國 立 清 華 大 學
生物資訊與結構生物研究所
Institute of Bioinformatics and structural Biology,
National Tsing Hua University
博士論文
TDP-43 結構與纖維化之研究
TDP-43 domain structure and aggregation studies
研究生：王宜婷 撰
指導教授：袁小琀 博士
中華民國一百年一月
CONTENTS
CONTENTS
I
CONTENT OF TABLES
III
CONTENT OF FIGURES
IV
論文摘要
1
ABSTRACT
2
1. INTRODUCTION
3
2. MATERIALS AND METHODS
6
2.1 Cloning of human TDP-43 constructs
6
2.2 Expression and purification of recombinant hTDP-43
6
2.3 GST pull-down assays
7
2.4 Small angle X-ray scattering(SAXS) experiments and data analysis
8
2.5 Fibril formation
9
2.6 Electron microscopy
9
2.7 Thioflavin T (ThT) binding assay
10
2.8 Anti-amyloid fiber dot blotting
10
2.9 X-ray fiber diffraction
10
3. RESULTS
12
3.1 Domain structure analysis of human TDP-43
12
3.2 Overexpression and purification of human TDP-43
12
3.3 Recombinant TDP-43s forms a homodimer
13
3.4 The oligomer states of TDP-43 determined by SAXS
13
3.5 Overall shape of TDP-43s and RRM1 in solution
14
3.6 The RRM2 peptides form fibrils
15
I
4. DISCUSSION
17
4.1 TDP-43 is a dimeric protein interacting via RRM1 domain
17
4.2 Fibrogenesis of TDP-43 RRM2 peptides
18
4.3 Model of TDP-43 proteinopathy
19
REFERENCES
44
APPENDIX
50
II
CONTENT OF TABLES
Table 1. Experimental and theoretical molecular weights (MW) and
scattering parameters for human TDP-43
III
21
CONTENT OF FIGURES
Figure 1-1. Proposed physiological roles of TDP-43.
22
Figure 1-2. Sequence alignment of human and mouse TDP-43.
23
Figure 1-3. The high versatility of RRM-RRM interactions.
24
Figure 1-4. A model of TDP proteinopathy.
25
Figure 1-5. TDP-43 25-kDa pathological C-terminal fragments.
26
Figure 1-6. The crystal structure of RRM2.
27
Figure 3-1. Secondary structure prediction of hTDP-43 by PSIPRED.
28
Figure 3-2. Functional domain assignment of hTDP-43 by PROSITE.
29
Figure 3-3. Constructs of human TDP-43 made in this thesis.
30
Figure 3-4. SDS–PAGE analysis of purified TDP-43 proteins.
31
Figure 3-5. Gel filtration analysis of purified TDP-43s protein.
32
Figure 3-6. GST pull-down assays suggest that the TDP-43 forms dimer
via RRM1 domain.
33
Figure 3-7. Small Small-angle X-ray scattering (SAXS) profiles of
TDP-43.
34
Figure 3-8. Small-angle X-ray scattering (SAXS) models of TDP-43 and
RRM1.
35
Figure 3-9. Fibrils of RRM2-3 by electron microscopy.
36
Figure 3-10. Fibrils of RRM2-5 by electron microscopy.
37
Figure 3-11. Fibrils of D1s by electron microscopy.
38
Figure 3-12. ThT binding assay of TDP-43 3 and 5 fibrils.
39
Figure 3-13. Dot blotting assays of 3 and5 fibrils.
40
Figure 3-14. X-ray fiber diffraction pattern of 3 fibrils.
41
IV
Figure 4-1. Aggregation propensities of TDP-43 analyzed by Tango.
42
Figure 4-2. The aggregation model of TDP-43.
43
V
論文摘要
TAR 去氧核糖核酸結合酶-43 (TAR DNA-binding protein 43)在正常細胞是一
個多功能的蛋白質，扮演轉錄因子以及調控信使核糖核酸剪接 (mRNA splicing)
的角色。TDP-43 結構包含兩個核糖核酸結合區 (RNA binding motif; RRM)，
RRM1 以及 RRM2，和一個富含甘氨酸的尾端區域 (C-termainal glycin rich
region)。在病理細胞內，TDP-43 形成包含體 (inclusion)，導致某些神經退化性
疾病。在包含體內的 TDP-43 被證實其前端 (N-terminus) 已被去除，而形成一個
大小約為 25 kDa 的片段，此片段包含部分的 RRM2 區域和 glycin rich 區域組成。
TDP-43 如何由正常的功能性蛋白，轉變成纖維狀的異常包含體，其致病機制目
前尚未得知。為了了解 TDP-43 的結構區排列方式，我們使用小角度 X 光散射法
取得了低解析度的 TDP-43s (包含 RRM1 及 RRM2 功能區)結構。再加上 GST
pull-down 檢驗法的得到的實驗數據，研究結果顯示 TDP-43 是以二聚體形式存
在，以頭對頭的方式使兩個 RRM1 功能區做結合，另兩個 RRM2 功能區朝外延
展。此外我們發現 RRM2 結構區裡的3 和5 有能力形成纖維。這些纖維並不能
被 thioflavin T 澱粉樣蛋白纖維染劑 (amyloid-binding dye)，以及抗澱粉樣蛋白纖
維抗體(anti-amyloid antibody) 所辨識，此結果和病理上的 TDP-43 纖維是一致
的。因此，綜合上述結果我們推測在移除 RRM1 功能區和 RRM2 1 後，不正常
摺疊的 RRM2 使3 和5 暴露出來，因而造成 TDP-43 蛋白質不正常堆疊和病理
上纖維的形成。
1
ABSTRACT
TAR DNA-binding protein 43 (TDP-43) is a multifunctional protein, acting as a
transcriptional factor and a splicing regulator in healthy cells. TDP-43 contains two
RNA binding domains, RRM1 and RRM2, and a C-terminal tail region rich in glycine
residues (glycine rich region). In pathological cells, TDP-43 forms inclusions linking to
several neurodegenerative diseases. These TDP-43 inclusions are N-terminally
truncated 25 kDa fragments, which contain partial RRM2 domain and glycine rich
region. The pathogenic mechanism of how the normal functional TDP-43 is truncated
and induced to abnormal filamentous inclusions remains unknown. To investigate the
domain organization of TDP-43, we solved the overall low resolution structure of a
truncated form of TDP-43 with only RRM1 and RRM2 domains (TDP43s) by small
angle X-ray scattering (SAXS). Together with GST pull-down assays, we showed that
the TDP43s dimerized in a head-to-head arrangement, with two RRM1 domains
interacting with each other, and two RRM2 domains flanking outward. Moreover, we
found that 3 and 5 strands in RRM2 were able to form filaments. These filamentous
fibrils did not bind to the amyloid-binding dye thioflavin T, neither anti-amyloid
antibody, consistent with the previous finding for the non-amyloid TDP-43 fibrils
identified in the patient brains. Taken together these results suggest that after the
removal of RRM1 domain and 1 strand in RRM2, 3 and 5 are exposed and thus they
may form pathogenic filamentous inclusions. This finding suggests a potential link
and a new direction for the future study of TDP-43 proteinopathies.
