K63-linked, linear polyubiquitin, and monoubiquitin binding of the D311N and D311G CC2-LZ domains. (A) Pull-down activity of His-tagged CC2-LZ WT, D311N, and D311G mutants for linear (left) and K63-linked (right) tetraubiquitin chains. His-tagged CC2-LZ WT, D311N, and D311G mutants were incubated for 1 hour at a concentration of 8μM with various concentrations of linear (7μM, 2μM, 1μM, 0.5μM, and 0.25μM) or K63-linked tetraubiquitin chains (3.5μM, 1.7μM, 0.9μM, 0.7μM, and 0.4μM). After 3 washes, the amount of bound tetraubiquitin chains was determined by Coomassie staining followed by densitometry. C1 and C2 indicate controls with beads alone and immobilized with a His-tagged protein control, respectively (both represent nonspecific ubiquitin binding); MW, molecular weight (in kilodaltons). (B) Fluorescence titration of F312W, F312W/D311N, and F312W/D311N mutants (10μM) in the presence of monoubiquitin (mono-Ub) or I44A monoubiquitin. Lines represent the fit to each binding curve, giving the affinity (K_d) or the relative affinity with respect to the WT (^mutK_D/^WTK_D) for mono-Ub (right panel).