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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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Crystal structure of RRM2–DNA complex. (A) A ribbon model of RRM2 dimer bound to single-stranded DNAs. Molecule a (in blue) is related to molecule b (in red) by a 2-fold crystallographic symmetry axis. DNA molecules are displayed as stick models. A classical RRM domain contains four β-strands (β2-β3-β1-β5) as marked by the rectangle on the top of the structure; however, TDP-43 RRM2 has an extra β4 strand next to β5. (B) A pair of hydrogen bonds are formed between the main-chain atoms of Asp247 (Asp247-O to Asp247-N), and a pair of hydrogen bonds are formed between Glu245 (Oε2) and Ile249 (N) between the two antiparallel β4 strands to stabilize the RRM2 dimeric structure. (C) The RRM2 domain (monomer a) also interacts with the neighboring 2-fold symmetry-related molecule (monomer e). This view is rotated ∼90 degrees vertically to that of panel A. (D) Four hydrogen bonds were formed between monomer a and e: Gln209 (Nε2) to Cys212 (O), and Glu208 (Oε1) to Cys (Sγ).
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Figure 4: Crystal structure of RRM2–DNA complex. (A) A ribbon model of RRM2 dimer bound to single-stranded DNAs. Molecule a (in blue) is related to molecule b (in red) by a 2-fold crystallographic symmetry axis. DNA molecules are displayed as stick models. A classical RRM domain contains four β-strands (β2-β3-β1-β5) as marked by the rectangle on the top of the structure; however, TDP-43 RRM2 has an extra β4 strand next to β5. (B) A pair of hydrogen bonds are formed between the main-chain atoms of Asp247 (Asp247-O to Asp247-N), and a pair of hydrogen bonds are formed between Glu245 (Oε2) and Ile249 (N) between the two antiparallel β4 strands to stabilize the RRM2 dimeric structure. (C) The RRM2 domain (monomer a) also interacts with the neighboring 2-fold symmetry-related molecule (monomer e). This view is rotated ∼90 degrees vertically to that of panel A. (D) Four hydrogen bonds were formed between monomer a and e: Gln209 (Nε2) to Cys212 (O), and Glu208 (Oε1) to Cys (Sγ).

Mentions: RRM2 was a tetramer in low salt conditions, however, RRM2 appeared as a dimer in the crystals grown from acidic high-salt conditions. A gel filtration analysis further confirmed that RRM2 indeed dissociated into dimers under crystallization conditions of 0.1 M phosphate-citrate and 2.0 M (NH4)2SO4 at pH 4.2 (data not shown). The ribbon model of RRM2 dimer is shown in Figure 4A, where RRM2 molecules a and b are related to each other by a crystallographic 2-fold symmetry. A typical RRM contains a four-stranded β-sheet (23), however, RRM2 in TDP-43 has an additional β4 next to β5 (β2-β3-β1-β5-β4). Thus, the RRM2 has a αβ sandwich structure containing a five-stranded β-sheet packed with two α-helices. The dimeric interface is formed mainly through the interactions of the two antiparallel β4 strands (residues 245–250), with a buried solvent accessible surface area of 685 Å2. Two hydrogen bonds were formed between the main-chain atoms of Asp247 (Asp247-O to Asp247-N, a to b and b to a), and two more hydrogen bonds were formed between Glu245 (Oε2) and Ile249 (N) in the two antiparallel β4 strands to stabilize the dimeric structure (Figure 4B). The RRM2 dimer thus has an extended β-sheet consisting of 10 antiparallel β-strands. The RRM2 molecule (monomer a) also interacted with another 2-fold symmetry-related RRM2 molecule (monomer e) via β2 and α1 (Figure 4C and D). Four hydrogen bonds (a to e and e to a) were formed between the side-chain atoms in α1 helix: Gln209 (Nε2) to Cys212 (O), and Gln208 (Oε1) to Cys212 (Sγ). The buried surface between monomer a and e was 901 Å2, slightly higher than the interface between monomer a and b.Figure 4.