2
1. INTRODUCTION
TDP-43 is a ubiquitously expressed nuclear protein highly conserved in various
species, including human, mouse, Drosophila, and Caenorhabditis elegans (Wang et
al., 2004). It was first identified as a 43 kDa protein that binds to the HIV-1 virus
transactive response (TAR) DNA sequence and acts as a transcriptional repressor (Ou
et al., 1995). Subsequently, TDP-43 has been shown to mediate exon skipping of cystic
fibrosis transmembrane conductance regulator(CFTR) and apolipoprotein A-II (apo
A-II) genes (Buratti et al., 2005; Buratti et al., 2001). TDP-43 has also been reported to
play roles in micro-RNA processing, mRNA transportation and stress granules
(summarized in Figure.1-1) (Lagier-Tourenne et al., 2010). Therefore, TDP-43 is a
multi-functional protein serving various roles in transcription regulation, splicing,
RNA processing, RNA transportation and translation regulation.
TDP-43 consists of two tandem RNA recognition domains (RRM1 and RRM2) and
a C-terminal glycine-rich region (Figure.1-2) with a domain architecture similar to
heterogeneous nuclear ribonucleoproteins (hnRNP) family proteins, which are also
involved in multiple levels of RNA processing, including transcription, splicing and
translation (Krecic and Swanson, 1999). The RRM domain can serve as a RNA binding
module or/and engage in RRM-protein or RRM-RRM interactions (Maris et al., 2005).
Although the RRM domain is one of the most common and well characterized protein
domains in eukaryotes, it is difficult to predict the mode of protein and RNA
recognition by RRMs due to the high variability of these interactions (Cléry et al.,
2008). Several protein structures with two tandem RRM domains have been
determined, including hnRNP A1 (Xu et al., 1997), HuD (Wang and Hall, 2001), and
FBP-interacting repressor (FIR) (Crichlow et al., 2008). The RRM domains in these
3
proteins have distinct domain interactions and orientations for the different purposes of
their biological functions (Figure.1-3). Therefore, it is important to reveal the domain
arrangement of TDP-43, because it may help to elucidate its molecular function.
A breakthrough study showed that TDP-43 is a primary protein component of
ubiquitin-positive tau-negative neuronal inclusions in amyotrophic lateral sclerosis
(ALS) and frontotemporal lobar degeneration (FTLD-U) (Arai et al., 2006; Neumann
et al., 2006). Subsequently TDP-43 inclusions were identified in various forms of
neurodegenerative disorders, including Lewy body disease, Parkinson's disease,
Alzheimer's disease (AD), and Corticobasal degeneration (Gendron et al., 2010). These
disease-specific TDP-43 inclusions are fibrillar like, hyper phosphosphorylated,
ubiquitinated and proteolytically cleaved into N-terminal truncated ~25 kDa fragments.
Recent studies further showed that the 25-kDa C-terminal fragments (CTF) of TDP-43
form toxic inclusions in human cell lines (Igaz et al., 2008; Zhang et al., 2009). In
transgenic mice, the amount of TDP-43 CTF accumulated in cell correlated with the
disease progression, suggesting that the accumulation of aberrant TDP-43 CTF may
lead to neuron dysfunction (Wils et al., 2010) (summarized in Figure1-4).
Two potential cleavage sites of the 25-kDa CTF were described, one located at
Arg208, characterized by the N-terminal sequencing of the urea extracts from FTLD-U
patient brains (Igaz et al., 2009), the other located at Asp219, identified as a caspase-3
cleavage site (Dormann et al., 2009; Nishimoto et al., 2010; Zhang et al., 2007). Both
cleavage sites result in C-terminal fragments with a truncated RRM2 domain and
glycine rich domain (Figure1-5). Previously studies in TDP-43 proteinopathy mainly
focused on the glycine rich region, since most of the TDP-43 mutations in ALS are
located in the glycine-rich region (Kabashi et al., 2008). Biochemistry data also suggest
4
that parts of glycine-rich region, D1 (287-332), are capable of forming twisted fibrils in
vitro (Chen et al., 2010).
Nevertheless, little is known for the involvement of the TDP-43 RRM2 domain in
protein aggregation. The crystal structure of TDP-43 RRM2 domain, as shown in our
previous report (Kuo et al., 2009), contains two α-helices pack against a five-stranded
anti-parallel -sheet with a2-3-1-5-4 topology (Figure1-6). Since the 1 strand
is located at the center of the -sheet, removal of the 1 may lead to destabilizing and
unfolding of the RRM2 domain. Indeed, A broad range of human diseases, including
the amyloidoses and many neurodegenerative diseases are raised from the failure of
protein folding (Chiti and Dobson, 2006).
To understand the TDP-43 proteinopathy, here we used small angle X-ray scattering
(SAXS) to study the native structure of TDP-43. We found that TDP-43 formed a dimer
with two RRM1 domains interacting in the dimeric interface. The fibrilgenesis study
supported that the peptides of RRM2 domain may play a role in protein aggregation and
fiber formation. Base on these results, we propose a model of unfolding of the TDP-43
RRM2 domain that leads to protein aggregation. This result provides a new direction
for the study of TDP-43 proteinopathy.
5
2. MATERIALS AND METHODS
2.1 Cloning of human TDP-43 constructs
The cDNA of the full-length human TDP-43 was obtained from RT-PCR method.
Five constructs for the expression of truncated forms of human TDP-43 were made by
PCR method, including hTDP-43 △N (residues 101-414), hTDP-43 △NRRM1
(residues 192-414), hTDP-43s (residues 101-265), hRRM1 (residues 101-191) and
hRRM2 (residues 192-265). The amplified target gene fragments were purified by
electrophoresis, eluted by the Gel extraction kit (Qiagen), and constructed into a
pQE30 expression vector (Qiagen) with BamHI and HindIII restriction sites. The
N-terminal glutathione-S-transferase (GST) tagged TDP-43s was cloned into a
pGEX-4T-1 expression vector (GE Healthcare) followed by the same procedure.
2.2 Expression and purification of recombinant hTDP-43
The N-terminal tagged recombinant plasmids were transformed into Escherichia
coli M15 strain for protein expression. The transformed bacteria were grown in LB
medium supplemented with 50 g/ml ampicillin, cultured for 4 hours at 37ºC to
OD600 ~ 0.5. Protein expression was induced by 0.8 mM IPTG at 18 ºC for 22 hours.
The harvested cells were resuspended in buffer A (100 mM NaCl, 10 mM
-mercaptoethanol and 50 mM Hepes pH 7.6) and passed through a microfluidizer to
disrupt the cells.
For His-tagged proteins, cell extracts were applied to a Ni-NTA affinity column
(Qiagen) equilibrated with buffer A. His-tagged TDP-43 proteins were eluted with a
linear gradient of the same buffer containing imidazole at increasing concentrations
6
from 0 to 0.5 M. Eluted TDP-43 were dialyzed overnight against 10 mM
-mercaptoethanol and 50 mM Hepes pH 7.6. Afterwards, protein samples were loaded
to a HiTrap heparin column (HiTrap SP column for RRM2) (GE Healthcare), and
eluted using 0 to 1 M NaCl gradient. Finally, the proteins samples were further
dialyzed against A buffer, and injected into a Superdex 200 gel filtration
chromatography column (GE Healthcare).
The GST-TDP-43s fusion protein was immobilized on the GST column with
glutathione-sepharose beads (GE Healthcare) and then eluted from the column with
GST elution buffer (100 mM NaCl, 10 mM -mercaptoethanol, 10 mM reduced
glutathione and 50 mM Hephs pH 7.6), followed by dialysis against buffer A to remove
glutathione.
To test the purity of the proteins, protein samples were mixed with sample loading
buffer (35 mM Tris-HCl, pH 7.0, 1 mM EDTA, 2% SDS, 4% sucrose, 0.002%
bromophenol blue and 6 mM -mercaptoethanol) under a reducing condition. The
protein sample was then subjected for SDS-PAGE electrophoresis in 12.5%
polyacrylamide gel, staining with coomassie blue. The purified proteins were then
concentrated to a suitable concentration with Centri prep (millipore) and stored in 4°C.
2.3 GST pull-down assays
GST-tagged hTDP43-s were incubated respectively with His-tagged hRRM1 and
hRRM2 in 10 mM -mercaptoethanol, 500 mM NaCl and 50 mM Hepes pH 7.6 at 4°C
overnight. The protein samples were mixed gently with 25 l glutathione-sepharose
beads (GE Healthcare) for 1 hour at 4°C. After mixing, the beads were washed three
7
times and eluted with 500 mM NaCl, 10 mM -mercaptoethanol, 10 mM reduced
glutathione and 50 mM Hepes pH 7.6.
Eluted samples were collected, separated using 12.5% SDS-PAGE, and transferred
onto a PVDF membrane for Western blot. GST-tagged TDP-43s and His-tagged hRRM
proteins were probed with anti-GST and anti-His antibodies (Novagen), respectively.
Protein bands were detected by chemiluminescence with an ECL luminescence kit
(Amersham) and visualized by luminescence image analyzer Fuji LAS-1000plus
(Fujifilm).
2.4 Small angle X-ray scattering(SAXS) experiments and data analysis
The synchrotron radiation X-ray scattering data of TDP-43s were collected at
beamline BL23A, National Synchrotron Radiation Research Center (NSRRC), Taiwan.
The sample-to-detector distance was 2.4 m with a wavelength of 0.103 nm (12 keV) to
cover the scattering vector (q) range from 0.05 to 3.1 nm-1, where q = sinθ/λ. The
q-axis was calibrated by the scattering pattern of silver-behenate salt. Protein samples
were prepared in 150 mM NaCl, 10 mM -mercaptoethanol and 50 mM Hepes pH 7.6
and concentrations ranged from 1 to 5 mg /ml for TDP-43-s and from 2.5 to 8 mg/ml for
hRRM1 and hRRM2. Samples were loaded into a 3-mm mica 4-loading rocking cell,
and the exposure time was optimized so as to exclude the interference of protein
aggregations resulted from radiation damage (100 to 300 s).
