Structural insights into host cell adhesion by Toxoplasma gondii MIC4 by Ben Cowper Imperial College London Department of Life Sciences Submitted (2012) in partial fulfilment of the requirements for the degree of Doctorate of Philosophy Declaration I hereby declare that the research described in this thesis is the result of my own work, except where indicated in the text. Information derived from the published and unpublished work of others has been acknowledged and a list of references is provided. Ben Cowper 2 Abstract Toxoplasma gondii is a highly pervasive protozoan parasite, capable of infecting almost all mammals, including humans. Infection can have fatal consequences for immuno-suppressed individuals, whilst transmission to a developing fetus can induce a spontaneous abortion in pregnant females. T. gondii is perceived to be a model organism in the study of its phylum, Apicomplexa, which includes the Plasmodium species which cause malaria. Apicomplexans are obligate intracellular parasites, in which a strong host cell attachment is established through surface microneme proteins (MICs). Numerous MICs have been identified in T. gondii, many forming multi-adhesive complexes, such as TgMIC1/4/6, within which TgMIC4 is known to possess host cell binding activity within its C-terminal apple-5 & 6 (A56) domains. Prior to these studies, recombinant TgMIC4-A56 was produced yielding a partially-folded protein capable of binding to galactose. Herein the folded component has been identified as A5, and the solution structure of this domain has been solved via NMR spectroscopy. Carbohydrate microarray experiments have confirmed an ability to bind galactosyl-terminated oligosaccharides, whilst NMR and ITC experiments have enabled extensive characterisation of TgMIC4-A5 binding to a range of ligands. The domain binds particularly strongly to a pentasaccharide fragment from GM1 ganglioside; a potential in vivo receptor. Additionally, chemical shift perturbation data and intermolecular NOEs have been used to drive molecular docking of TgMIC4-A5 and lacto-N-biose (Galβ1→3GlcNac). The resulting structure suggests that the mechanism of galactose discrimination by TgMIC4-A5 is similar to that of other galactose-specific lectins. Combined with collaborating studies, this work aids our overall understanding of TgMIC4 function and encourages speculation as to the precise roles of the protein within T. gondii. In addition to a probable contributory role in the initial stages of host cell invasion, the protein may modulate events downstream of this process, through interactions with galactosylated receptors. 3 Table of Contents List of figures ............................................................................................................................... 9 List of tables .............................................................................................................................. 13 Acknowledgements .................................................................................................................... 15 Chapter 1: Biological Introduction .............................................................................................. 20 1.1. An introduction to Toxoplasma gondii ............................................................................................ 21 1.1.1. Identification and classification................................................................................................ 21 1.1.2. Life cycle ................................................................................................................................... 22 1.2. Toxoplasmosis ................................................................................................................................. 25 1.2.1. Disease transmission and consequences ................................................................................. 25 1.2.2. Disease prevalence and prevention ......................................................................................... 27 1.3. Apicomplexan host cell invasion ..................................................................................................... 30 1.3.1. Cell ultrastructure and morphology ......................................................................................... 30 1.3.2. The glideosome; a unique system for motility ........................................................................ 32 1.3.3. The moving junction ................................................................................................................. 34 1.3.4. The model of Apicomplexan host cell invasion ........................................................................ 34 1.3.5. Targeting host cell invasion ...................................................................................................... 37 1.4. Apicomplexan microneme proteins ................................................................................................ 37 1.4.1. Oligomeric microneme protein complexes ............................................................................. 39 1.4.2. Apple/PAN domains ................................................................................................................. 41 1.4.3. The TgMIC1/4/6 complex ......................................................................................................... 47 1.5. Project aims & objectives ................................................................................................................ 53 Chapter 2: An Introduction to Nuclear Magnetic Resonance (NMR) Spectroscopy ........................ 54 2.1. Introduction ..................................................................................................................................... 55 2.2. The origins of NMR signals .............................................................................................................. 55 2.2.1. Nuclear spin angular momentum and magnetism ................................................................... 55 2.2.2. Larmor precession and resonance ........................................................................................... 56 2.3. The excitation of NMR signals ......................................................................................................... 57 2.3.1. The vector model ..................................................................................................................... 58 2.3.2. The rotating frame ................................................................................................................... 58 2.3.3. The effect of a pulse ................................................................................................................. 59 4 2.3.4. Chemical shifts ......................................................................................................................... 60 2.3.5 Relaxation .................................................................................................................................. 62 2.3.6. NMR signal detection ............................................................................................................... 65 2.3.7. Putting it together; pulse-acquire NMR ................................................................................... 65 2.4. Nuclear coupling .............................................................................................................................. 66 2.4.1. Scalar coupling (and decoupling) ............................................................................................. 66 2.4.2. Dipolar coupling ....................................................................................................................... 67 2.5. The nuclear Overhauser effect (NOE) .............................................................................................. 67 2.6. Multi-dimensional NMR .................................................................................................................. 70 2.6.1. Two-dimensional techniques ................................................................................................... 70 2.6.2. Three-dimensional NMR .......................................................................................................... 75 2.7. Summary .......................................................................................................................................... 78 Chapter 3: Identification and production of a stable, adhesive fragment from TgMIC4-A56 .......... 79 3.1. Introduction ..................................................................................................................................... 80 3.2. Considerations for MIC protein production .................................................................................... 80 3.2.1. pET-32 Xa/LIC vector ................................................................................................................ 81 3.2.2. Origami cells ............................................................................................................................. 82 3.3. Materials & methods ....................................................................................................................... 82 3.3.1. Sequence analysis .................................................................................................................... 82 3.3.2. Gene cloning of TgMIC4-A5 and TgMIC4-A6 ...........................................................................