Structural Studies of C9orf72-SMCR8-WDR41 Protein Complex Valeria Shkuratova Department of Biochemistry McGill University, Montreal A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Valeria Shkuratova, 2020 Table of Contents Abstract ............................................................................................................................................ 3 Résumé ............................................................................................................................................ 4 Acknowledgment ............................................................................................................................. 5 Author Contribution ........................................................................................................................ 6 List of Abbreviations ....................................................................................................................... 7 List of Figures .................................................................................................................................. 9 List of Tables ................................................................................................................................... 9 Introduction ................................................................................................................................... 10 1. Amyotrophic Lateral Sclerosis (ALS) ............................................................................... 10 2. Frontotemporal Dementia (FTD) ...................................................................................... 13 3. C9orf72 gene mutation ...................................................................................................... 15 4. Cellular functions of C9orf72 protein and formation of CSW complex ........................... 18 5. CSW structure. .................................................................................................................. 20 6. Project goals ...................................................................................................................... 21 Results ........................................................................................................................................... 22 1. Purification optimization ................................................................................................... 22 1.1. Optimization of expression and purification of His-tagged constructs ..................... 22 1.2. Optimization of expression and purification of GFP-tagged constructs ................... 26 2. C9orf72-SMCR8-WDR41 complex forms a trimer .......................................................... 28 3. Initial crystallization trials for CSW and CS complexes ................................................... 29 4. HDX-MS analysis for CS and CSW ................................................................................. 30 5. CSW unstructured regions are essential for the formation of the complex ....................... 38 1 6. Crystallization trials for CSW mutants .............................................................................. 41 Discussion ...................................................................................................................................... 42 Materials and Methods .................................................................................................................. 47 1. Protein constructs .............................................................................................................. 47 2. Cloning into pFastBac ....................................................................................................... 47 2.1. Restriction enzyme digestion .................................................................................... 47 2.2. Ligation and transformation ...................................................................................... 48 3. Protein expression in Sf9 insect cells ................................................................................ 48 4. Protein purification with Ni-affinity beads. ....................................................................... 48 5. Expression and purification of GFPnb .............................................................................. 49 6. Preparation of GFPnb-coupled beads ................................................................................ 50 7. Generation of GFP tagged constructs ................................................................................ 50 7.1. C9orf72-SMCR8 construct ........................................................................................ 50 7.2. C9orf72-SMCR8-WDR41 construct ......................................................................... 51 8. Protein purification with GFPnb-coupled beads ............................................................... 52 9. Sedimentation-Velocity Analytical Ultracentrifugation (SV-AUC) ................................. 53 10. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) ................................ 53 11. Generation of deletion mutants for CSW complex. ...................................................... 54 References ..................................................................................................................................... 55 2 Abstract Hexanucleotide repeat expansions in the C9orf72 gene are the leading cause for the development of two neurodegenerative diseases: amyotrophic lateral sclerosis and frontotemporal dementia. Previous studies have identified that C9orf72 binds SMCR8 and WDR41 to form a stable complex, which was shown to be important during autophagy in neuronal cells. However, the exact functions of the complex and its structural features are mainly unknown. Here we established a purification protocol for C9orf72-SMCR8-WDR41 protein complex. We constructed a single plasmid containing all three proteins for efficient expression in Sf9 insect cells. The two- step purification using GFP-nanobody-coupled beads was optimized to yield high quantities of pure trimeric complex (up to 7.5 mg for 1 L of cell culture). Crystallization trials were unsuccessful, suggesting a requirement for other techniques such as cryoEM for solving the complex structure. Studies using HDX-MS revealed that WDR41 binds to SMCR8 DENN domain but not to C9orf72. However, the conformational changes occur in both SMCR8 and C9orf72. Additionally, we identified unstructured regions within the SMCR8 linker region and WDR41 that could be important for complex assembly. All these results are in agreement with the recently published structure of the C9orf72-SMCR8-WDR41 complex. 3 Résumé Les expansions répétées hexanucléotidiques du gène C9orf72 sont la principale cause du développement de deux maladies neurodégénératives: la sclérose latérale amyotrophique et la démence fronto-temporale. Des études antérieures ont montré que C9orf72 se lie à SMCR8 et WDR41 pour former un complexe stable. Ce complexe s’est révélé important lors de l’autophagie dans les cellules neuronales. Cependant, les fonctions exactes du complexe et ses caractéristiques structurelles sont principalement inconnues. Ici, nous avons établi un protocole de purification pour le complexe protéique C9orf72-SMCR8-WDR41. Nous avons construit un seul plasmide contenant les trois protéines pour une expression efficace dans les cellules d'insectes Sf9. La purification en deux étapes à l'aide de billes couplées à la nanoparticule GFP a été optimisée pour maximiser le rendement du complexe trimérique pur (jusqu’à 7,5 mg pour 1 L de culture cellulaire). Les essais de cristallisation menés ont échoué, suggérant une exigence pour d'autres techniques tels que cryoEM pour résoudre la structure du complexe. Des études utilisant HDX- MS ont révélé que WDR41 se lie au domaine DENN du SMCR8 mais pas à C9orf72. Cependant, les changements de conformation se produisent à la fois dans SMCR8 et C9orf72. De plus, nous avons identifié des régions non structurées au sein dans la région de liaison SMCR8 et WDR41 qui peuvent être importantes pour l'assemblage du complexe. Tous ces résultats sont en accord avec la structure du complexe C9orf72-SMCR8-WDR41 récemment publiée. 4 Acknowledgment I wish to express my gratitude to my supervisor Dr. Kalle Gehring for accepting me into his lab and allowing me to work on this new exciting project. His constant involvement and immense knowledge and valuable suggestions helped to drive my project forward. I would like to thank Dr. Guennadi Kozlov for training me when I joined the lab as a master’s student and for guiding me throughout the project. I would like to thank Katalin Kocsis Illes for joining me on the project and helping with insect cell cultures and virus generation. I also wish to thank my research advisory committee members Dr. Peter McPherson and Dr. Bhushan Nagar for their valuable insights and guidance along the way. A special thank you to George Sung, Seby Chan, Rayan Fakih for their support, guidance and help. I am also grateful to all past and present Gehring’s lab members for their warm welcome and support during these years. I am pleased that I had the opportunity to know them and to conduct my research in such a friendly environment. I would like to acknowledge our collaborators on this project: Dr. John Burke and Brandon Moeller for carrying out HDX-MS experiments. I am also extremely