The Unusual Structure of the Ubiquitin-Like Domain of the Protein

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.

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

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 self-diffusion measurements

Figure 3.8: Overlaid 1H–15N HSQC spectra of 15N-labeled UBL 2-85 at 50 μM (red)

(256 scans) and 220 μM (blue) (16 scans) in the NMR buffer 20 mM PB, pH 6.6

10 List of Tables

Table 1.1: Sacsin mutations around the world

Table 1.2: Common ubiquitin-like domains

Table 3.1: Refinement statistics

11 Chapter I: Introduction

1.1 Autosomal Recessive Spastic Ataxia of Charlevoix Saguenay

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease associated with clinical features such as early onset signs of spasticity in the lower limbs, gait ataxia with a tendency to fall, slower motor development, muscle wasting and slurred speech (Bouchard, Barbeau et al. 1978;

Bouchard 1991; Bouchard, Richter et al. 1998). The disease was first discovered among the inhabitants of the Charlevoix-Saguenay region of northeastern Quebec, Canada, with a carrier frequency estimated to 1/22, but later cases have been found elsewhere in the world too (Bouchard, Barbeau et al. 1978; De Braekeleer, Giasson et al. 1993). One study from Quebec reports the patients become wheelchair bound at a mean age of 41 and the mean age at death is 51 years (Bouchard 1991). Usually, the earliest sign of ARSACS is spasticity in the lower limbs, which appears between the ages of 12 and 24 months when the patients begin to walk (Bouchard 1991; Bouchard, Richter et al. 1998; Criscuolo,

Banfi et al. 2004; Grieco, Malandrini et al. 2004; Takiyama 2006; Fogel and Perlman

2007; Ouyang, Segers et al. 2008; Vermeer, Meijer et al. 2008; Bouhlal, El Euch-Fayeche et al. 2009; Bouhlal, Amouri et al. 2011). There are patients that have slower progression of the disease in whom limb spasticity appears later than infancy, and there have also been cases in which leg spasticity has been completely absent (Shimazaki, Takiyama et al. 2005; Shimazaki, Sakoe et al. 2007; Bouhlal, Zouari et al. 2008). Patients have slurred speech in childhood and explosive speech in adulthood. In Quebec patients, one of the main clinical manifestations of ARSACS is prominent retinal myelinated fibers but this

12 feature is frequently absent in reports on non-Quebec patients (El Euch-Fayache, Lalani et al. 2003; Takiyama 2006; Bouhlal, Amouri et al. 2011). Electromyography has shown signs of axonal degeneration in the peripheral nervous systems, chronic neurogenic atrophy in some patients, and severe denervation in the distal muscles by the time the patients are in their late twenties (Garcia, Criscuolo et al. 2008). Motor nerve conduction velocities are usually moderately reduced. Despite slower motor development in with

ARSACS, no intellectual impairment has been observed in Quebec preschool patients.

However, patients at the elementary school level are generally slow to learn; they are slower to master handwriting, while the verbal IQ even on reaching adulthood is usually within normal limits. MRI studies have shown cerebellar atrophy more visible in the upper cerebellar vermis and associated with spinal atrophy (Kamada, Okawa et al. 2008;

Bouhlal, Amouri et al. 2011).

Being a rare disease, ARSACS lacks ample scientific research, and we are still far from curing it. Some oral medications, such as Baclofen, are used to control spasticity and prevent other symptoms (Bouhlal, Amouri et al. 2011). Studies on ARSACS would not only increase our understanding of the disease, which would help in developing treatment methodology, but also enhance our knowledge of neurodegeneration, role of chaperone systems in preventing misfolding and aggregation of ataxia-related proteins, and mitochondrial involvement in neurodegenerative disorders. As many ataxia-related proteins are linked, it would also help in bridging the pathways involved in different forms of ataxia.

13 1.2 ARSACS genetics

Defects in the SACS gene were identified as the genetic cause for the disease. The SACS gene is located on chromosome 13q12.12 and transcribes a nine exon transcript, which codes for a 4579 amino acid (520 kDa) protein called (Engert, Berube et al. 2000; Mrissa,

Belal et al. 2000). More than 70 different mutations have been identified worldwide with only evidence of founder effect found in the Quebec population (Table 1.1). Nearly all the mutations are spread over the whole of exon 9 without any evidence of hot spot mutation regions or genotype-phenotype correlation (Bouhlal, Amouri et al. 2011). The majority of

Quebec patients have a single mutation, a single nucleotide deletion (6594delT) (Engert,

Dore et al. 1999).

1.3 Biological and molecular features

The prominent neurological features include cerebellar atrophy and loss of cerebellar

Purkinje cells. (Bouchard 1991; Martin, Bouchard et al. 2007; Bouhlal, Amouri et al.

2011; Girard, Lariviere et al. 2012) (Figure 1.1). Recent studies have pointed towards malfunctioning in mitochondrial dynamics in a number of neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s disease. Mitochondrial dynamics involves constant changes in the mitochondrial morphology through two processes called mitochondrial fission and fusion. Mitochondrial fusion is the merging of two mitochondria and is considered to be a highly protective process, allowing the mitochondria to tolerate mitochondrial DNA mutations (Chen and Chan 2009).

Mitochondrial fission, the complementary process of fusion, is the division of mitochondria and is required to maintain their number and function

Table 1.1: Sacsin mutations around the world. Table taken from (Bouhlal, Amouri et al. 2011).

16 (Otera and Mihara 2011). Mitochondrial dynamics plays an important role in cellular quality control, and defects in either fission or fusion have detrimental effects on the cell including the supply of energy. Since neurons are in high demands of energy, they are highly sensitive to defects in mitochondrial function and dynamics, and neuronal cell pathology has been associated with mitochondrial dysfunction (Lin and Beal 2006; Chen and Chan 2010; Cho, Nakamura et al. 2010; Su, Wang et al. 2010; Westermann 2010;

Schon and Przedborski 2011). Proteins called mitofusins 1 and 2 and Opa1 are GTPases that are present on the mitochondrial outer and inner membrane respectively, and they are involved in mitochondrial fusion. Sacsin has been shown to interact with Drp1 (dynamin- related protein 1), which is a large GTPase required in mitochondrial fission (Otsuga,

Keegan et al. 1998; Smirnova, Shurland et al. 1998; Smirnova, Griparic et al. 2001; Chen and Chan 2010; Reddy, Reddy et al. 2011; Girard, Lariviere et al. 2012). Sacsin knockdown causes a highly interconnected and functionally impaired mitochondrial network, suggesting a loss of mitochondrial fission efficiency. Also, fibroblasts from

ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission (Girard, Lariviere et al. 2012). In a similar manner to sacsin, Drp1 disruption also leads to excessively interconnected mitochondria (Smirnova, Shurland et al. 1998; Smirnova, Griparic et al. 2001). Since sacsin (residues 1-1368) is known to interact with Drp1, it can be speculated that sacsin might play a role in the oligomeric assembly of Drp1 around the fission site of mitochondria, and defects in sacsin lead to improper assembly of Drp1 and thus the inefficient fission and the mitochondrial defects

(de Castro, Martins et al. 2011; Yamano and Youle 2011; Girard, Lariviere et al. 2012).

