Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex.
BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RINGRING heterodimer. The BRCA1BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn(2+)-loops and central alpha-helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Peter S
PS
Brzovic
Jennifer R
JR
Keeffe
Hiroyuki
H
Nishikawa
Keiko
K
Miyamoto
David
D
Fox
Mamoru
M
Fukuda
Tomohiko
T
Ohta
Rachel
R
Klevit
D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D019281:Dimerization; D019398:Genes, BRCA1; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers
D019313:BRCA1 Protein; D002352:Carrier Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases