Abstract
Cancer-predisposing missense mutations in the RING domain of BRCA1 primarily target Zn(2+)-liganding residues. Here we report on the structural consequences of such mutations introduced into the second Zn(2+) site (Site II) of the BRCA1 RING domain and their effect on the interaction with the BARD1 RING domain. Each of the BRCA1 Site II mutants still interact and form a stable heterodimer with BARD1. Limited proteolysis of BRCA1/BARD1 complexes, monitored by matrix-assisted laser desorption ionization time-of-flight spectrometry, show that the mutations cause a local structural perturbation that is primarily confined to the second Zn(2+) binding loop of the BRCA1 subunit. These findings are consistent with the structure of the BRCA1/BARD1 heterodimer, which shows this region is well removed from the helices required for dimerization with BARD1. Instead, the mutations alter a region of BRCA1 that appears to be required for interaction with ubiquitin-conjugating enzymes.
Highlights
Cancer-predisposing missense mutations in the RING domain of BRCA1 primarily target Zn2؉-liganding residues
Using limited proteolysis in conjunction with MALDI-TOF mass spectrometry, we demonstrate that the structural effects of these BRCA1 Site II mutations are primarily localized to the second Zn2ϩ binding loop
BRCA1 and BARD1 interact by formation of an extensive 4-helix bundle formed by helices N- and C-terminal to the central RING motifs of both subunits
Summary
E3, ubiquitin ligase; E2, ubiquitinconjugating enzyme; MALDI, matrix-assisted laser desorption ionization; ESI, electrospray ionization; TOF, time-of-flight; wt, wild type; Endo, endoproteinase. BARD1 1–115 BRCA1 1–112 BRCA1 1–112 BRCA1 1–112 BRCA1 1–112 BRCA1 1–112 BRCA1 1–112. 13121.1 12960.3 12928.3 12894.3 12914.2 12928.3 12928.3 a Each construct has two extra residues at both the N-terminal (AS) and C-terminal (GK) ends, introduced as a result of the cloning procedures. Within the central RING motif of BRCA1, a region not involved in the BRCA1-BARD1 dimerization interface. BRCA1 Site II mutations cause a local structural perturbation and do not abolish one function of the BRCA1 RING domain, the ability to form a specific complex with BARD1. Site II cancer-predisposing mutations may alter a binding surface required for BRCA1 to interact with a ubiquitin-conjugating enzyme
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