SAXS data were analyzed by ATSAS 2.4 program suite (X. Wang & Hall, 2001).
The scattering intensity of the buffer was subtracted and the radius of gyration (Rg) and
forward intensity at zero angle I(0) were estimated by PRIMUS (Konarev et al., 2003).
8
The different protein concentration curves were scaled and averaged. The pairwise
distance distribution functions P(r) and the maximum dimension (Dmax) were
calculated using the program GNOM (Svergun, 1992). Ten independent dummy
residue models with P2 symmetry were obtained from Dammin (Svergun, 1999) and
averaged by DAMAVER (Volkov and Svergun, 2003) to generate the ab initio
envelopes. Using the crystal structure of mRRM2 as a searching model, rigid-body
modeling was performed by the program SAXREF (Petoukhov and Svergun, 2005).
2.5 Fibril formation
Peptides of hRRM2 fragments 2 (MDVFIPKPF), 3 (RAFAFVT), 4 (GFDLII),
5 (ISNHISN) and glycine rich domain fragments D1s (FGAFSIN) were synthesized
by MDBio, Inc. Peptide samples (5 mg/ml) were incubated in 20 mM phosphate buffer
(pH7), sonicated for 10 min, and centrifuged for 5 min at 16,100g to remove insoluble
materials. The protein samples was set aside in room temperature for two weeks for
fibril formation and subjected for examination in electron microscopy.
2.6 Electron microscopy
The peptide solutions were examined by using transmission electron microscopy
Tecnai G2 Spirit TWIN（FEI Company）at 120 kV. The protein sample (2 l) was
placed on a 300 square mesh carbon-coated, glow-discharged grids (Electron
Microscopy Scinece) and allowed to be dried by air (~15 min). The grid was washed 6
times with water, and then stained by 2% uranyl acetate (Polyscience. Inc) for 1min
and allowed to be air dried.
9
2.7 Thioflavin T (ThT) binding assay
The fluorescence of fibril samples was measured in 20 mM phosphate buffer, pH7 with
100 M Thioflavin T. The ThT solution was freshly prepared and filtered through a
0.22-m filter (Millipore) before used. The fibril solutions and ThT solution were
mixed together with a 1:1 ratio for five minutes at room temperature. Samples were
excited at 442 nm, and the fluorescence emission intensity was record from 455 to 600
nm using a Carv Eclipse Fluorescence Spectrophothmeter (Varian).
2.8 Anti-amyloid fiber dot blotting
Afibrils used for positive control was a gift from Dr. Yun-Ru Chen (Chen and
Glabe, 2006). TDP-43 fibril samples and Afibrils (2 l) were applied onto
nitrocellulose membrane, and allowed to air dry. Non-specific bindings were blocked
by 5% non-fat milk in TBST solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and
0.1% Tween 20) at room temperature for 30 min. After brief washing with TBST, the
membrane was dipped into 5% milk-TBST with diluted 1:1000 anti-amyloid fibrils OC
antibody (Millipore) and incubated with gentle shaking for 1 hour, and followed by
washing with TBST. Blots were then incubated with anti-rabbit antibody (Amersham)
at a dilution of 1:5000 for 1hour, washed 3 times with TBST, and developed by ECL
luminescence kit (Amersham) and visualized by luminescence image analyzer Fuji
LAS-1000plus (Fujifilm).
2.9 X-ray fiber diffraction
The 3 fibril samples (2 l) were suck into a 0.7-mm diameter glass capillary
10
(Charles Supper Company). The capillary tube was then sealed by melted wax at the
end, and was fixed on clay to keep the capillary positioning in a horizontal way.
Sample was allowed to air dry in room temperature for approximately one day. The
sample was mounted on a goniometer and collected using in hose X-ray equipment
(Saturn 944+CCD Detector with FR-E+ SuperBright X-ray generator).
11
3. RESULTS
3.1 Domain structure analysis of human TDP-43
To characterize the secondary and domain structure of human TDP-43, the
secondary
structure
of
hTDP-43
was
predicted
by
PSIPRED
(http://bioinf.cs.ucl.ac.uk/psipred/) (Jones, 1999), and functional domain assignment
were performed by PROSITE (http://expasy.org/prosite/) (Sigrist et al., 2010) and
SMART (http://smart.embl.de/smart/) (Letunic et al., 2009) (Figure 3-1; Figure 3-2).
The results showed that the TDP-43 contains four regions: (1) N-terminal domain
(residues 1-100) with unknown function; (2) RRM1 domain (residues 104-191), which
is a RNA binding domain; (3) RRM2 domain (residues 192-262), which is the second
RNA binding domain; (4) Glycine-rich C-terminus (274-414), contains many glycine
residues, mainly a random coil region.
3.2 Overexpression and purification of human TDP-43
Several TDP-43 constructs were made for this study, including hTDP-43 △N
(residues 101-414), hTDP-43△NRRM1 (residues 192-414), hTDP-43s (residues
101-265), hRRM1 (residues 101-191) and hRRM2 (residues 192-265) (Figure 3-3).
These truncated TDP-43 were overexpressed in E. coli M15 strain and purified by
chromatographic methods. Among these, proteins containing glycine rich domain,
hTDP-43 △N and hTDP-43△NRRM1, were highly unstable, and were degraded
within 3 days after purification. The recombinant proteins of hTDP-43s, hRRM1 and
hRRM2 were considerably more stable and suitable for further studies. The molecular
masses of hTDP-43s, hRRM1 and hRRM2 were 20.6, 11.1 and 10.3 kDa, respectively.
12
The homogeneity of the protein samples were examined by SDS-PAGE, shown a
single band with high homogeneity (Figure 3-4).