It has also been observed that siRNA-mediated sacsin knockdown enhanced the toxicity

17 of the cells expressing GFP-ataxin-1[82Q] leading to reduction in the number of cells expressing GFP-ataxin-1[82Q], suggesting that sacsin has a protective function against mutant ataxin-1 (Parfitt, Michael et al. 2009). This also suggests a link between

ARSACS and other forms of ataxia. It is usual to find that common interacting partners are shared by many of the proteins involved in inherited ataxias, indicating that they are functionally linked. Also, there are cases of overlapping pathways of molecular pathogenesis in some types of ataxia. Theses molecular pathogenesis includes perturbations in normal protein homeostasis that can give rise to cellular responses from molecular chaperone and ubiquitin–proteasome system (UPS) (Lim, Hao et al. 2006;

Parfitt, Michael et al. 2009).

1.4 Sacsin

Sacsin is an unusually large protein. Sacsin is expressed in various tissues, but the highest level of expression is in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization is predominantly cytoplasmic with a mitochondrial component. The N-terminus of sacsin contains a ubiquitin-like (UBL) domain that shares 43% identity over 65 residues with the N-terminal UBL domain of

Rad23 (Parfitt, Michael et al. 2009). The UBL domain is followed by three repeats of a large complex supra-domain termed the “sacsin repeating region” (SRR), and the first

SRR is known to have ATPase activity (Anderson, Siller et al. 2010). The three SRR repeats are followed by a domain that has low similarity to the XPCB domain from

Figure 1.1: Loss of cerebellar Purkinje cells in sacsin KO mice: left: Immunohistochemical sections of cerebellum from 120-day-old sacsin KO mice (SACS- /-) and wild-type littermates (SACS+/+) are compared to show the age-dependent loss of cerebellar Purkinje cells. Arrows point to Purkinje cell bodies or regions in which Purkinje cells are absent; right: Cell counts revealing the number of Purkinje cells in cerebellum from 120-day-old and 200-day-old sacsin KO mice (SACS-/-) and wild-type littermates (SACS+/+). Figure and legend taken from (Girard, Lariviere et al. 2012).

19 HHR23A, a human homologue of Rad23 (Kamionka and Feigon 2004). It is still debatable whether it is a real XPCB domain or not. At its C-terminus, the sacsin protein contains a region that has similarity to the DnaJ domain of the Hsp40 family of Hsp70 co- chaperones, which is immediately followed by a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain. (Engert, Berube et al. 2000; Grynberg,

Erlandsen et al. 2003; Kozlov, Denisov et al. 2011) (Figure 1.2). Due to the presence of the DnaJ domain, and similarity to the ATP binding domain of HSP90 in the SRRs of sacsin, it has been proposed to play a role in the chaperone machinery, but the underlying basis of the neurological defects is still largely unknown (Parfitt, Michael et al. 2009;

Anderson, Siller et al. 2010). Also, most of the identified and predicted domains of sacsin are not structurally and functionally characterized. There are vast stretches with different predicted secondary structure elements that do not show significant similarity with any of the known proteins.

8-79 90-505 1455-1921 2523-2922 3657-3740 4306-4366 4443-4576

Figure 1.2: Human sacsin domains: ubiquitin-like domain (UBL), sacsin-repeat regions (SRR) 1, 2 and 3, XPCB domain, DnaJ-domain, and higher euakryotes and prokaryotes nucleotide binding domain. The start and end residues numbers for different domains with in the full-length human sacsin protein are marked.

DnaJ domain of sacsin is a helix bundle in its NMR structure, as expected for the Hsp40 family members (Figure 1.3) (pdb id: 1iur). It is shown to be a functional J-domain using

20 a bacterial in vivo complementation assay (Parfitt, Michael et al. 2009). Also J-domain was shown to stimulate the ATPase activity of Hsp70 in vitro (Anderson, Siller et al.

2010). It is also known that J-domains have been associated with several neurodegenerative disorders such as Parkinson's disease and polyglutamine expansion diseases such as Huntington's disease (Chuang, Zhou et al. 2002; Durrenberger, Filiou et al. 2009; Hageman, Rujano et al. 2010; Rose, Novoselov et al. 2011). The recently solved crystal structure of the HEPN domain of sacsin shows the domain is a dimer and has a large positively charged cavity at the dimer interface that binds nucleotides (Kozlov,

Denisov et al. 2011) (Figure 1.3). Sacsin region 1–1456 has been demonstrated to have direct chaperone activity towards the model client protein firefly luciferase (FLuc). This region of sacsin is also capable of maintaining client polypeptides in folding competent states and could cooperate with members of the bacterial Hsp70 system to achieve high refolding yields (Anderson, Siller et al. 2011).

1.5 Ubiquitin–proteasome pathway and ubiquitin-like domains

The ubiquitin–proteasome pathway is an important pathway that is involved in the maintenance of the proper levels of cellular proteins by protein degradation as required by the cell. The molecular machine called proteasome is a large multi-subunit protein complex that maintains cellular homeostasis by contributing to the turnover of short-lived proteins, as well as providing housekeeping functions, such as degradation of misfolded proteins. The proteasomal degradation of misfolded or damaged secretory and non- secretory proteins is an ubiquitin and ATP-dependent process. There are two major

DnaJ

HEPN Figure 1.3: Schematic representations of the DnaJ and HEPN domains of sacsin (pdb id: 1Iur and 3O10): Dna-J is helix bundle with the typical three helices as found in the J-domains. HEPN is a dimer, with each molecule consisting of five parallel helices, and the nucleotides bind at the dimer interface. The structures are colored blue to red.

22 subunits in the 26S proteasome holoenzyme: the 19S regulatory particle (RP) and the 20S core particle (CP) (Voges, Zwickl et al. 1999). The 20S CP is the subunit responsible for proteolysis. The 19S RP complex, which, directly or indirectly, binds ubiquitin and ubiquitinated substrates, is thought to be involved in recognition and processing of the ubiquitinated target proteins, and is necessary for their subsequent degradation

(Deveraux, Ustrell et al. 1994; Elsasser, Gali et al. 2002; Husnjak, Elsasser et al. 2008).

Multiple chains of ubiquitin, a highly conserved 76 amino acid polypeptide, are covalently attached to the protein substrate (termed ubiquitination), which is mediated by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and substrate recognizing enzyme E3 (Figure 1.4). Thus the ubiquitin-tagged (ubiquitinated target protein substrate is ready to be recognized and degraded by the 26S proteasome assembly

(Fang and Weissman 2004; Grabbe and Dikic 2009). Ubiquitination of a protein substrate is required in a number and wide variety of cellular processes, such as antigen processing, apoptosis, biogenesis of organelles, cell cycle and division, DNA transcription and repair, differentiation and development, immune response and inflammation, neural and muscular degeneration, morphogenesis of neural networks, modulation of cell surface receptors, ion channels and the secretory pathway, response to stress and extracellular modulators, ribosome biogenesis, viral infection. Thus, now it is understood that ubiquitination of a protein substrate can have both conventional (degradative) and unconventional (non-degradative) functions as required by the cell.

Ubiquitin structure is marked with the globular β-grasp superfold, and later when other polypeptides were discovered with similar β-grasp superfold, they were classified as

Figure 1.4: Ubiquitination of a substrate for proteasomal degradation: ubiquitin is a small 76 amino-acid protein which is evolutionarily highly conserved in eukaryotic cells. Ubiquitination of proteasomal substrate proteins is performed by a complex system consisting of ubiquitin activating (E1) enzymes, ubiquitin-conjugating (E2) enzymes and substrate recognition proteins (E3 enzymes) (Figure and legend taken from the MIT open course, Ubiquitination: The Proteasome and Human Disease, MIT Course Number: 7.340, Fall 2004 http://ocw.mit.edu/courses/biology/7-340-ubiquitination-the- proteasome-and-human-disease-fall-2004/).

24 ubiquitin-like (UBL) domains (or ULD), and the whole family was named as the UBL family. Variations in the ubiquitin fold (or β-grasp superfold) have been found with varied functions too (Grabbe and Dikic 2009) (Figure 1.5). Some members of the UBL family include: small ubiquitin-like modifier (SUMO), interferon-stimulated gene-15

(ISG15), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8), human leukocyte antigen F- associated (FAT10), autophagy-8 (ATG8), autophagy-12 (ATG12), Fau ubiquitin-like protein (FUB), MUB (membrane-anchored UBL), ubiquitin fold-modifier-1 (UFM1) and ubiquitin-like protein-5 (UBL5). Despite the presence of the same ubiquitin fold, usually the UBLs share modest to low sequence identity with ubiquitin, for example SUMO shares only 18% sequence identity with ubiquitin. These ubiquitin-like proteins have evolved to function distinctly while conserving the ubiquitin fold (Table 1.2) (Welchman,

Gordon et al. 2005). Some of the UBLs are independent polypeptides like ubiquitin and play similar role of labeling a protein substrate for diverse regulatory activities rather than tagging the substrate for proteasomal degradation, for example SUMO-1 modification often acts antagonistically to that of ubiquitination and serves to stabilize protein substrates, and ISG15 plays role in immune response and interferon signal transduction.

But there also proteins, for example proteasomal shuttle factors such as Rad23 and Dsk2

(Dominant suppressor of Kar1)/ Ubiquilin (Grabbe and Dikic 2009), E3 ligases such as

Parkin and Elongin B, the chaperone cofactors Bag1 and Scythe, which have an integral

UBL domain along with other domains (Hartmann-Petersen and Gordon 2004). In eukaryotes there are protein in which these integral UBL domains are often found in combination with UBA (ubiquitin-associated domains). These proteins include Rad23 and

25 Dsk2 ubiquilin proteins, DDI1 (DNA damage inducible) proteins, which all have an N- terminal UBL. Through their UBL domain RAD23, Dsk2/ubiquilin, and DDI1 all bind directly to both Rpn10/S5a and Rpn1, two subunits of the 19S proteasomal regulatory particle (Figure 1.6) (Schauber, Chen et al. 1998; Kleijnen, Shih et al. 2000; Wilkinson,

Seeger et al. 2001; Saeki, Saitoh et al. 2002; Kleijnen, Alarcon et al. 2003; Grabbe and

Dikic 2009).

Ubiquitin and the UBL domains have a hydrophobic patch, for example in ubiquitin the patch comprises Leu8, Val70, and Ile44, and this hydrophobic patch is responsible for interaction with the proteasome (Haririnia, Verma et al. 2008). In some UBL domains, these hydrophobic patches have consensus sequences and are called proteasome- interacting motifs (Upadhya and Hegde 2003).

1.6 Sacsin UBL

The existence of an integral UBL domain at the N-terminus of sacsin was first revealed by bioinformatics analyses. The sacsin UBL shares 43% identity over 65 residues with the N-terminal UBL domain of Rad23. The sacsin UBL also contains part of a potential consensus sequence for proteasome interaction; the consensus sequence is found within ubiquitin and the UBL domains such as Nedd8 and the N-terminus of Rad23. A weak interaction between the sacsin UBL domain and the proteasome was observed by co- immunoprecipitating the 20S proteasomal alpha subunit C8 with the sacsin UBL domain.

The interaction was further weakened when the experiment was repeated with the sacsin

UBL domain double mutants L58/D60A and R50A/L51A, which implicated these

Figure 1.5: Variations of the ubiquitin β-grasp superfold: Structural comparison of the ubiquitin-like folds characterized to date, based on published structural information provided by NMR- and crystallography-based studies as follows: (1) ubiquitin1 (PDB id 1UBQ), (2) SUMO-1146 (PDB id 1A5R), (3) the UBX domain in p47147 (PDB id 1I42), (4) the PB1-domain in p62148 (PDB id 1PFJ), and (5) the UBL in Rad23149 (PDB id 1OQY). (Figure and legend taken from (Grabbe and Dikic 2009).

Table 1.2: Examples of ubiquitin-like domains, their substrates, sequence identity to ubiquitin and attributed function. Figure taken from (Welchman, Gordon et al. 2005).

Figure 1.6: Ubiquitin and integral UBL domains (ULDs) in proteasomal degradation: role of ULD/UBA proteins in proteasomal degradation. After the sequential activity of ubiquitin E1 activating, E2 conjugating, E3 ligating, and E4 elongating enzymes, polyubiquitinated substrates are recognized by the UBA domain in the proteasomal shuttle factors, RAD23, ubiquilin family proteins, and DDI1. Subsequently, utilizing their integral ULD domains, the shuttle factors directly interacts with multiple sites on the proteasome (Rpn1, Rpn10, and Rpn13), thus serving to target their ubiquitinated cargo for degradation. Figure and legend taken from (Grabbe and Dikic 2009).

29 residues in the interaction (Figure 1.7) (Parfitt, Michael et al. 2009).

1.7 Objective

Jean-François Trempe, a previous lab member, was able to crystallize the native UBL domain (residues 2-85), but the phase problem was not solved because neither molecular replacement (MR) nor isomorphous replacement (IR) worked. So, the idea of selenomethionine incorporation and solving the phase problem by multi-wavelength anomalous diffraction (MAD) was a feasible option, but the UBL 2-85 in sacsin does not have any methionine residues in its sequence. In order to overcome this difficulty, different plasmid constructs of the UBL domain were created with mutations of single leucines at different positions to methionine: L26M, L58M, L67M and L78M.

The main aim of the research work presented in this thesis was to obtain well-diffracting selenomethionine-labeled UBL domain crystals from one or more of these constructs and to obtain the sacsin UBL domain structure by solving the phase problem by using anomalous signal from selenomethionine from the MAD data. As mentioned earlier UBL domains are often found with novel functions in diverse regulatory activities, including a role in the ubiquitin-proteasome pathway. Parfitt, Michael et al. 2009 reported the interaction of the sacsin UBL with the 20S proteasomal alpha subunit C8. The interaction, as can be viewed in the Figure 1.7, appears rather weak and a high-resolution structure of the sacsin UBL can provide insight into the nature of its interaction with proteasome.

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 and legend taken from (Parfitt, Michael et al. 2009).

31 Chapter II: Materials and Methods

2.1 Expression constructs

UBL residues 2-85 and UBL-L78M 2-85 sequences in the multicloning site of the plasmid pGEX-6p1, with N-terminal GST tag, were obtained from a previous lab member, Jean-François Trempe.

2.2 Bacterial transformation

For plasmid amplification, 1.5 μL of plasmid DNA was added to 120 μL of Escherichia coli DH5-alpha competent cells in an eppendorf tube. For 20 minutes, the mixture was incubated on ice, heat shocked at 42°C for 30 to 45 seconds, then placed on ice for 3-5 minutes. For recovery of the bacterial cell, 800 μL of LB was added and the culture was incubated at 37°C on a shaker at 200 rpm for 1 hour. Centrifuging the culture at 4000 rpm for 2 minutes pelleted the bacteria, and 700 μL of supernatant was removed. The pellet was resuspended in the remaining solution, then the suspension was plated on LB with ampicillin and incubated at 37° C overnight.

Four single colonies were chosen at random and grown in 5 mL of LB with ampicillin at

37° C overnight. The plasmids were extracted from the bacteria and purified using Qiagen spin miniprep kit. The constructs were confirmed by sequence analysis, and then transformed into E. coli BL21 and/or DL41 (DE3) (methionine auxotroph) cells by the procedure just stated.

