Figure 3.

Ubiquitin Binding by BRCA1-A

(A) Upper panel: binding of di-ubiquitin to the BRCC45/MERIT40 subcomplex measured by biolayer interferometry. The top panel shows the pseudo first-order association constant (K_obs) plotted against K63-linked Ub₂ concentration. The dissociation equilibrium constant (K_d) of 17 uM was determined from the ratio of k_off and k_on. Lower panel: association and dissociation phases in the raw kinetic trace for Ub₂ binding to a probe linked to the BRCC45/MERIT40 complex (blue) and a non-derivatized probe (orange).

(B) Superposition of the AMSH-LP/K63-Ub₂ complex with the docked Abro1/BRCC36 structure shows the approximate positions of the proximal (teal) and distal (purple) Ub molecules. As previously noted (Zeqiraj et al., 2015), the Ins-2 loop that packs against the proximal Ub in AMSH-LP (yellow) is absent in BRCC36.

(C) The alignment in (A) and the structure of K63-linked di-ubiquitin were used to model an extended Ub chain on the proximal and distal sides of the cleavage site into the 2×4 complex (white surface). Abraxas and BRCC36 are shown in gold and blue, respectively.