Modelling the structure and interactions of leukocyte integrins Kyle-Richard Dawson Submitted in fulfilment of the requirements for the degree of Magister Scientiae: Biochemistry (Msc) in the Faculty of Science at the Nelson Mandela University 9 April 2019 Prof Vaughan Oosthuizen i Declaration I know that plagiarism is wrong. Plagiarism is to use another’s work and pretend that it is one’s own. I have used the Bioinformatics convention for citation and referencing. Each contribution to, and quotation in, this dissertation from the work(s) of other people has been attributed, and has been cited and referenced. This dissertation is my own work. This work has not been submitted to any institution other than Nelson Mandela University. I have not allowed, and will not allow, anyone to copy my work with the intention of passing it off as his or her own work. Signature ______________________________ Date __________________________________ ii Acknowledgements I would like to express my sincere gratitude and appreciation to: My supervisor, Prof V Oosthuizen, for his positive attitude and guidance and the National Research Foundation (NRF) for financial assistance. I would also like to thank Dr R Hatherley, Dr Vuyani Moses and Prof O Bishop for their guidance in the field of Bioinformatics. I would also like to thank the following; Abigail Sephton, Liza Findt, Eugen Schnautz, Jan Batelka, Travis Dugmore, Martin Dorfling and Blake Callahan for the encouragement, occasional idea and helpful assistance in maintaining my composure. iii Contents Abbreviations and acronyms ................................................................................................................ vii List of Figures ......................................................................................................................................... ix List of Tables .......................................................................................................................................... xi Abstract ................................................................................................................................................... 1 1. Integrin proteins ............................................................................................................................. 2 1.1. Structure ................................................................................................................................. 2 1.2. Activation and regulation ........................................................................................................ 5 1.3. Functions ................................................................................................................................. 6 1.3.1. Cell signalling ................................................................................................................... 6 1.3.2. Cell survival ........................................................................................................................... 7 1.3.3. Cell proliferation ................................................................................................................... 7 1.3.4. Cell differentiation ................................................................................................................ 9 1.3.5. Leukocyte recruitment, activation and adhesion ................................................................. 9 1.3.6. Integrin ligands .................................................................................................................... 11 2. Homology modelling ..................................................................................................................... 13 2.1. Comparative homology modelling overview ........................................................................ 14 2.1.1. Template searching ....................................................................................................... 14 2.1.2. Selecting templates ....................................................................................................... 16 2.1.3. Sequence alignment ...................................................................................................... 17 2.1.4. Model construction ....................................................................................................... 20 2.2. Modelling programs .............................................................................................................. 23 2.2.1. MODELLER ..................................................................................................................... 23 2.2.2. MEDELLER ..................................................................................................................... 23 2.2.3. PRIMO ........................................................................................................................... 25 2.2.4. Phyre2 ........................................................................................................................... 25 2.2.5. I-TASSER ........................................................................................................................ 27 2.3. Errors in homology modelling ............................................................................................... 28 2.4. Determining model accuracy ................................................................................................ 28 2.4.1. PROSA............................................................................................................................ 28 2.4.2. Verify 3D ........................................................................................................................ 29 2.4.3. PROSESS ........................................................................................................................ 29 2.5. Docking programs ................................................................................................................. 31 2.5.1. HADDOCK2.2 ................................................................................................................. 31 2.5.2. CLUSPRO ....................................................................................................................... 33 2.5.3. AutoDock Vina ............................................................................................................... 35 iv 3. Problem statement, approach and objectives for this study ....................................................... 37 4. Methods ........................................................................................................................................ 41 4.1. Obtaining target sequences .................................................................................................. 41 4.2. Modelling using online servers ............................................................................................. 41 4.2.1. PRIMO ........................................................................................................................... 42 4.2.2. I-TASSER and Phyre2 ..................................................................................................... 42 4.2.3. MEDELLER ..................................................................................................................... 42 4.3. MODELLER ............................................................................................................................. 42 4.3.1. Generating alignment files, PDB files and python scripts ............................................. 43 4.3.2. Modelling the complete protein ................................................................................... 43 4.3.3. Determining optimal model iterations ......................................................................... 43 4.3.4. Determining optimal refinement iterations ................................................................. 44 4.3.5. Determining optimal template numbers ...................................................................... 44 4.3.6. Very_slow against slow_large refinement .................................................................... 44 4.4. Modelling monomeric subunits ............................................................................................ 44 4.4.1. Initial modelling ............................................................................................................ 44 4.4.2. Separating “closed” and “open” templates .................................................................. 45 4.4.3. Modelling with secondary structure arguments .......................................................... 45 4.4.4. Modelling with forced transmembrane regions ........................................................... 45 4.4.5. Fragmented integrin modelling .................................................................................... 45 4.5. Final modelling ...................................................................................................................... 45 4.5.1. BLASTp........................................................................................................................... 46 4.5.2. Separation of “closed” and “open” templates ............................................................. 46 4.5.3. Preparing multiple runs under varying template numbers .......................................... 46 4.5.4. Generating alignment files ...........................................................................................