32 2.3 Protein expression

To generate unlabeled protein, the cells are grown either in the usual LB media or the auto-inducible media (Studier 2005). To generate 15N-labeled protein, the cells were

15 grown in M9 minimal media containing NH4Cl. To generate slenomethionine-labeled protein, the cells were grown in the LeMaster media (Hendrickson, Horton et al. 1990).

100 mL of starter culture with 100 μg/mL of ampicillin were incubated overnight shaking at 200 rpm at 37°C. 10 mL of culture was diluted in 1L of LB or auto-inducible media. 50 mL of culture was diluted in 1L of 15N-minimal media and the LeMaster media. In the LB and the minimal media (both 15N and LeMaster), bacteria were induced for protein production using isopropyl-thiogalactopyranoside (IPTG) at a final concentration of 1 mM once the optical density (OD) at 600 nm was 0.8. The incubation temperature was then lowered to 25°C or 30°C and sustained at 200 rpm for another 4-6 hours or the temperature was lowered to 20°C and sustained at 200 rpm overnight. In the auto- inducible media, the temperature was lowered to 20°C and sustained at 200 rpm overnight. The cells were then harvested by centrifugation for a period of 20 minutes at

4000 rpm. The pellets were then either stored at -20° C or resuspended in the lysis buffer

(TBS pH 7.4 + 250 μL of 1M PMSF + 400 μL of lysozyme (2 μg/μL)), with or without

0.5mM EDTA and the suspension stored at -20°C or -80°C.

2.4 Protein purification

The lysis buffer suspension of the cells is taken out of the freezers and incubated in water bath at room temperature for 1 hour. The suspensions were sonicated on ice using 7 x 10 second pulses 3 times on a Fisher Scientific Conic Dismembrator Model 500 at 50%

33 amplitude. The lysate is then centrifuged at 18000 rpm for 45 minutes at 4°C. The supernatant was the filtered using MILLEX HV 0.45 μm filter unit by MILLIPORE. 2 mL of glutathione sepharose beads were equilibrated in TBS, pH 7.4. The beads are added to the filtered supernatant in a Falcon tube and incubated for 45 minutes at 4 °C with gentle rocking. The mixture was added to a gravity-column, allowed to settle and then the filtrate allowed to flow through the column. The beads were washed three times with 5 CV of TBS. The GST-tagged UBL-L78M 2-85 is eluted with elution buffer (TBS,

30mM glutathione, pH 8.0) 4-5 times with 2 mL of elution buffer each time. The eluate was concentrated to 3 mg/mL using Amicon Ultra centrifugal filters by Millipore

(Centricon tubes) with10K MWCO. The eluate was poured in a dialysis bag to remove glutathione, 50 mL of 2mg/mL and PreScission protease was added to the dialysis bag to cleave off the GST tag, leaving an extra GPLGS sequence at the N-terminus of UBL-

L78M 2-85. Thus purified UBL-L78M 2-85 means that it has an extra GPLGS sequence at its N-terminus. Thus glutathione removal and Prescission protease cleavage were carried out simultaneously overnight in the dialysis bag. UBL-L78M 2-85 was found both in the supernatant and the pellet after centrifuging (13,500 r.p.m for 10 minutes) the cleaved product. The supernatant was collected and the precipitate was stored at -20 ºC or

-80 ºC. Then, a 16/60 S75 gel filtration column was equilibrated in HBS buffer (10 mM

HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) at 1.0 mL/min at 4ºC in a GE Healthcare

HPLC or an Akta Prime FPLC system. The supernatant of the cleaved GST+ UBL-L78M

2-85 solution was filtered using 0.45 μm filters and injected on the gel filtration column at

1.0 mL/min, and 2 mL fractions were collected and monitored at 258 nm and/or 280 nm.

The pellet of the the cleaved GST+ UBL-L78M 2-85 was dissolved in 8M guanidium

34 chloride in HBS buffer, and gradual dialysis was carried out to obtain refolded UBL-

L78M 2-85 in solution. The refolding was efficient, as the NMR spectra overlaid perfectly when the refolded one was compared with the one purified from the supernatant of the cleaved GST+ UBL-L78M 2-85 after running the gel filtration column.

UBL 2-85, 15N-labelled and unlabelled, both were purified in similar manner as that of

UBL-L78M 2-85.

2.5 SDS-PAGE

SDS-PAGE was used to analyze protein purity at various steps of purification. Fractions under denaturing and reducing conditions were taken, and samples were prepared by mixing 6 μL of fraction sample and 2 μL of 4x loading buffer for the Phastgels and 18 μL of fraction sample and 6 μL of 4x loading buffer for the usual 12% SDS PAGE. The samples are incubated for a period of 3-5 minutes at 95° C before loading on the polyacrylamide gels. The 20% polyacrylamide Phastgels were run on a GE Healthcare /

Amersham Pharmacia PhastSystem Automated Electrophoresis Development Unit at

110V. The usual staining and destaining procedure was followed to view the protein bands.

2.6 Protein crystallization

The fractions collected from 16/60 S75 gel filtration column run were pooled together and pure UBL-L78M 2-85 was concentrated to 3.2 mg/mL using the Centricon tubes. 1.5

μL of protein solution was added to 1.5 μL of the mother liquor for setting crystallization wells. Crystals grew within 16 hours at 22ºC. For data collection, glycerol 25% was

35 added for cryoprotection and the crystals flashcooled in a N2 cold stream.

2.7 Structure solution and refinement

Diffraction data from the selenomethionine-labeled crystals of UBL-L78M 2-85 were collected using using three wavelengths at the selenium adsorption edge, inflection and remote positions on an ADSC Quantum- 210 CCD detector (Area Detector Systems

Corp.) at beamline F2 at the Cornell High-Energy Synchrotron Source (CHESS). Phenix was used to solve the structure, using Autosol and the peak/inflection data sets (Adams,

Afonine et al. 2010). The UBL-L78M 2-85 structure served as the model for molecular replacement for the previously collected data on the native UBL 2-85 crystals. The partial model thus obtained was extended manually with the program Coot and improved by refinement using REFMAC and model refitting, followed by TLS (translation-libration- screw) refinement (Murshudov, Vagin et al. 1999; Winn, Murshudov et al. 2003; Emsley and Cowtan 2004).

2.8 NMR Spectroscopy

All NMR experiments were recorded at 30 °C on a Bruker Avance 600-MHz spectrometer. 1H–15N HSQC spectra of a protein is a signature of the protein. A conformational change in the protein can be easily viewed as shift of peaks in its 1H–15N

HSQC spectra. 1H–15N HSQC spectra of 15N-labeled UBL 2-85 were acquired for the following two cases:

1. 220 μM 15N-UBL 2-85, 20 mM PB, pH 6.6 (16 scans)

36 2. 50 μM 15N-UBL 2-85, 20 mM PB, pH 6.6 (256 scans)

Pulsed field gradient NMR self-diffusion measurements were carried out using the stimulated echo technique with a gradient duration of 3.5 ms and diffusion time of 250 ms

(Stejskal and Tanner 1965; Altieri, Hinton et al. 1995). Lysozyme and ubiquitin were used as reference samples for the self-diffusion measurements.

The Stokes-Einstein equation gives the diffusion coefficient as:

D = KT/6πηRh

D = diffusion coefficient

K = Boltzmann constant

T = temperature

η = coefficient of viscosity

Rh = hydrodynamic radius

and the Stejskal and Tanner’s equation for the PFG-NMR experiment is

2 2 2 ln(Io/I) = γ G δ (Δ – δ/3)D

I = peak intensity

Io = initial peak intensity

γ = gyromagnetic constant

G = gradient strength

δ = gradient duration

Δ = diffusion time

37 D = diffusion coefficient

Data point for Stejskal and Tanner’s equation are determined by varying the gradient

2 2 2 strength in the PFG-NMR self-diffusion experiments. The curve of ln(Io/I) vs γ G δ (Δ –

δ/3) is a straight line, whose slope is a measure of diffusion coefficient, which on the basis of Stokes-Einstein equation gives a measure of the hydrodynamic radius or the size of the protein. Thus PFG-NMR self-diffusion experiment is useful in determining the oligomeric state of a protein in solution.

38 Chapter III: Results and Discussion

3.1 Protein Purification

UBL-L78M 2-85 and UBL 2-85 were purified by the procedure described in “Materials and Methods.” Expression, purification and crystallization trials were tried for different

L-to-M mutants of the sacsin UBL. L78M was the only promising construct, thus the results are presented only for the L78M construct. The fractions of the “cells lysate,”

“flow down” from the gravity column, the third “wash” by the washing buffer (TBS), and different batches of “eluate” were run on an SDS Phastgel to analyze the purification process in steps (Figure 3.1). The efficiency of the cleavage by the PreScission protease to chop off the GST tag was also analysed on an SDS Phastgel. The band for UBL-L78M

2-85 was viewed on the gel at around 10 kDa (Figure 3.1).

The cleaved product was injected into a 16/60 S75 gel filtration column, and thus GST was separated from UBL-L78M 2-85. The UBL-L78M 2-85 had a broad peak and kept eluting from 90 minutes to 110 minutes at the flow rate of 1 mL/min. The fractions thus collected were run on a 12% SDS gel to analyze the purity (Figure 3.2). As can been seen on the gel, the UBL-L78M 2-85 obtained from the final purification step had a high degree of purity and was suitable for crystallization trials and NMR spectroscopy.

Figure 3.1: SDS analysis of the purification steps of UBL-L78M 2-85: left gel: from left to right, fractions from the “cell lysate,” the “flow-down” from the gravity column, the third “wash” by the washing buffer, different batches of “eluate” with GST-tagged

UBL domain of sacsin, and molecular weight markers. right gel: from left to right, GST-tag “cleaved” off by PreScission protease, the “fusion” protein before cleaving of the GST-tag, and molecular weight markers.

Figure 3.2: HPLC chromatogram and SDS-PAGE analysis for the final purification step of UBL-L78M 2-85: UBL eluted from 90 to 110 minutes at the flow rate of 1 mL/min, and the SDS-PAGE showing the purified UBL domain at around 10 kDa.

41 3.2 The unusual structure of the sacsin UBL

The best diffracting UBL-L78M crystals were obtained in the mother liquor condition:

100 mM bicine, 250mM NaCl, 1mM lead acetate, glycerol 10%, pH 8.6 (Figure 3.3). The crystals diffracted up to 2.4 Å at the CHESS synchrotron. The space group was C2221, the unit cell dimensions 52 x 63 x 89 Å, and there were two molecules of UBL-L78M 2-

85 per asymmetric unit. Lead acetate was only used to achieve higher order diffraction on the basis of empirical trials. Lead atom was not incorporated in the unit cell and thus not visible in the structure. The structure of UBL-L78M 2-85 was obtained using the methods described in “Materials and Methods.” The UBL-L78M 2-85 structure served as the model for molecular replacement for the previously collected data on the native UBL 2-

85 crystals. The native crystals had diffracted up to 2.1 Å. Thus the final UBL structure was solved up to the resolution of 2.1 Å and further refinement was achieved as described in “Materials and Methods.” The refinement statistics is shown in Table 3.1.

Figure 3.3: UBL-L78M 2-85 crystals.

Resolution (Å) 2.1

Rwork/Rfree 0.220/0.270

RMS deviations bond length (Å) – 0.009

bond angles (°) – 1.121

Ramachandran Preferred (135, 97.12%)

Allowed (3, 2.16%)

Outliers (1, 0.72%)(C72 A)

FOM 0.792

Table 3.1: Refinement statistics.

Surprisingly, the structure of the UBL of sacsin, thus obtained, is a swapped dimer with the exchange of the N-terminus between the two monomers (Figure 3.4). This is unusual as the proteins that are UBLs, or that contain UBLs, do not dimerize via the UBL domain, with the only exception known is that of ATG12, which is a swapped dimer in its crystal structure (Suzuki, Yoshimoto et al. 2005) (Figure 3.5). Also, the hydrophobic patch responsible for interaction with proteasome in ubiquitin and the usual UBL domains is occluded in the sacsin UBL due to the dimer formation (Figure 3.4).

Figure 3.4: Unusual UBL swapped dimer (left), and occlusion of the hydrophobic patch (red) responsible for interaction with proteasome in usual UBL domains (right).

Figure 3.5: Swapped dimer of ATG12 (pdb id: 1WZ3).

Figure 3.6: Cys17-Cys20 disulfide bond formation in the sacsin UBL domain.

44 Since domain swapping can occur as a crystallographic artifact, it was vital to know if the sacsin UBL exists as a dimer in solution too or is just an artifact of crystallization (Liu and Eisenberg 2002). A Cys17-Cys20 disufide bond is present near the region of exchange of the N-terminus between the two monomers in the crystal structure (Figure

3.6). This disulfide bond is not present in the other UBL domains. So, it was also important to study the effect of reduction of these disulfide bonds on the dimerization of the sacsin UBL. PFG-NMR self-diffusion measurements were carried out on the sacsin

2 2 2 UBL to determine its oligomeric state. The slope of the ln(Io/I) vs γ G δ (Δ – δ/3) curve for the sacsin UBL was compared with that of ubiquitin (~9 kDa) and lysozyme (~15 kDa) (Figure 3.7). It is evident from these measurements that the sacsin UBL exists as dimer in solution too. Similar results were obtained when 10 mM β-mercaptoethanol was present in the protein solution in the NMR tube and PFG-NMR self-diffusion measurements were carried out again in the presence of this reducing agent, suggesting that the reducing environment, which is sufficient to reduce the Cys17-Cys20 disulfide bond, has no effect on the state of dimerization of the sacsin UBL. The effect of concentration of the sacsin UBL on its dimerization was also studied by overlaying the

1H–15N HSQC spectra of 15N-labeled UBL 2-85 acquired at two different concentrations.

The two spectra acquired at the concentration of 50 μM (256 scans) and 220 μM (16 scans) overlaid very well, suggesting tht no transition took place in between the dimer/monomer in the range of concentration of 50 – 220 μM (Figure 3.8). Thus the sacsin UBL is a dimer both in crystal and in solution. It is worth mentioning that the C- terminus of sacsin also appears to be a dimer via the HEPN domain (Kozlov, Denisov et

45 al. 2011). This is also intriguing, as it suggests that sacsin dimerizes both from its N- terminal and C-terminal, which is again not a usual phenomenon.

46 ln(Io/I)

γ2G2δ2(Δ – δ/3)

Figure 3.7: Pulsed field gradient NMR self-diffusion measurements: the translational diffusion coefficient of a molecule, measured as the slope of the straight line obtained from the experiment, depends on the size of the molecule. The size of a dimer can be differentiated from the size of a monomer by using standard molecules (lysozyme and ubiquitin in this case). The ratio of the translational diffusion coefficients of ubiquitin and the sacsin UBL obtained from these measurements is 0.74, which is very close to the theoretical monomer to dimer ratio of 0.75, thus confirming the existence of the sacsin UBL dimer in solution too. The experiment is based on the diffusion NMR experiments conceived by Stejskal and Tanner 1965.

Figure 3.8: Overlaid 1H–15N HSQC spectra of 15N-labeled UBL 2-85 at 50 μM (red) (256 scans) and 220 μM (blue) (16 scans) in the NMR buffer 20 mM PB, pH 6.6: the well-overlaid peaks of the protein at two different concentrations suggest that there is no concentration dependent transition in between the dimer/monomer in the tested range.

48 Chapter IV: Conclusion

ARSACS is a neurodegenerative disease that can be caused by missense, nonsense or frameshift mutations in the SACS gene, which was discovered around a decade ago.

ARSACS patients have a wide range of ataxia and spasticity related symptoms. The defects in the SACS gene have been associated with different neuroanatomical features such as progressive loss of cerebellar Purkinje cells, functionally impaired and hyperfused mitochondrial network. The SACS gene codes for an extremely large protein called sacsin, which is highly expressed in larger neurons. The protein sacsin has multiple domains, some predicted and others studied, and stretches of unidentified predicted secondary structure elements in between the domains in its sequence. The N-terminus of sacsin was predicted to have a UBL domain by bioinformatics analyses as shown by

Parfitt, Michael et al. 2009. They also showed the interaction between the sacsin UBL domain and the proteasome by co-immunoprecipiation studies. This interaction appears to be fairly weak as can be seen in the form of the faint band of the proteasomal subunit C-8 in the Figure 1.7.

As a central aim of the research work leading to this thesis, the structure of the sacsin

UBL domain was solved to understand the nature of the interaction between the sacsin

UBL domain and the proteasome. Selenomethionine incorporation in the sacsin UBL domain was achieved, and the anomalous signal from selenium from the MAD data was used to solve the sacsin UBL domain structure. This 2.4 Å structure thus obtained with selenomethionine incorporation served as molecular replacement model to finally solve

49 the 2.1 Å structure of the wild type/native UBL. Surprisingly, the structural of the UBL domain is unusual, as it happens to be a swapped dimer with the exchange of the N- terminus between the two monomers, which is not usually seen in the UBLs. Also, the hydrophobic patch responsible for interaction with proteasome in usual UBL domains is occluded in the sacsin UBL due to the dimer formation. This would make the interaction with the proteasome impossible in the dimer form, and a dimer to monomer switching mechanism would be required for explaining the proteasomal interaction reported by

Parfitt, Michael et al. 2009. PFG-NMR self diffusion measurements eliminated the possibility of the swapping being an artifact of crystallization, as the diffusion coefficient measurements in solution were consistent with the dimer seen in the crystal structure.

Thus confirming the existence of the sacsin UBL as a dimer in solution. No shift in the peaks was observed in between the two 1H–15N HSQC spectra of 15N-labeled UBL 2-85 acquired at two different concentrations, suggesting the existence of the dimer is independent of the protein concentration in the tested range.

The domain swapping seen in the sacsin UBL domain raises further questions and possibilities. Does the domain-swapped sacsin UBL dimer exist only in solution or also in vivo in the full-length sacsin? As mentioned in the purification scheme, on cleaving off the GST tag, the UBL gets distributed in between the precipitate and the solution. Is it the monomer, which, owing to its exposed hydrophobic patch, goes into the precipitate, with only the dimeric form left in the solution? Also, by dissolving the precipitate in 8M guanidium chloride followed by gradual dialysis, a fraction of soluble protein and another of protein precipitate were obtained again. The soluble protein thus obtained was dimeric

50 as confirmed by the NMR spectra (data not shown). Can under certain cellular conditions, such as sacsin acting as a part of the chaperone machinery, a transition between the monomer/dimer take place? Can sacsin form amyloids as is the case with the protein human cystatin-C, in which domain swapping can lead to oligomerization and amyloid deposition. This process is greatly accelerated with a naturally occurring L68Q variant which causes the disease called hereditary Cystatin-C amyloidosis (HCCA); the patients can have cystatin-C dimers present in their blood too (Bjarnadottir, Nilsson et al. 2001;

Janowski, Kozak et al. 2005; Lin, Liu et al. 2007; Liu, Lin et al. 2007). The normal prion proteins are also known to swap their domains leading to the pathological aggregated form (Hafner-Bratkovic, Bester et al. 2011).

The protein sacsin also dimerizes at its C-terminal via the HEPN domain. This also opens the possibility of sacsin forming rings that can help in the formation of the oligomeric

Drp1 assembly around the mitochondrial fission site. Sacsin region 1-1368 is known to interact with Drp1, which is a vital GTPase in mitochondrial fission. Sacsin disruption and Drp1 disruption both cause similar mitochondrial defects. Or can sacsin form an oligomeric chain by involving dimerization at both N-terminus and C-terminus?

The only known case of swapped dimerization in the UBL domains so far is that of

ATG12, but Suzuki, Yoshimoto et al. 2005 observed both monomeric and dimeric forms for ATG12 in the purification scheme. Also, they don’t believe the dimeric form of

ATG12 is biologically relevant. Domain-swapped oligomers for other proteins such as the

SH2 domain of Grb2, the SH3 domain of p47phox and barnase have also been observed,

51 but the biological significance of domain swapping is still not well studied (Zegers,

Deswarte et al. 1999; Schiering, Casale et al. 2000; Yuzawa, Suzuki et al. 2004). The possibilities discussed above need to be examined in the future studies to throw light on the possible role of the sacsin UBL swapped dimer, if any. It is not surprising to see the

UBL domains being associated with novel functions as studies on new UBL domains are carried out, and it might be a possibility with the sacsin UBL domain too.

52 References

Adams, P. D., P. V. Afonine, et al. (2010). "PHENIX: a comprehensive Python-based system for macromolecular structure solution." Acta crystallographica. Section D,

Biological crystallography 66(Pt 2): 213-221.

Altieri, A. S., D. P. Hinton, et al. (1995). "Association of Biomolecular Systems Via

Pulsed-Field Gradient Nmr Self-Diffusion Measurements." Journal of the American

Chemical Society 117(28): 7566-7567.

Anderson, J. F., E. Siller, et al. (2010). "The sacsin repeating region (SRR): a novel

Hsp90-related supra-domain associated with neurodegeneration." Journal of molecular biology 400(4): 665-674.

Anderson, J. F., E. Siller, et al. (2011). "The neurodegenerative-disease-related protein sacsin is a molecular chaperone." Journal of molecular biology 411(4): 870-880.

Anesi, L., P. de Gemmis, et al. (2011). "Two novel homozygous SACS mutations in unrelated patients including the first reported case of paternal UPD as an etiologic cause of ARSACS." Journal of molecular neuroscience : MN 43(3): 346-349.

Bayer, P., A. Arndt, et al. (1998). "Structure determination of the small ubiquitin-related modifier SUMO-1." Journal of molecular biology 280(2): 275-286.

Bjarnadottir, M., C. Nilsson, et al. (2001). "The cerebral hemorrhage-producing cystatin

C variant (L68Q) in extracellular fluids." Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of

Amyloidosis 8(1): 1-10.

Bouchard, J. P. (1991). "Hereditary neuropathies and spinocerebellar atrophies."

Handbook of clinical neurology / edited by P.J. Vinken and G.W. Bruyn 16: 451–459.

Bouchard, J. P., A. Barbeau, et al. (1978). "Autosomal recessive spastic ataxia of

Charlevoix-Saguenay." The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques 5(1): 61-69.

Bouchard, J. P., A. Richter, et al. (1998). Autosomal recessive spastic ataxia of

Charlevoix-Saguenay. Neuromuscular disorders : NMD. 8: 474-479.

