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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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BARD1 ankyrin-BRCT fragment samples a broad range of conformations in solution. Frequency of occurrence of RG (A) and Dmax (B) values for the optimized ensemble (empty boxes) compared to those of the pool of 10000 randomly generated conformations (filled boxes). The optimized ensemble, representing the BARD1 C-terminal domain in solution, samples a broad range of both RG and Dmax, comparable to those of the pool. The C-terminal domain of BARD1 is therefore described as conformationally flexible in solution. The RG and Dmax ranges are however both systematically shorter than for those in the pool. This may be due to a partially folded rather than completely flexible linker. (C) The conformational flexibility of the BARD1 C-terminal domain in solution is shown by the optimized ensemble that best represents the experimental SAXS curve. The 19 models within the ensemble were aligned on their BRCT domains. The BRCT repeat is shown in red, the flexible linker is in blue, and ankyrin domains are in gray.
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fig5: BARD1 ankyrin-BRCT fragment samples a broad range of conformations in solution. Frequency of occurrence of RG (A) and Dmax (B) values for the optimized ensemble (empty boxes) compared to those of the pool of 10000 randomly generated conformations (filled boxes). The optimized ensemble, representing the BARD1 C-terminal domain in solution, samples a broad range of both RG and Dmax, comparable to those of the pool. The C-terminal domain of BARD1 is therefore described as conformationally flexible in solution. The RG and Dmax ranges are however both systematically shorter than for those in the pool. This may be due to a partially folded rather than completely flexible linker. (C) The conformational flexibility of the BARD1 C-terminal domain in solution is shown by the optimized ensemble that best represents the experimental SAXS curve. The 19 models within the ensemble were aligned on their BRCT domains. The BRCT repeat is shown in red, the flexible linker is in blue, and ankyrin domains are in gray.

Mentions: Given that the C-terminal region is a multidomain protein joined by a proteolytically labile linker and that conformational heterogeneity was suggested from analysis of the p(r) function, the solution scattering profile was first tested for interdomain flexibility using the ensemble optimization method53. An ensemble, optimized against the experimental SAXS curve using a genetic algorithm, was selected from a pool of 10000 randomly generated C-terminal domain conformers. Values of RG and Dmax for the ensemble were compared against those calculated from the pool. In the case where the molecule adopts a limited set of conformations in solution, the ensemble is expected to sample a narrow range of RG and Dmax relative to the values in the pool. However, if the molecule is flexible, sampling a large number of interdomain conformations in solution, the distribution of RG and Dmax values will be correspondingly broad, more comparable to those of the pool53,64. The C-terminal domain of BARD1 resembles the later case and thus exhibits conformational heterogeneity in solution via its flexible linker (Figure 5A,B). All models in the optimized ensemble are shown aligned on their BRCT domains in Figure 5C. The theoretical scattering curve of the ensemble fits that of the experimental scattering curve with a χ2 = 2.2 (Figure 2A). For comparison, the calculated p(r) function of the most compact model generated by the EOM (RG = 20.5 Å, Dmax = 69.4 Å, χ = 13) has a single peak and no extended tail (Figure 2B).


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)

BARD1 ankyrin-BRCT fragment samples a broad range of conformations in solution. Frequency of occurrence of RG (A) and Dmax (B) values for the optimized ensemble (empty boxes) compared to those of the pool of 10000 randomly generated conformations (filled boxes). The optimized ensemble, representing the BARD1 C-terminal domain in solution, samples a broad range of both RG and Dmax, comparable to those of the pool. The C-terminal domain of BARD1 is therefore described as conformationally flexible in solution. The RG and Dmax ranges are however both systematically shorter than for those in the pool. This may be due to a partially folded rather than completely flexible linker. (C) The conformational flexibility of the BARD1 C-terminal domain in solution is shown by the optimized ensemble that best represents the experimental SAXS curve. The 19 models within the ensemble were aligned on their BRCT domains. The BRCT repeat is shown in red, the flexible linker is in blue, and ankyrin domains are in gray.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: BARD1 ankyrin-BRCT fragment samples a broad range of conformations in solution. Frequency of occurrence of RG (A) and Dmax (B) values for the optimized ensemble (empty boxes) compared to those of the pool of 10000 randomly generated conformations (filled boxes). The optimized ensemble, representing the BARD1 C-terminal domain in solution, samples a broad range of both RG and Dmax, comparable to those of the pool. The C-terminal domain of BARD1 is therefore described as conformationally flexible in solution. The RG and Dmax ranges are however both systematically shorter than for those in the pool. This may be due to a partially folded rather than completely flexible linker. (C) The conformational flexibility of the BARD1 C-terminal domain in solution is shown by the optimized ensemble that best represents the experimental SAXS curve. The 19 models within the ensemble were aligned on their BRCT domains. The BRCT repeat is shown in red, the flexible linker is in blue, and ankyrin domains are in gray.
Mentions: Given that the C-terminal region is a multidomain protein joined by a proteolytically labile linker and that conformational heterogeneity was suggested from analysis of the p(r) function, the solution scattering profile was first tested for interdomain flexibility using the ensemble optimization method53. An ensemble, optimized against the experimental SAXS curve using a genetic algorithm, was selected from a pool of 10000 randomly generated C-terminal domain conformers. Values of RG and Dmax for the ensemble were compared against those calculated from the pool. In the case where the molecule adopts a limited set of conformations in solution, the ensemble is expected to sample a narrow range of RG and Dmax relative to the values in the pool. However, if the molecule is flexible, sampling a large number of interdomain conformations in solution, the distribution of RG and Dmax values will be correspondingly broad, more comparable to those of the pool53,64. The C-terminal domain of BARD1 resembles the later case and thus exhibits conformational heterogeneity in solution via its flexible linker (Figure 5A,B). All models in the optimized ensemble are shown aligned on their BRCT domains in Figure 5C. The theoretical scattering curve of the ensemble fits that of the experimental scattering curve with a χ2 = 2.2 (Figure 2A). For comparison, the calculated p(r) function of the most compact model generated by the EOM (RG = 20.5 Å, Dmax = 69.4 Å, χ = 13) has a single peak and no extended tail (Figure 2B).

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