3.3 Recombinant TDP-43s forms a homodimer
The oligomeric state of hTDP-43s was analyzed by S-200 gel filtration
chromatography. By comparison with marker proteins profile, the estimated molecular
weight was approximated 40 kDa, suggesting that the recombinant hTDP-43s was a
homodimer (Figure 3-5). To further clarify the intermolecular interaction of TDP-43, a
GST-tagged hTDP-43s was made for GST pull-down assays. GST-hTDP43s were
mixed with His-tagged RRM1 or RRM2, and loaded into glutathione beads, followed by
intense washing to remove the non-specific binding. The pull-down solution was probed
by anti-His antibodies showing that RRM1 interacted with TDP-43s, but RRM2 did not.
This result suggests that TDP-43 forms a homodimer via the interactions between
RRM1 domains (Figure 3-6).
3.4 The oligomer states of TDP-43 determined by SAXS
To determine the domain arrangement of TDP-43s, small angle X-ray scattering
(SAXS) was performed. SAXS profiles for His-tagged TDP-43, hTDP-43s, hRRM1
and hRRM2, were collected for scattering vector values (q) ranging from 0.02 to 0.3 Å-1.
The forward intensity at zero angle I(0) is proportional to the molecular mass of the
scattering intensity, together with radius of gyration (Rg), that makes SAXS a perfect
method to determine the oligomeric state of the proteins (Putnam et al.). The I(0) and
Rg values were calculated by PRIMUS (Konarev et al., 2003), and then the
13
experimental molecular masses of the proteins were calculated directly from I(0). The
theoretical R(g) value of one RRM domain (crystal structure of RRM2, pdb entry
3D2W) was 14 Å and two RRM domain (crystal structure of hnRNPA1, pdb entry
1UP1) was 19 Å, calculated by CRYSOL (Svergun et al., 1995). The SAXS data
showed that TDP-43s and RRM1 were homodimers, and RRM2 was monomers (Table
1). These results were consistent with our gel filtration analyses and GST pull-down
data as shown above.
Taken together these results suggest that TDP-43s is a
homodimer and the dimeric interactions are mediated through RRM1 domains.
The raw data I(q) in SAXS can be converted into Pair-distance distribution
functions P(r) by the Fourier transform. P(r) represents all of the distances between
each electrons within the macromolecular structure. The P(r) function of TDP-43s,
RRM1 and RRM2 revealed a bell shape distributions, which were typically seen for a
globular protein, with a maximum dimension (Dmax) of 95, 65 and 55 Å, respectively
(Figure 3-7; Table 1).
3.5 Overall shape of TDP-43s and RRM1 in solution
In order to understand the domain organization of TDP-43, low resolution dummy
atom models were built using the Ab initio shape determination program DAMMIN
(Svergun, 1999). Ten independent reconstructions were performed to yield
reproducible models. Models with normalized spatial discrepancy value (NSD)
value<1 were chosen and averaged by DAMAVER (Volkov and Svergun, 2003). The
final models are display in Figure 3-8 showing an elongated structure with a bulged
region in the center and slim regions at the two sides.
14
The crystallographic and NMR structures of individual domains (RRM1, pdb entry
2CQG; RRM2, pdb entry 3D2W) were solved. Thus, an alternative approach for
modeling TDP-43s and RRM1 structures were accomplished by using the rigid body
refinement program SASREF. The molecular model from SASREF were similar to the
envelop model obtained from DAMMIN. The two RRM1 domains formed a
globular-shaped dimer, which can be fitted into the bulged region in the TDP-43s
structure. Therefore, taken together these and GST pull-down results, suggest that
TDP-43s has a dimeric structure with RRM1 interacting with each other and RRM2
franking outward (Figure 3-8).
3.6 The RRM2 peptides form fibrils
Based on the GST-pull down assays and SAXS studies, we have shown that the
RRM2 domain did not interact with each other in TDP-43. It is possible that the
truncation of the N-terminal domain of TDP-43 releases free RRM2 domain for
abnormal aggregation. To test if any of the strands in RRM2 domain can contribute
to protein aggregation, each  strand of RRM2 was synthesized for fiber formation
study. Solutions of RRM2 2, 3, 4 and 5 were incubated in the phosphate buffer at
room temperature for 2 weeks. As revealed by negative stained electron microscope,
only 3 and 5 formed fibrils (Figure 3-9; Figure 3-10). Both 3 and 5 form sheet like,
long (>1m) and straight fibrils. The diameters of the 3 and 5 filaments were 5 and 7
nm, thinner than the pathology TDP-43 fibrils (11 nm) (Thorpe et al., 2008).
A previous study reported that a 40 amino-acid glycine rich region D1 forms fibrils
in phosphate buffer (Chen et al., 2010). Compared with those disease fibrils as
15
described in literature, forming of fibrils can be achieved by shorted sequence (Rochet
and Lansbury, 2000). Thus, a shorter D1 (D1s) was synthesized by the prediction of
WALTZ (http://waltz.vub.ac.be/) (Maurer-Stroh et al., 2010), an online server for
finding amylogenic regions in protein sequence. The D1s was synthesized and
investigated for fiber formation followed the same procedure as described above.
Unlike fibrils from RRM2 3 and 5, the D1s formed wider (11 nm) and twisted fibrils
(Figure 3-11).
The TDP-43 pathologenic fiber extracted from patient brains intriguingly cannot be
stained by amyloid detecting dyes, such as Thioflavin T (ThT) (Neumann, 2009). The
filament solutions of 3 and 5 were tested by ThT binding assays (Figure 3-12) and a
dot blotting assay probing by amyloid fiber structure-specific antibody, OC (Figure
3-13). An amyloid fiber made of A was used for a positive control (Naiki et al.,
1989). Both assays showed that 3 and 5 fibrils cannot be stained by ThT or blotted
by amyloid-specific antibodies, suggesting that the 3 and 5 fibers were structurally
dissimilar to the amyloid fibrils.
