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Structural insights into TDP-43 in nucleic-acid binding and domain interactions.

Kuo PH, Doudeva LG, Wang YT, Shen CK, Yuan HS - Nucleic Acids Res. (2009)

Bottom Line: The crystal structure reveals the basis of TDP-43's TG/UG preference in nucleic acids binding.It also reveals that RRM2 domain has an atypical RRM-fold with an additional beta-strand involved in making protein-protein interactions.These studies thus characterize the recognition between TDP-43 and nucleic acids and the mode of RRM2 self association, and provide molecular models for understanding the role of TDP-43 in cystic fibrosis and the neurodegenerative diseases related to TDP-43 proteinopathy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Taipei, Taiwan, ROC.

ABSTRACT
TDP-43 is a pathogenic protein: its normal function in binding to UG-rich RNA is related to cystic fibrosis, and inclusion of its C-terminal fragments in brain cells is directly linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Here we report the 1.65 A crystal structure of the C-terminal RRM2 domain of TDP-43 in complex with a single-stranded DNA. We show that TDP-43 is a dimeric protein with two RRM domains, both involved in DNA and RNA binding. The crystal structure reveals the basis of TDP-43's TG/UG preference in nucleic acids binding. It also reveals that RRM2 domain has an atypical RRM-fold with an additional beta-strand involved in making protein-protein interactions. This self association of RRM2 domains produced thermal-stable RRM2 assemblies with a melting point greater than 85 degrees C as monitored by circular dichroism at physiological conditions. These studies thus characterize the recognition between TDP-43 and nucleic acids and the mode of RRM2 self association, and provide molecular models for understanding the role of TDP-43 in cystic fibrosis and the neurodegenerative diseases related to TDP-43 proteinopathy.

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Domain structures and assembly of TDP-43 proteins. (A) TDP-43 has two RRM domains, RRM1 and RRM2, and a C-terminal glycine-rich domain. In this study, three truncated TDP-43 proteins were constructed: TDP-43s, RRM1 and RRM2. (B) Amino-acid sequences of mouse and human TDP-43 in the RRM1 and RRM2 domains. The secondary structures listed above the sequences are derived from the crystal structure of RRM2–DNA complex. (C) The purity of TDP-43 truncated proteins was assayed by 12.5% SDS–PAGE. (D) RRM2 appeared as a tetramer in the native 20% PAGE gel. (E) Gel filtration (Superdex 200) profiles of TDP-43 truncated proteins show that TDP-43s, RRM1 and RRM2 all had a molecular weight of ∼40 kDa, suggesting that the recombinant TDP-43s was a homodimer, and RRM1 and RRM2 were homotetramers. (F) The GFP-fused TDP-43, with a molecular weight of 69 kDa, was expressed in human 293T cells for size exclusion chromatography analysis. The fractionated cell extract eluted from a Superdex 200 column were blotted by TDP-43 antibodies. GFP-TDP-43 was mainly eluted with a size of a dimer. The molecular weight markers are: aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa).
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Figure 1: Domain structures and assembly of TDP-43 proteins. (A) TDP-43 has two RRM domains, RRM1 and RRM2, and a C-terminal glycine-rich domain. In this study, three truncated TDP-43 proteins were constructed: TDP-43s, RRM1 and RRM2. (B) Amino-acid sequences of mouse and human TDP-43 in the RRM1 and RRM2 domains. The secondary structures listed above the sequences are derived from the crystal structure of RRM2–DNA complex. (C) The purity of TDP-43 truncated proteins was assayed by 12.5% SDS–PAGE. (D) RRM2 appeared as a tetramer in the native 20% PAGE gel. (E) Gel filtration (Superdex 200) profiles of TDP-43 truncated proteins show that TDP-43s, RRM1 and RRM2 all had a molecular weight of ∼40 kDa, suggesting that the recombinant TDP-43s was a homodimer, and RRM1 and RRM2 were homotetramers. (F) The GFP-fused TDP-43, with a molecular weight of 69 kDa, was expressed in human 293T cells for size exclusion chromatography analysis. The fractionated cell extract eluted from a Superdex 200 column were blotted by TDP-43 antibodies. GFP-TDP-43 was mainly eluted with a size of a dimer. The molecular weight markers are: aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa).

Mentions: The TARDBP gene is highly conserved in human, mouse, Drosophila melanogaster and Caenorhabditis elegans (21). Sequence analysis identified two RNA-recognition motifs, RRM1 and RRM2, and a C-terminal glycine-rich domain in TDP-43 (Figure 1), similar to the domain organization of heterogeneous nuclear ribonucleoproteins (hnRNP) family proteins such as hnRNP A1 and A2/B1 (22). RRMs are common RNA-binding motifs, with two highly conserved hexameric and octameric segments denoted respectively as ribonucleoprotein 2 (RNP2) and ribonucleoprotein 1 (RNP1) (23). The conserved RNP segments in TDP-43 are involved in binding to TAR DNA sequences (4) and RNA sequences with UG-repeats (8,24). The glycine-rich C-terminal tail of TDP-43 can interact with the hnRNP family proteins to form the hnRNP-rich complex involved in splicing inhibition (25). Mutation analyses further suggest that the TDP-43 glycine-rich domain is essential for the CFTR exon 9-skipping activity (21,24,25).Figure 1.