Bouhlal, Y., R. Amouri, et al. (2011). "Autosomal recessive spastic ataxia of Charlevoix-

Saguenay: an overview." Parkinsonism & related disorders 17(6): 418-422.

Bouhlal, Y., G. El Euch-Fayeche, et al. (2009). "A novel SACS gene mutation in a Tunisian family." Journal of molecular neuroscience : MN 39(3): 333-336.

54 Bouhlal, Y., M. Zouari, et al. (2008). "Autosomal recessive ataxia caused by three distinct gene defects in a single consanguineous family." Journal of neurogenetics 22(2): 139-148.

Chen, H. and D. C. Chan (2010). "Physiological functions of mitochondrial fusion."

Annals of the New York Academy of Sciences 1201: 21-25.

Cho, D. H., T. Nakamura, et al. (2010). "Mitochondrial dynamics in cell death and neurodegeneration." Cellular and molecular life sciences : CMLS 67(20): 3435-3447.

Chuang, J. Z., H. Zhou, et al. (2002). "Characterization of a brain-enriched chaperone,

MRJ, that inhibits Huntingtin aggregation and toxicity independently." The Journal of biological chemistry 277(22): 19831-19838.

Criscuolo, C., S. Banfi, et al. (2004). "A novel mutation in SACS gene in a family from southern Italy." Neurology 62(1): 100-102.

De Braekeleer, M., F. Giasson, et al. (1993). "Genetic epidemiology of autosomal recessive spastic ataxia of Charlevoix-Saguenay in northeastern

Quebec." Genetic epidemiology 10(1): 17-25.

de Castro, I. P., L. M. Martins, et al. (2011). "Mitochondrial quality control and

Parkinson's disease: a pathway unfolds." Molecular neurobiology 43(2): 80-86.

55 Deveraux, Q., V. Ustrell, et al. (1994). "A 26 S protease subunit that binds ubiquitin conjugates." The Journal of biological chemistry 269(10): 7059-7061.

Durrenberger, P. F., M. D. Filiou, et al. (2009). "DnaJB6 is present in the core of Lewy bodies and is highly up-regulated in parkinsonian astrocytes." Journal of neuroscience research 87(1): 238-245.

El Euch-Fayache, G., I. Lalani, et al. (2003). "Phenotypic features and genetic findings in sacsin-related autosomal recessive ataxia in Tunisia." Archives of neurology 60(7): 982-

988.

Elsasser, S., R. R. Gali, et al. (2002). "Proteasome subunit Rpn1 binds ubiquitin-like protein domains." Nature cell biology 4(9): 725-730.

Emsley, P. and K. Cowtan (2004). "Coot: model-building tools for molecular graphics."

Acta crystallographica. Section D, Biological crystallography 60(Pt 12 Pt 1): 2126-2132.

Engert, J. C., P. Berube, et al. (2000). "ARSACS, a spastic ataxia common in northeastern Quebec, is caused by mutations in a new gene encoding an

11.5-kb ORF." Nature genetics 24(2): 120-125.

56 Engert, J. C., C. Dore, et al. (1999). "Autosomal recessive spastic ataxia of Charlevoix-

Saguenay (ARSACS): high-resolution physical and transcript map of the candidate region in chromosome region 13q11." Genomics 62(2): 156-164.

Estaquier, J. and D. Arnoult (2007). "Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis." Cell death and differentiation 14(6): 1086-1094.

Fang, S. and A. M. Weissman (2004). "A field guide to ubiquitylation." Cellular and molecular life sciences : CMLS 61(13): 1546-1561.

Fogel, B. L. and S. Perlman (2007). "Clinical features and molecular genetics of autosomal recessive cerebellar ataxias." Lancet neurology 6(3): 245-257.

Garcia, A., C. Criscuolo, et al. (2008). "Neurophysiological study in a Spanish family with recessive spastic ataxia of Charlevoix-Saguenay." Muscle & nerve 37(1): 107-110.

Gervais, V., V. Lamour, et al. (2004). "TFIIH contains a PH domain involved in DNA nucleotide excision repair." Nature structural & molecular biology 11(7): 616-622.

Girard, M., R. Lariviere, et al. (2012). "Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay

(ARSACS)." Proceedings of the National Academy of Sciences of the United States

57 of America 109(5): 1661-1666.

Grabbe, C. and I. Dikic (2009). "Functional roles of ubiquitin-like domain (ULD) and ubiquitin-binding domain (UBD) containing proteins." Chemical reviews 109(4): 1481-

1494.

Grieco, G. S., A. Malandrini, et al. (2004). "Novel SACS mutations in autosomal recessive spastic ataxia of Charlevoix-Saguenay type." Neurology 62(1): 103-106.

Grynberg, M., H. Erlandsen, et al. (2003). "HEPN: a common domain in bacterial drug resistance and human neurodegenerative proteins." Trends in biochemical sciences 28(5):

224-226.

Guernsey, D. L., M. P. Dube, et al. (2010). "Novel mutations in the sacsin gene in ataxia patients from Maritime Canada." Journal of the neurological sciences 288(1-2): 79-87.

Hafner-Bratkovic, I., R. Bester, et al. (2011). "Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion." The Journal of biological chemistry 286(14): 12149-12156.

Hageman, J., M. A. Rujano, et al. (2010). "A DNAJB chaperone subfamily with

HDAC-dependent activities suppresses toxic protein aggregation." Molecular cell

37(3): 355-369.

Haririnia, A., R. Verma, et al. (2008). "Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins." Journal of molecular biology

375(4): 979-996.

Hartmann-Petersen, R. and C. Gordon (2004). "Integral UBL domain proteins: a family of proteasome interacting proteins." Seminars in cell & developmental biology 15(2):

247-259.

Hendrickson, W. A., J. R. Horton, et al. (1990). "Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure." The EMBO journal

9(5): 1665-1672.

Husnjak, K., S. Elsasser, et al. (2008). "Proteasome subunit Rpn13 is a novel ubiquitin receptor." Nature 453(7194): 481-488.

Janowski, R., M. Kozak, et al. (2005). "3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets." Proteins 61(3): 570-578.

Kamada, S., S. Okawa, et al. (2008). "Autosomal recessive spastic ataxia of Charlevoix-

Saguenay (ARSACS): novel compound heterozygous mutations in the SACS gene."

Journal of neurology 255(6): 803-806.

Kamionka, M. and J. Feigon (2004). "Structure of the XPC binding domain of hHR23A reveals hydrophobic patches for protein interaction." Protein science : a publication of the

Protein Society 13(9): 2370-2377.

Kleijnen, M. F., R. M. Alarcon, et al. (2003). "The ubiquitin-associated domain of hPLIC-2 interacts with the proteasome." Molecular biology of the cell 14(9): 3868-3875.

Kleijnen, M. F., A. H. Shih, et al. (2000). "The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome." Molecular cell 6(2): 409-419.

Kozlov, G., A. Y. Denisov, et al. (2011). "Structural basis of defects in the sacsin

HEPN domain responsible for autosomal recessive spastic ataxia of

Charlevoix-Saguenay (ARSACS)." The Journal of biological chemistry

286(23): 20407-20412.

Lim, J., T. Hao, et al. (2006). "A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration." Cell 125(4): 801-814.

Lin, M. T. and M. F. Beal (2006). "Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases." Nature 443(7113): 787-795.

60 Lin, Y. M., H. L. Liu, et al. (2007). "Molecular dynamics simulations to investigate the domain swapping mechanism of human cystatin C." Biotechnology progress 23(3): 577-

584.