To verify the structure of TDP-43 fibrils, X-ray fiber diffraction experiments were
performed. Partially aligned -amyloid fibrils give a typical diffraction pattern with
meridional reflections at 4.68 Å and equatorial reflections at 9.8 Å, signifying a
cross-beta structure. The meridional reflections correspond to the spacing of adjacent
 strands which are parallel to the fibril axis. The equatorial reflections represent the
spacing of stacked -sheets perpendicular to the fiber axis (Serpell and Fraser, 1999).
The x-ray diffraction patterns from 3 fibrils showed two rings at a spacing of 4.7 Å
and 9.8 Å (Figure 3-14), similar to the pattern of amyloid fibrils.
16
4. DISCUSSION
4.1 TDP-43 is a dimeric protein interacting via RRM1 domain
Several tandem RRM motifs protein structures have been reported, including
hnRNP A1 (Xu et al., 1997), Hrp1 (Pérez-Cañadillas, 2006), FIR (Crichlow et al.,
2008), HuD (Wang and Hall, 2001), Sxl (Handa et al., 1999). The SAXS data revealed
that the TDP-43 exhibited a novel domain organization with head-to-head oriented
RRM1 and separated RRM2 domains, that has not been reported in previous studies of
any tandem RRM structure.
Although TDP-43 belongs to the hnRNP superfamily, unlike hnRNP A1 which
forms dimers only when bound with single-stranded nucleic acids, TDP-43 forms
dimer naturally without the binding of nucleic acids. This stable dimeric RRM1
structure may allow the recognition of longer nucleotide sequences. As the binding
length of a single RRM domain ranging from 2 to 8 nucleotides, combination of two
RRMs allows the extension of recognition for long sequences (Cléry et al.). Our
previous data shown that the mTDP-43s prefers binding with six UG repeats (12
nucleotides; Kd=14 nM) rather than four UG repeats (8 nucleotides; Kd=115 nM)
(Kuo et al., 2009), indicating that the TDP-43 needs two subunits dimerized for nucleic
acid recognition.
The function of RRM2 is still unclear. Recent study showed that the binding
between TDP-43 and FUS/TLS requires both glycine-rich and RRM2 domain of
TDP-43 (Kim et al., 2010), suggesting that the RRM2 domain may participate in
protein-protein interaction. Thus, the outward position of RRM2 may facilitate the
recruiting of TDP-43 interacting proteins.
17
4.2 Fibrogenesis of TDP-43 RRM2 peptides
The pathogenic TDP-43 inclusions are N-terminally truncated 25-kDa fragments,
which contain the partial RRM2 domain and glycine rich domain. In this study, we
tasted the fiber formation ability of the peptides from RRM2  strand region and the
glycine rich region. We found that the peptides from both regions were able to form
filaments, implying that both the truncated RRM2 domain and the glycine rich domain
may be involved in TDP-43 fiber formation.
The RRM2 β3 and β5 filaments showed a similar characteristic to that of disease
TDP-43 fibrils that they are failing to be stained by amyloid-detecting dye Thioflavin
T. To learn more about the structure of 3 and 5 filaments, an amyloid fiber
structure-specific antibody OC was used to validate the structure feature of these fibrils.
The β3 and β5 filaments cannot be recognized by the OC antibody suggesting that these
filaments were structurally different to amyloid fibers. On the other hand, the X-ray
fiber diffraction revealed that the 3 filaments have the signature diffraction pattern of
amyloid fibers at 4.7 and 9.8 Å. This result implies that 3 filaments may share some
similar structural features to those of amyloid fibers. We thus suggested that the
TDP-43 filaments have unique features that they are “amyloid-like filaments” which
however cannot be stained by ThT.
Judging from the diameter of TDP-43 filaments, the 3 and 5 fibrils (Fiesel et al.)
were thinner than the TDP-43 fibrils in patient brains (Fiesel et al., 2010). The
difference in thickness may result from different hierarchical assembly state in fiber
formation (Stromer and Serpell, 2005). Amyloid fibrils (10 nm in diameter) are
18
suggested to be composed of four narrower fibrillar units (3 nm in diameter), so called
protofilament, to making up the mature fibril (Blake and Serpell, 1996). The 3 and 5
fibrils form sheet-like arrangement, resemble to the protofilaments of amyloid fibrils
(Figure 3-9; Figure 3-10) (Stromer and Serpell, 2005). The protofilaments of 3 and 5
likely lack the critical interactions between protofilaments, due to the incomplete short
-sheet used in this study. Longer peptides sequences, such as a combination of 3 and
5 or an extended sequence with 2 and 4, are necessary to be further synthesized
and tested for fiber formation.
To confirm the aggregate probity of RRM2 domain in 25 kDa fragment, the
sequence of the C-terminal fragment, including hRRM2 and glycine rich domain
(192-414), were subject to a web server, TANGO (http://tango.crg.es/) for analysis.
The TANGO algorithm has been shown to have an accuracy of more than 80% in
predicting the aggregation propensities, and correctly predicted several human disease
related peptide segments, such as Alzheimer
-peptide and transthyretin
(Fernandez-Escamilla et al., 2004). The result showed that the 3 of hRRM2 (228-235)
has high propensities in beta aggregation >90% (Figure 4-1). Taken together with our
experiment data, we believe that the truncated RRM2 domain may take part in
aggregation and fiber formation.