Structural insights into TDP-43 in nucleic-acid binding and domain interactions.

Kuo PH, Doudeva LG, Wang YT, Shen CK, Yuan HS - Nucleic Acids Res. (2009)

Domain structures and assembly of TDP-43 proteins. (A) TDP-43 has two RRM domains, RRM1 and RRM2, and a C-terminal glycine-rich domain. In this study, three truncated TDP-43 proteins were constructed: TDP-43s, RRM1 and RRM2. (B) Amino-acid sequences of mouse and human TDP-43 in the RRM1 and RRM2 domains. The secondary structures listed above the sequences are derived from the crystal structure of RRM2–DNA complex. (C) The purity of TDP-43 truncated proteins was assayed by 12.5% SDS–PAGE. (D) RRM2 appeared as a tetramer in the native 20% PAGE gel. (E) Gel filtration (Superdex 200) profiles of TDP-43 truncated proteins show that TDP-43s, RRM1 and RRM2 all had a molecular weight of ∼40 kDa, suggesting that the recombinant TDP-43s was a homodimer, and RRM1 and RRM2 were homotetramers. (F) The GFP-fused TDP-43, with a molecular weight of 69 kDa, was expressed in human 293T cells for size exclusion chromatography analysis. The fractionated cell extract eluted from a Superdex 200 column were blotted by TDP-43 antibodies. GFP-TDP-43 was mainly eluted with a size of a dimer. The molecular weight markers are: aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa).
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Related In: Results  -  Collection

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Figure 1: Domain structures and assembly of TDP-43 proteins. (A) TDP-43 has two RRM domains, RRM1 and RRM2, and a C-terminal glycine-rich domain. In this study, three truncated TDP-43 proteins were constructed: TDP-43s, RRM1 and RRM2. (B) Amino-acid sequences of mouse and human TDP-43 in the RRM1 and RRM2 domains. The secondary structures listed above the sequences are derived from the crystal structure of RRM2–DNA complex. (C) The purity of TDP-43 truncated proteins was assayed by 12.5% SDS–PAGE. (D) RRM2 appeared as a tetramer in the native 20% PAGE gel. (E) Gel filtration (Superdex 200) profiles of TDP-43 truncated proteins show that TDP-43s, RRM1 and RRM2 all had a molecular weight of ∼40 kDa, suggesting that the recombinant TDP-43s was a homodimer, and RRM1 and RRM2 were homotetramers. (F) The GFP-fused TDP-43, with a molecular weight of 69 kDa, was expressed in human 293T cells for size exclusion chromatography analysis. The fractionated cell extract eluted from a Superdex 200 column were blotted by TDP-43 antibodies. GFP-TDP-43 was mainly eluted with a size of a dimer. The molecular weight markers are: aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa).
Mentions: The TARDBP gene is highly conserved in human, mouse, Drosophila melanogaster and Caenorhabditis elegans (21). Sequence analysis identified two RNA-recognition motifs, RRM1 and RRM2, and a C-terminal glycine-rich domain in TDP-43 (Figure 1), similar to the domain organization of heterogeneous nuclear ribonucleoproteins (hnRNP) family proteins such as hnRNP A1 and A2/B1 (22). RRMs are common RNA-binding motifs, with two highly conserved hexameric and octameric segments denoted respectively as ribonucleoprotein 2 (RNP2) and ribonucleoprotein 1 (RNP1) (23). The conserved RNP segments in TDP-43 are involved in binding to TAR DNA sequences (4) and RNA sequences with UG-repeats (8,24). The glycine-rich C-terminal tail of TDP-43 can interact with the hnRNP family proteins to form the hnRNP-rich complex involved in splicing inhibition (25). Mutation analyses further suggest that the TDP-43 glycine-rich domain is essential for the CFTR exon 9-skipping activity (21,24,25).Figure 1.

Bottom Line: The crystal structure reveals the basis of TDP-43's TG/UG preference in nucleic acids binding.It also reveals that RRM2 domain has an atypical RRM-fold with an additional beta-strand involved in making protein-protein interactions.These studies thus characterize the recognition between TDP-43 and nucleic acids and the mode of RRM2 self association, and provide molecular models for understanding the role of TDP-43 in cystic fibrosis and the neurodegenerative diseases related to TDP-43 proteinopathy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Taipei, Taiwan, ROC.

ABSTRACT
TDP-43 is a pathogenic protein: its normal function in binding to UG-rich RNA is related to cystic fibrosis, and inclusion of its C-terminal fragments in brain cells is directly linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Here we report the 1.65 A crystal structure of the C-terminal RRM2 domain of TDP-43 in complex with a single-stranded DNA. We show that TDP-43 is a dimeric protein with two RRM domains, both involved in DNA and RNA binding. The crystal structure reveals the basis of TDP-43's TG/UG preference in nucleic acids binding. It also reveals that RRM2 domain has an atypical RRM-fold with an additional beta-strand involved in making protein-protein interactions. This self association of RRM2 domains produced thermal-stable RRM2 assemblies with a melting point greater than 85 degrees C as monitored by circular dichroism at physiological conditions. These studies thus characterize the recognition between TDP-43 and nucleic acids and the mode of RRM2 self association, and provide molecular models for understanding the role of TDP-43 in cystic fibrosis and the neurodegenerative diseases related to TDP-43 proteinopathy.

Show MeSH
Related in: MedlinePlus