The unusual structure of the ubiquitin-like domain of the protein sacsin Harshit Pande Department of Biochemistry McGill University, Montreal April 2012 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science. © Harshit Pande, 2012 Table of Contents Acknowledgments 3 Abstract (English) 4 Résumé (French) 5 List of Abbreviations 6 List of Figures 9 List of Tables 11 Chapter I: Introduction 12 1.1 Autosomal Recessive Spastic Ataxia of Charlevoix Saguenay 12 1.2 ARSACS genetics 14 1.3 Biological and molecular features 14 1.4 Sacsin 18 1.5 Ubiquitin-proteasome pathway and ubiquitin-like domains 21 1.6 Sacsin UBL 26 1.7 Objective 30 Chapter II: Materials and Methods 32 2.1 Expression constructs 32 2.2 Bacterial transformation 32 2.3 Protein expression 33 2.4 Protein purification 33 2.5 SDS-PAGE 35 2.6 Protein crystallization 35 1 2.7 Structure solution and refinement 36 2.8 NMR Spectroscopy 36 Chapter III: Results and Discussion 39 3.1 Protein Purification 39 3.2 The unusual structure of the sacsin UBL 42 Chapter IV: Conclusion 49 References 53 2 Acknowledgements First of all, I would like to thank my supervisor Dr. Kalle Gehring for giving me the opportunity to work in his laboratory, insightful guidance in the research work, help with NMR experiments, and for interesting discussions on topics as varied as the theory of NMR, brain-teasing puzzles, pointers, pool, Star Trek, and Hollywood movies. I am also grateful to Dr. Jason Young and Dr. Anthony Mittermaier for their thoughtful advice on experiment designs, and experimental help from Dr. Young’s laboratory. Dr. Jean-François Trempe has been very important for my research work, as he was working on the project before me, but even after leaving the laboratory he helped a lot with the research strategy, experimental set up, suggestions on relevant literature and data collection at the synchrotron. I would like to thank Dr. Guennadi Kozlov, Dr. Marie Ménade, Dr. Véronique Sauvé and Angelika Rosenauer for helping with various laboratory techniques, their suggestions and expertise in different experiments, and wonderful discussions on different areas. It was a great time spent in the company of Jingwei Xie, Christian Baran, Sara Bastos, Juliana Muñoz, Marjan Seirafi, Edna Matta-Camacho, Sebastian Murphy, Vasudha Khurana, Vivek Sharma and Irina Gulerez, with whom I not only discussed research issues and a wide range of other general topics but also had fun at several memorable occasions. Vasudha also helped in conducting experiments as a summer student. I am really glad that my parents always encouraged my interest in the things I wanted to do, and they always extended full support. My father has been very important in igniting my interest in science at a young age. Finally, I am thankful to the Canadian Institutes of Health Research for the funding. 3 Abstract Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a progressive neurodegenerative disorder that first presents in early childhood, with prominent symptoms of spasticity in limbs, gait ataxia, slower motor development, muscle wasting and slurred speech. The disease is due to mutations in the SACS gene, which codes for sacsin, a large multidomain protein found in neurons. Loss of sacsin function is associated with progressive loss of cerebellar Purkinje cells and hyperfused mitochondrial network, most likely due to loss of the mitochondrial fission efficiency. Most of sacsin is structurally and functionally uncharacterized. Sacsin is believed to play a role as a chaperone for promoting the folding of ataxia-related proteins. Bioinformatics analysis showed that sacsin contains an integral ubiquitin-like domain (UBL) domain, which was subsequently shown to weakly interact with the proteasomal subunit C-8 in the co- immunoprecipitation studies. The research work described in this thesis includes the incorporation of selenomethionine in the UBL sequence, crystallization of the selenomethionine-labeled UBL domain to produce well-diffracting crystals, and determination of the structure of the UBL domain by using the anomalous scattering signal from selenium. The UBL structure obtained is unusual as it is a swapped dimer formed by the exchange of the N-terminal portions of two molecules. The existence of dimer was confirmed in solution by PFG-NMR self-diffusion experiments. The hydrophobic patch that is usually responsible for interaction of the UBL domain with other proteins is occluded in the swapped dimer, which suggests that the sacsin UBL domain does not bind the proteasome as a dimer. 4 Résumé L’ataxie récessive spastique autosomale de Charlevoix-Saguenay (ARSACS) est une maladie neurodégénérative progressive dont les symptômes se présentent dès la petite enfance. Les symptômes les plus importants sont la spasticité dans les membres, l’ataxie, le développement plus lent des fonctions motrices, une atrophie musculaire et des troubles de l'élocution. La maladie est due à des mutations dans le gène SACS qui code pour la sacsine, une grande protéine multidomaine qui se trouve dans les neurones. La perte de fonction dans la sacsine est associée à la perte progressive des cellules de Purkinje du cervelet et à un réseau mitochondrial hyperfusionné, probablement due à une réduction de la fission mitochondriale. La structure et la fonction de la sacsine sont mal caractérisées. La sacsine est censée jouer un rôle de chaperon pour la promotion du repliement des protéines impliquées dans l’ataxie. Une analyse bioinformatique a montré que la sacsine contient un domaine homologue à l’ubiquitine (UBL). Des études de co- immunoprécipitation ont démontré que le domaine UBL interagirait avec la sous-unité du protéasome C-8. Le travail de recherche décrit dans cette thèse comprend l'incorporation de la sélénométhionine dans le domaine UBL, sa cristallisation et la détermination de sa structure à haute résolution par diffraction de rayons X et le signal de diffusion anomale du sélénium. La structure obtenue est inhabituelle et se présente comme un dimère formé par l'échange des parties N-terminales des deux molécules. L'existence du dimère a été confirmée en solution par des expériences d'auto-diffusion en RMN. Le site hydrophobique, qui est habituellement responsable de l'interaction des domaines UBL avec leurs ligands, est obstrué dans le dimère échangé ce qui suggère que le domaine UBL de la sacsine ne lie pas le protéasome sous forme de dimère. 5 List of Abbreviations ARSACS - Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay ATG12 - autophagy 12 ATP - adenosine triphosphate CHESS - Cornell High-Energy Synchrotron Source CNS - central nervous system CP - core particle CV - column volume DNA - deoxyribonucleic acid Drp1 - dynamin-related protein 1 E. coli - Escherichia coli EDTA - ethylenediaminetetraacetic acid FPLC - fast protein liquid chromatography GCL - granule cell layer GFP - green fluorescent protein GST - glutathione S-transferase HBS - HEPES-buffered saline HEPN - higher eukaryotes and prokaryotes nucleotide-binding domain HPLC - high pressure liquid chromatography HSQC - heteronuclear single quantum correlation IPTG - isopropyl-thiogalactopyranoside IQ - intelligence quotient IR - isomorphous replacement 6 ISG15 - interferon-stimulated gene-15 KD - knock down KO - knockout LB - lysogeny broth MAD - multi-wavelength anomalous dispersion ML - molecular layer MR - molecular replacement MRI - magnetic resonance imaging MWCO - molecular weight cut-off NMR - nuclear magnetic resonance PAGE - polyacrylamide gel electrophoresis PB - phosphate buffer PDB - protein data bank PFG- NMR - pulsed field gradient NMR PMSF - phenylmethylsulfonyl fluoride Rad23 - radiation-sensitive mutant 23 RP - regulatory particle SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis siRNA - small interfering RNA SRR - sacsin repeat region SUMO - small ubiquitin-like modifier TBS - Tris-buffered saline UBA - ubiquitin-associated domains 7 UBL - ubiquitin-like ULD - ubiquitin-like domain UPS - ubiquitin-proteasome system XPCB - Xeroderma pigmentosum complementation group 8 List of Figures Figure 1.1: Loss of cerebellar Purkinje cells in sacsin KO mice Figure 1.2: Human sacsin domains Figure 1.3: DnaJ and HEPN domain Figure 1.4: Ubiquitination of a substrate for proteasomal degradation Figure 1.5: Variations of the ubiquitin β-grasp superfold Figure 1.6: Ubiquitin and integral UBL domains (ULDs) in proteasomal degradation Figure 1.6: (A) Sequence alignment of the sacsin UBL with ubiquitin and other UBLs. (B) Co-immunoprecipitation of the proteasomal subunit C-8 with the N-terminus of sacsin, containing the UBL domain Figure 1.7: (A) Sequence alignment of the sacsin UBL with ubiquitin and other UBL domains. Sacsin residues that match key hydrophobic amino acids of the consensus sequence, which may play a role in proteasomal targeting are indicated by asterisks (B) Co-immunoprecipitation of the proteasomal subunit C-8 with the N-terminus of sacsin, containing the UBL domain, and the double mutants Figure 3.1: SDS analysis of the purification steps of UBL-L78M 2-85 Figure 3.2: HPLC chromatogram and SDS analysis for the final purification step of UBL-L78M 2-85 Figure 3.3: UBL-L78M 2-85 crystals Figure 3.4: Unusual UBL swapped dimer, and occlusion of the the hydrophobic patch responsible for interaction with proteasome in usual UBL domains Figure 3.5: Swapped dimer of ATG12 9 Figure 3.6: Cys17-Cys20 disulfide bond formation in the sacsin UBL domain Figure 3.7: PFG-NMR