4.3 Model of TDP-43 proteinopathy
Proteins misfolding disease (or protein conformation disease) are all started from the
misfolding of the native functional conformation state (Chiti and Dobson, 2009). The
conformation changes can be induced by shifting from native oligomeric state to
19
abnormal monomeric state. For example, the transthyretins (TTR) is normally a
homotetramer protein. When it dissociates into monomiric state, the locally unfolded
monomer assembles into amyloid fibril (Quintas et al., 2001) and leads to several
diseases, including familial amyloidotic polyneuropathy (FAP), senile systemic
amyloidosis (SSA) and familial amyloid cardiomyopathy (FAC) (Benson and Kincaid,
2007).
Proteolysis is another way commonly seen to trigger protein misfolding in amyloid
fibril proteins. For instance, A derived from amyloid precursor protein (APP) by
cleavage of secretases (Teplow, 1998). Internal cleavage products of gelsolin result in
the exposure of its β strands, which are normally buried in the core of gelsolin, and
produce a 71-residue fragment that forms amyloid fibrils in humans (Ratnaswamy et al.,
1999).
The crystal structure of TDP-43 RRM2 domain (Kuo et al., 2009) gave us a hint that
the cleavage in the middle of RRM2 may lead to conformational change and
misfolding of the protein. Base on the data shown above, we propose a model of
TDP-43 misfolding and aggregation. In native state, TDP-43 is dimerized via the
RRM1 domain with the two RRM2 domains separated and flanking at the two sides. In
disease state, the RRM1 domain and the 1 strand in RRM2 are removed by the
cleavage, and afterwards TDP-43 dissociates into dysfunctional monomer. The
exposed RRM2 3, 5 and/or glycine rich domain D1s may then be cooperated in
protein aggregation, and consequently form the pathogenic fibrils (Figure 4-2).
20
Calculated
MW from SAXS
MW (kDa)
(kDa)
TDP43s
20
RRM1
RRM2
Rg (Å)
Dmax (Å)
Oligomer state
41
23.9
95
Dimer
11
20
18.4
65
Dimer
10
12
14.9
55
Monomer
Table 1. Experimental and theoretical molecular weights (MW) and scattering
parameters for human TDP-43.
*TDP43, residues 101 to 265; RRM1, resides 101 to 191; RRM2 residues 192 to 265.
21
Figure 1-1. Proposed physiological roles of TDP-43.
TDP-43 is a multi-functional protein. In nuclei, TDP-43 binds DNA functioning as a
transcription regulator and binds RNA participating in mRNA alternative splicing. In
cytosol, TDP-43 plays roles in mRNA transportation to synaptic sites and mRNA
inactivation in processing (P)-bodies.
22
Figure 1-2. Sequence alignment of human and mouse TDP-43.
The amino acid sequences of human TDP-43 and mouse TDP-43 (mTDP-43) are
aligned with high sequence identity (97%). The two RRM domains and glycine-rich
regions are boxed.
23
Figure 1-3. The high versatility of RRM-RRM interactions.
Structures of HuD (PDB entry: 1FXL), hnRNP A1 (PDB entry: 1U1Q) and FIR
RRMs (PDB entry: 2QFJ) in complex with their ssRNA/DNA substrates. The nucleic
acids are colored in yellow. HuD is a monomeric protein and it binds RNA with a
high affinity using two RRM domains (RRM1 and RRM2). hnRNP A1 forms dimer
with head-to-tail conformation. While the FIR dimerized with head-to-head
orientation with RRM1 participates in nucleic acid binding and RRM2 contributes to
protein-protein interactions.
24
Figure 1-4. A model of TDP proteinopathy.
TDP-43 protein is improperly processed and/or translocated from the nucleus to the
cytoplasm and forms detergent-insoluble urea-soluble inclusions. TDP-43 filaments
are hyper phosphosphorylated, ubiquitinated and proteolytically cleaved into an
N-terminal truncated ~25 kDa fragments.
25
Figure 1-5. TDP-43 25-kDa pathological C-terminal fragments.
After cleavage, the TDP-43 C-terminal fragments contain a truncated RRM2 domain
and glycine rich domain. The proposed cutting sites of 25-kDa pathological
C-terminal fragments, identified by N-terminal sequencing (amino acid 208) and
caspase-3 cleavage (amino acid 220), are marked by arrows.
26
Figure 1-6. The crystal structure of RRM2.
Overall structure of the TDP-43 RRM2 domain contains two α-helices and five
-strands arranged in a topology of 2-3-1-5-4 (pdb entry 3D2W).
27
Figure 3-1. Secondary structure prediction of hTDP-43 by PSIPRED.
The prediced secondary structures are shown in pink rods for α-helices, yellow arrows
for β-strands and lines for coil structures. The blue bars represent confidence level of
the prediction. Sequence from 262 to 414 has merely no secondary structure,
suggesting a long and flexible region in the tail of the TDP-43.
28
Figure 3-2. Functional domain assignment of hTDP-43 by PROSITE.
The domains architecture of TDP-43 was analyzed by PROSITE. The TDP-43
contains an N-terminal function-unknown domain (colored in gold) and two RNA
binding domain, RRM1 and RRM2 (colored in green), and a glycine rich region in
C-terminal end (colored in blue).
29
Figure 3-3. Constructs of human TDP-43 made in this thesis.
Several constructs were made for this study. The domain of N-terminal, RRM1,
RRM2, glycine-rich are shown in purple, green, yellow and blue, respectively.
30
Figure 3-4. SDS–PAGE analysis of purified TDP-43 proteins.
The purified recombinant TDP-43 proteins were analyzed by SDS-PAGE. All the
proteins are highly homogeneous with a single band. The molecular masses of
hTDP-43s, hRRM1 and hRRM2 are 20.6, 11.1 and 10.3 kDa, respectively. Markers
(M) (kD) are shown as indicated.
31
Figure 3-5. Gel filtration analysis of purified TDP-43s protein.