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)

Crystal structure of RRM2–DNA complex. (A) A ribbon model of RRM2 dimer bound to single-stranded DNAs. Molecule a (in blue) is related to molecule b (in red) by a 2-fold crystallographic symmetry axis. DNA molecules are displayed as stick models. A classical RRM domain contains four β-strands (β2-β3-β1-β5) as marked by the rectangle on the top of the structure; however, TDP-43 RRM2 has an extra β4 strand next to β5. (B) A pair of hydrogen bonds are formed between the main-chain atoms of Asp247 (Asp247-O to Asp247-N), and a pair of hydrogen bonds are formed between Glu245 (Oε2) and Ile249 (N) between the two antiparallel β4 strands to stabilize the RRM2 dimeric structure. (C) The RRM2 domain (monomer a) also interacts with the neighboring 2-fold symmetry-related molecule (monomer e). This view is rotated ∼90 degrees vertically to that of panel A. (D) Four hydrogen bonds were formed between monomer a and e: Gln209 (Nε2) to Cys212 (O), and Glu208 (Oε1) to Cys (Sγ).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Crystal structure of RRM2–DNA complex. (A) A ribbon model of RRM2 dimer bound to single-stranded DNAs. Molecule a (in blue) is related to molecule b (in red) by a 2-fold crystallographic symmetry axis. DNA molecules are displayed as stick models. A classical RRM domain contains four β-strands (β2-β3-β1-β5) as marked by the rectangle on the top of the structure; however, TDP-43 RRM2 has an extra β4 strand next to β5. (B) A pair of hydrogen bonds are formed between the main-chain atoms of Asp247 (Asp247-O to Asp247-N), and a pair of hydrogen bonds are formed between Glu245 (Oε2) and Ile249 (N) between the two antiparallel β4 strands to stabilize the RRM2 dimeric structure. (C) The RRM2 domain (monomer a) also interacts with the neighboring 2-fold symmetry-related molecule (monomer e). This view is rotated ∼90 degrees vertically to that of panel A. (D) Four hydrogen bonds were formed between monomer a and e: Gln209 (Nε2) to Cys212 (O), and Glu208 (Oε1) to Cys (Sγ).
Mentions: RRM2 was a tetramer in low salt conditions, however, RRM2 appeared as a dimer in the crystals grown from acidic high-salt conditions. A gel filtration analysis further confirmed that RRM2 indeed dissociated into dimers under crystallization conditions of 0.1 M phosphate-citrate and 2.0 M (NH4)2SO4 at pH 4.2 (data not shown). The ribbon model of RRM2 dimer is shown in Figure 4A, where RRM2 molecules a and b are related to each other by a crystallographic 2-fold symmetry. A typical RRM contains a four-stranded β-sheet (23), however, RRM2 in TDP-43 has an additional β4 next to β5 (β2-β3-β1-β5-β4). Thus, the RRM2 has a αβ sandwich structure containing a five-stranded β-sheet packed with two α-helices. The dimeric interface is formed mainly through the interactions of the two antiparallel β4 strands (residues 245–250), with a buried solvent accessible surface area of 685 Å2. Two hydrogen bonds were formed between the main-chain atoms of Asp247 (Asp247-O to Asp247-N, a to b and b to a), and two more hydrogen bonds were formed between Glu245 (Oε2) and Ile249 (N) in the two antiparallel β4 strands to stabilize the dimeric structure (Figure 4B). The RRM2 dimer thus has an extended β-sheet consisting of 10 antiparallel β-strands. The RRM2 molecule (monomer a) also interacted with another 2-fold symmetry-related RRM2 molecule (monomer e) via β2 and α1 (Figure 4C and D). Four hydrogen bonds (a to e and e to a) were formed between the side-chain atoms in α1 helix: Gln209 (Nε2) to Cys212 (O), and Gln208 (Oε1) to Cys212 (Sγ). The buried surface between monomer a and e was 901 Å2, slightly higher than the interface between monomer a and b.Figure 4.

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