Liu, Y. and D. Eisenberg (2002). "3D domain swapping: as domains continue to swap."

Protein science : a publication of the Protein Society 11(6): 1285-1299.

Liu, H. L., Y. M. Lin, et al. (2007). "Molecular dynamics simulations of human cystatin

C and its L68Q varient to investigate the domain swapping mechanism." Journal of biomolecular structure & dynamics 25(2): 135-144.

Martin, M. H., J. P. Bouchard, et al. (2007). "Autosomal recessive spastic ataxia of

Charlevoix-Saguenay: a report of MR imaging in 5 patients." AJNR. American journal of neuroradiology 28(8): 1606-1608.

McMillan, H. J., M. T. Carter, et al. (2009). "Homozygous contiguous gene deletion of

13q12 causing LGMD2C and ARSACS in the same patient." Muscle & nerve 39(3): 396-

399.

MIT Course Number: 7.340 http://ocw.mit.edu/courses/biology/7-340-ubiquitination-the-proteasome-and-human- disease-fall-2004/

61 Mrissa, N., S. Belal, et al. (2000). "Linkage to chromosome 13q11-12 of an autosomal recessive cerebellar ataxia in a Tunisian family." Neurology 54(7): 1408-1414.

Murshudov, G. N., A. A. Vagin, et al. (1999). "Efficient anisotropic refinement of macromolecular structures using FFT." Acta crystallographica. Section D, Biological crystallography 55(Pt 1): 247-255.

Ogawa, T., Y. Takiyama, et al. (2004). "Identification of a SACS gene missense mutation in ARSACS." Neurology 62(1): 107-109.

Otsuga, D., B. R. Keegan, et al. (1998). "The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast." The Journal of cell biology 143(2): 333-349.

Otera, H. and K. Mihara (2011). "Discovery of the membrane receptor for mitochondrial fission GTPase Drp1." Small GTPases 2(3): 167-172.

Ouyang, Y., K. Segers, et al. (2008). "Novel SACS mutation in a Belgian family with sacsin-related ataxia." Journal of the neurological sciences 264(1-2): 73-76.

Parfitt, D. A., G. J. Michael, et al. (2009). "The ataxia protein sacsin is a functional co- chaperone that protects against polyglutamine-expanded ataxin-1." Human molecular genetics 18(9): 1556-1565.

62 Reddy, P. H., T. P. Reddy, et al. (2011). "Dynamin-related protein 1 and mitochondrial fragmentation in neurodegenerative diseases." Brain research reviews 67(1-2): 103-118.

Richter, A. M., R. K. Ozgul, et al. (2004). "Private SACS mutations in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) families from Turkey."

Neurogenetics 5(3): 165-170.

Rose, J. M., S. S. Novoselov, et al. (2011). "Molecular chaperone-mediated rescue of mitophagy by a Parkin RING1 domain mutant." Human molecular genetics 20(1): 16-27.

Saeki, Y., A. Saitoh, et al. (2002). "Ubiquitin-like proteins and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis." Biochemical and biophysical research communications 293(3): 986-992.

Schauber, C., L. Chen, et al. (1998). "Rad23 links DNA repair to the ubiquitin/proteasome pathway." Nature 391(6668): 715-718

Schon, E. A. and S. Przedborski (2011). "Mitochondria: the next (neurode)generation."

Neuron 70(6): 1033-1053.

63 Schiering, N., E. Casale, et al. (2000). "Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex." Biochemistry 39(44): 13376-

13382.

Shimazaki, H., K. Sakoe, et al. (2007). "An unusual case of a spasticity-lacking phenotype with a novel SACS mutation." Journal of the neurological sciences 255(1-2):

87-89.

Shimazaki, H., Y. Takiyama, et al. (2005). "A phenotype without spasticity in sacsin related ataxia." Neurology 64(12): 2129-2131.

Smirnova, E., L. Griparic, et al. (2001). "Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells." Molecular biology of the cell 12(8): 2245-

2256.

Smirnova, E., D. L. Shurland, et al. (1998). "A human dynamin-related protein controls the distribution of mitochondria." The Journal of cell biology 143(2): 351-358.

Stejskal, E. O. and J. E. Tanner (1965). "Spin Diffusion Measurements: Spin Echoes in the Presence of a Time-Dependent Field Gradient." Journal of Chemical Physics 42(1):

288-+.

64 Studier, F. W. (2005). "Protein production by auto-induction in high density shaking cultures." Protein expression and purification 41(1): 207-234.

Su, B., X. Wang, et al. (2010). "Abnormal mitochondrial dynamics and neurodegenerative diseases." Biochimica Et Biophysica Acta 1802(1): 135-142.

Suzuki, N. N., K. Yoshimoto, et al. (2005). "The crystal structure of plant ATG12 and its biological implication in autophagy." Autophagy 1(2): 119-126.

Takiyama, Y. (2006). "Autosomal recessive spastic ataxia of Charlevoix-Saguenay."

Neuropathology : official journal of the Japanese Society of Neuropathology 26(4): 368-

375.

Terracciano, A., C. Casali, et al. (2009). "An inherited large-scale rearrangement in SACS associated with spastic ataxia and hearing loss." Neurogenetics 10(2): 151-155.

Upadhya, S. C. and A. N. Hegde (2003). "A potential proteasome-interacting motif within the ubiquitin-like domain of parkin and other proteins." Trends in biochemical sciences

28(6): 280-283.

Vermeer, S., R. P. Meijer, et al. (2008). "ARSACS in the Dutch population: a frequent cause of early-onset cerebellar ataxia." Neurogenetics 9(3): 207-214.

65 Vijay-Kumar, S., C. E. Bugg, et al. (1987). "Structure of ubiquitin refined at 1.8 A resolution." Journal of molecular biology 194(3): 531-544.

Voges, D., P. Zwickl, et al. (1999). "The 26S proteasome: a molecular machine designed for controlled proteolysis." Annual review of biochemistry 68: 1015-1068.

Walters, K. J., P. J. Lech, et al. (2003). "DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a." Proceedings of the National Academy of Sciences of the United States of America 100(22): 12694-12699.

Welchman, R. L., C. Gordon, et al. (2005). "Ubiquitin and ubiquitin-like proteins as multifunctional signals." Nature reviews. Molecular cell biology 6(8): 599-609.

Westermann, B. (2010). "Mitochondrial fusion and fission in cell life and death." Nature reviews. Molecular cell biology 11(12): 872-884.

Wilkinson, C. R., M. Seeger, et al. (2001). "Proteins containing the UBA domain are able to bind to multi-ubiquitin chains." Nature cell biology 3(10): 939-943.

Winn, M. D., G. N. Murshudov, et al. (2003). "Macromolecular TLS refinement in

REFMAC at moderate resolutions." Methods in enzymology 374: 300-321.

66 Yamano, K. and R. J. Youle (2011). "Coupling mitochondrial and cell division." Nature cell biology 13(9): 1026-1027.

Yuan, X., A. Shaw, et al. (2001). "Solution structure and interaction surface of the C- terminal domain from p47: a major p97-cofactor involved in SNARE disassembly."

Journal of molecular biology 311(2): 255-263.

Yuzawa, S., N. N. Suzuki, et al. (2004). "A molecular mechanism for autoinhibition of the tandem SH3 domains of p47phox, the regulatory subunit of the phagocyte NADPH oxidase." Genes to cells : devoted to molecular & cellular mechanisms 9(5): 443-456.

Zegers, I., J. Deswarte, et al. (1999). "Trimeric domain-swapped barnase." Proceedings of the National Academy of Sciences of the United States of America 96(3): 818-822.