Gel filtration (Superdex 200) profiles of TDP-43s. Comparing with the marker profile,
TDP-43 were eluted with a molecular weight between 43 kDa and 25 kDa, suggesting
that the recombinant TDP-43s is a homodimer (~ 40kDa).
32
Figure 3-6. GST pull-down assays suggest that the TDP-43 forms dimer via
RRM1 domain.
The GST-tagged TDP-43s was used as a bait to pull down the His-tagged RRM1 or
RRM2. The GST-tagged TDP-43s was incubated over night with His-tagged RRM1
and RRM2, and then pulled down by glutathione-sepharose beads. The result was
analyzed by Western blotting using anti-GST and anti-His antibodies.
33
Figure 3-7. Small Small-angle X-ray scattering (SAXS) profiles of TDP-43.
Figures illustrated in the left panel are the experimental scattering curves of TDP-43s,
RRM1 and RRM2, showing no signal of aggregations. The P(r) functions of TDP-43s,
RRM1 and RRM2, with a maximum dimension (Dmax) of 95, 65 and 55 Å,
respectively, are shown in the right panel.
34
Figure 3-8. Small-angle X-ray scattering (SAXS) models of TDP-43 and RRM1.
Dummy atom models of TDP-43s and RRM1 in solution determined from DAMMIN
(left panel) and the Rigid body modeling of TDP-43s and RRM1, using the
crystallographic structures of RRM1 (pdb entry 2CQG) and RRM2 (pdb entry 3D2W)
(middle panel) as the starting model. Superimpose of two models (right panel).
35
Figure 3-9. Fibrils of RRM2-3 by electron microscopy.
Solutions of RRM2 2, 3, 4, 5 and D1s were incubated in the 20 mM phosphate
buffer at room temperature for 2 weeks. All samples were visualized by electron
microscope. 3 form sheet-like, long (>1μm) and straight fibrils. The diameter of the
3 filament is 5 nm. The lower panel is an enlarged section, showing a sheet-like
arrangement resemble to those protofilaments of amyloid fibrils
36
Figure 3-10. Fibrils of RRM2-5 by electron microscopy.
5 form sheet-like, long (>1μm) and straight fibrils. The diameter of 5 filaments is 7
nm. The lower panel is an enlarged section, showing a sheet-like arrangement resemble
to those protofilaments of amyloid fibrils.
37
Figure 3-11. Fibrils of D1s by electron microscopy.
D1s is a shorter form of D1. D1s forms twisted fibrils similar to the D1 fibrils reported
earlier (Chen et al., 2010). The diameter of the D1s filament is 11 nm.
38
A


Figure 3-12. ThT binding assay of TDP-43 3 and 5 fibrils.
The Thioflavin T (ThT) fluorescence assays showed that only Ahad the fluorescence
emission signal, whereas and had no signal of binding to the ThT dyes. Awas
from amyloid beta-peptides 1-40. and  were the two peptides from the RRM2
domain in TDP-43.
39
Figure 3-13. Dot blotting assays of 3 and 5 fibrils.
A,  and  are dipped onto a nitrocellulose membrane to perform dot blotting assays.
The samples were probed by amyloid fiber structure specific antibody, OC. Only A
sample can be recognized by anti-OC, whereas and fiberscannot be recognized
by OC.
40
(a)
(b)
Figure 3-14. X-ray fiber diffraction pattern of 3 fibrils.
(a) The fiber diffraction pattern of 3 with the d-spacing of 4.7 Å and 9.8 Å. (b)
Architecture of the cross beta structure. Arrows represent β-strands.
41
Beta aggregation propensities
RRM2
Glycine rich
100
ß3
80
60
40
20
0
200
250
300
350
400
Residue number
Figure 4-1. Aggregation propensities of TDP-43 analyzed by Tango.
The β3 of RRM2 (228-235) has high propensities in beta aggregation (>90%) as
analyzed by Tango (Fernandez-Escamilla et al., 2004). The locations of β-strands in
RRM2 are illustrated as black boxes.
42
Figure 4-2. The aggregation model of TDP-43.
In native state, TDP-43 is dimerized via the RRM1 domain with the two RRM2
domains separated and flanking at the two sides. In disease state, the RRM1 and the
1 strand in RRM2 are removed by protein processing, and afterwards, the exposed 3
and 5 in RRM2 may abnormally aggregate into pathogenic fibrils.
43
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APPENDIX
Structural basis for the sequence preference of the nonspecific endonucleases
ColE7 and Vvn
Wang, Y.-T., Yang, W.-J., Li, C.-L., Doudeva, L. G. and Yuan*, H. S. (2007).
Structural basis for sequence-dependent cleavage by nonspecific endonucleases.
Nucleic Acid Res. 35, 584-594.
Wang, Y.-T., Wright, J. D., Doudeva, L. G., Chen, H.-C., Lim*, C. and Yuan*, H. S.
(2009). Design high-affinity nonspecific nucleases with altered sequence
preference. J. Am. Chem. Soc. 131, 17345-17353.
Structure determination and refinement of CRN-5 and Rrp46
Yang, C.-C, Wang, Y.-T., Hsiao, Y. Y., Doudeva, L. G., Kuo, P.-H., Chow, S. Y. and
Yuan*, H. S. (2010). Structural and biochemical characterization of CRN-5 and
Rrp46: an exosome component participating in apoptotic DNA degradation. RNA.
16, 1748-1759
Structure determination of TDP-43 RRM2 domain
Kuo, P,-S, Doudeva, L. G., Wang, Y.-T., Shen, C.-K. J. and Yuan*, H. S. (2009).
Structural insights into TDP-43 in nucleic acid binding and domain interactions.
Nucleic Acids Res. 37, 1799-1808
50