD high-energy collisional dissociation HOAt 1-hydroxy-7-azabenzotriazole HOBt hydroxybenzotriazole HPLC high-performance liquid chromatography HRMS high-resolution mass spectrometry HRP horseradish peroxidase HYPE Huntingtin interacting protein E IEF iso-electronic focussing IMAC immobilised metal-ion affinity chromatography IMPRS International Max Planck Research School IP immunoprecipitation KLH keyhole limpet hemocyanin KNT kanamycin nucleotidylyl transferase LAI-1 Legionella autoinducer 1 LAMP lysosome-associated membrane glycoprotein LCMS liquid chromatography-mass spectrometry LCV Legionella-containing vacuole Lqs Legionella quorum sensing MBS 3-maleimidobenzoyl-N-hydroxysuccinimide ester MICAL molecules interacting with CasL MS mass spectrometry MWCO molecular weight cut-off NAD+ nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide, reduced nhAd non-hydrolysable adenosyl sulfonamide NHS N-hydroxysuccinimide NMN nicotinamide mononucleotide NMR nuclear magnetic resonance P4M phosphatidylinositol-4-phosphate PAC polyaminocarboxylate PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PC phosphocholine PEG polyethylene glycol PG protective group vi Pi inorganic phosphate POI protein of interest PPM metal-dependent protein phosphatase PTM post-translational modification PVDF polyvinylidene fluoride PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Q-TOF quadrupole-time-of-flight RIPA radioimmunoprecipitation assay Rf retention factor RNA ribonucleic acid rt room temperature SAB (S)-2-amino-butyrate SDS sodium dodecyl sulfate SEC size-exclusion chromatography SG side chain group SPPS solid-phase peptide synthesis STAGE stop-and-go extraction TBAF tetra-n-butylammonium fluoride TBDPS tert-butyldiphenylsilyl TBHP tert-butylhydroperoxide TBME tert-butylmethyl ether TBS tris buffered saline TCA trichloroacetic acid TCEP tris(2-carboxyethyl)phosphine TFA trifluoroacetic acid THF tetrahydrofuran TIPS triisopropylsilane TLC thin-layer chromatography TMP trimethyl phosphate tRNA transfer RNA UTP uridine triphosphate UV ultraviolet vii viii List of Publications The following publications form the basis of this dissertation. In text, the publications are referred to by use of the corresponding roman numbers. I. Smit, C., Blümer J., Eerland, M. F., Albers, M. F., Müller, M. P., Goody, R. S., Itzen, A., & Hedberg, C. Efficient synthesis and applications of peptides containing adenylylated tyrosine residues. Angew. Chem. Int. Ed. 2011, 50, 9200–9204. II. Albers, M. F., van Vliet, B. & Hedberg, C. Amino acid building blocks for efficient Fmoc solid-phase synthesis of peptides adenylylated at serine or threonine. Org. Lett. 2011, 13, 6014–7. III. Hansen, T., Albers, M., Hedberg, C. & Sickmann, A. Adenylylation, MS, and proteomics - Introducing a ‘new’ modification to bottom-up proteomics. Proteomics 2013, 13, 955–963. IV. Albers, M. F. & Hedberg, C. Amino acid building blocks for Fmoc solid-phase synthesis of peptides phosphocholinated at serine, threonine,