The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50.
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.
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Ross A
RA
Edwards
Megan S
MS
Lee
Susan E
SE
Tsutakawa
R Scott
RS
Williams
I
I
Nazeer
F E
FE
Kleiman
John A
JA
Tainer
J N Mark
JN
Glover
D001665:Binding Sites; D039241:Cleavage Stimulation Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004249:DNA Damage; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D026723:Polyadenylation; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014961:X-Ray Diffraction
D039241:Cleavage Stimulation Factor; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases