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The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50.

Edwards RA, Lee MS, Tsutakawa SE, Williams RS, Nazeer I, Kleiman FE, Tainer JA, Glover JN - Biochemistry (2008)

Bottom Line: Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding.Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains.The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

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Structure of the tandem BRCT repeats of BARD1. (A) Overview of the BARD1 BRCT structure (orange) aligned with the structure of the BRCA1 BRCT (blue) bound to an optimized phosphopeptide target (green) (PDB accession code 1T2V). (B) Details of the phosphopeptide recognition surfaces of BRCA1 and BARD1, colored as in (A). Residues involved in peptide binding are labeled and shown as sticks.
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fig3: Structure of the tandem BRCT repeats of BARD1. (A) Overview of the BARD1 BRCT structure (orange) aligned with the structure of the BRCA1 BRCT (blue) bound to an optimized phosphopeptide target (green) (PDB accession code 1T2V). (B) Details of the phosphopeptide recognition surfaces of BRCA1 and BARD1, colored as in (A). Residues involved in peptide binding are labeled and shown as sticks.

Mentions: To probe the structure of the BARD1 C-terminus, we crystallized and determined the X-ray structure of the tandem BARD1 BRCT repeat domain (554−777). The BARD1 BRCT repeat structure was solved using molecular replacement methods and refined to 2.1 Å resolution (see Materials and Methods). The structure of BARD1 (554−777) is reminiscent of the BRCT repeats in BRCA1 and MDC1 with the two BRCT domains packing in the same head-to-tail manner (Figure 3A). The structure determined here is also essentially identical to the structure of a smaller BARD1 BRCT fragment recently determined by Birrane et al.55.


The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50.

Edwards RA, Lee MS, Tsutakawa SE, Williams RS, Nazeer I, Kleiman FE, Tainer JA, Glover JN - Biochemistry (2008)

Structure of the tandem BRCT repeats of BARD1. (A) Overview of the BARD1 BRCT structure (orange) aligned with the structure of the BRCA1 BRCT (blue) bound to an optimized phosphopeptide target (green) (PDB accession code 1T2V). (B) Details of the phosphopeptide recognition surfaces of BRCA1 and BARD1, colored as in (A). Residues involved in peptide binding are labeled and shown as sticks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654182&req=5

fig3: Structure of the tandem BRCT repeats of BARD1. (A) Overview of the BARD1 BRCT structure (orange) aligned with the structure of the BRCA1 BRCT (blue) bound to an optimized phosphopeptide target (green) (PDB accession code 1T2V). (B) Details of the phosphopeptide recognition surfaces of BRCA1 and BARD1, colored as in (A). Residues involved in peptide binding are labeled and shown as sticks.
Mentions: To probe the structure of the BARD1 C-terminus, we crystallized and determined the X-ray structure of the tandem BARD1 BRCT repeat domain (554−777). The BARD1 BRCT repeat structure was solved using molecular replacement methods and refined to 2.1 Å resolution (see Materials and Methods). The structure of BARD1 (554−777) is reminiscent of the BRCT repeats in BRCA1 and MDC1 with the two BRCT domains packing in the same head-to-tail manner (Figure 3A). The structure determined here is also essentially identical to the structure of a smaller BARD1 BRCT fragment recently determined by Birrane et al.55.

Bottom Line: Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding.Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains.The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

Show MeSH
Related in: MedlinePlus