TECHNISCHE UNIVERSITÄT MÜNCHEN Fakultät für Chemie Lehrstuhl für Biochemie Structural and biochemical characterization of the YaxAB pore- forming toxin from Yersinia enterocolitica Bastian Loong Wang Yung Shan Bräuning Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzende/-r: Prof. Dr. Matthias Feige Prüfende/-r der Dissertation: 1. Prof. Dr. Michael Groll 2. Prof. Dr. Franz Hagn 3. Prof. Dr. Michal Sharon (schriftliche Begutachtung), 4. Prof. Dr. Tanja Gulder (mündliche Prüfung) Die Dissertation wurde am 09.04.2018 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 23.05.2018 angenommen. Table of contents Chapter 1 ...................................................................................................................................................................... 11 Introduction ............................................................................................................................................................. 11 1.1 The enterobacteriaceae: successful pathogens with a broad host spectrum .................... 11 1.1.1 The human pathogen Yersinia enterocolitica ........................................................................ 12 1.1.2 The insect pathogen Photorhabdus luminescens .................................................................. 12 1.2 Pore-forming toxins (PFT) as virulence factors of enteropathogenic bacteria from the genera Enterobacteriaceae and Bacillus ...................................................................................................... 13 1.3 Structure and mechanism of PFTs ..................................................................................................... 15 1.3.1 Homomultimeric β-PFT ................................................................................................................. 16 1.3.2 Homomultimeric α-PFTs ............................................................................................................... 18 1.3.3 Heteromultimeric PFT.................................................................................................................... 20 1.4 The XaxAB family of heteromultimeric α-PFTs ............................................................................. 22 Bibliography ............................................................................................................................................................ 24 Chapter 2 ...................................................................................................................................................................... 31 Objectives.................................................................................................................................................................. 31 Chapter 3 ...................................................................................................................................................................... 32 Materials & Methods ............................................................................................................................................ 32 3.1 Materials ....................................................................................................................................................... 32 3.1.1 Standard laboratory chemicals ................................................................................................... 32 3.1.2 Material for molecular cloning and expression vectors .................................................... 32 3.1.3 Protein and DNA standards .......................................................................................................... 36 3.1.4 Bacterial strains and growth media .......................................................................................... 36 1 3.1.5 FPLC chromatography ................................................................................................................... 38 3.1.6 Protein crystallization .................................................................................................................... 38 3.1.7 Software ............................................................................................................................................... 39 3.2 General biochemical techniques ......................................................................................................... 39 3.2.1 Molecular cloning ............................................................................................................................. 39 3.2.2 Agarose gel electrophoresis ......................................................................................................... 42 3.2.3 DNA isolation and PCR clean-up ................................................................................................ 42 3.2.4 SDS-PAGE analysis ........................................................................................................................... 42 3.2.5 Determination of protein concentration ................................................................................. 44 3.3 Recombinant toxin expression and purification .......................................................................... 44 3.4 Protein crystallization............................................................................................................................. 45 3.5 X-ray data collection and structure determination ..................................................................... 46 3.6 Reconstitution and purification of YaxAB pores from human erythrocyte membranes .............................................................................................................................................................. 47 3.7 Negative-stain EM data acquisition and image processing ...................................................... 48 3.8 Preparation of the detergent-treated YaxAB complex for cryo-EM ...................................... 49 3.9 Cryo-EM: sample vitrification and data acquisition .................................................................... 49 3.10 Cryo-EM: image processing .................................................................................................................. 50 3.11 Modelling the YaxAB pore into the cryo-EM density .................................................................. 50 3.12 Generation of figures ............................................................................................................................... 51 3.13 Liposome floatation assays ................................................................................................................... 51 3.14 Erythrocyte membrane co-sedimentation assay ......................................................................... 52 3.15 Hemolysis assays ...................................................................................................................................... 52 3.16 MBP-tag localization and negative-stain analysis ........................................................................ 53 3.17 Crosslinking/mass spectrometry ....................................................................................................... 53 2 3.18 Native mass spectrometry ..................................................................................................................... 54 3.19 Analytical ultracentrifugation .............................................................................................................. 54 3.20 Multiple sequence alignment ............................................................................................................... 55 Bibliography ............................................................................................................................................................ 56 Chapter 4 ...................................................................................................................................................................... 59 Results ........................................................................................................................................................................ 59 4.1 Statement of contributions ................................................................................................................... 60 4.2 Cloning, protein expression and purification................................................................................. 61 4.2.1 Cloning .................................................................................................................................................. 61 4.2.2 Protein expression and purification ......................................................................................... 61 4.3 Protein crystallization and structure determination .................................................................. 63 4.3.1 Protein crystallization .................................................................................................................... 63 4.3.2 Experimental phasing of YaxA and PaxB by selenium SAD ............................................. 64 4.4 Crystal structure of YaxA, YaxB and PaxB ....................................................................................... 66 4.5 Comparison