Fig 6. Binding of TAX1BP1 zinc finger domains to ubiquitin and myosin VI.

Dependence of the chemical shift changes of resonances of residues in TAX1BP1 as a function of ubiquitin concentration: (A) and (B) overlay of sections of the ¹H/¹⁵N HSQC spectra of ¹⁵N labeled TAX1BP1 zinc fingers at different concentrations of unlabeled ubiquitin showing the concentration dependence of the peak positions of residues in each of the zinc fingers. (C) Graph showing the concentration dependence of these peaks together with two other peaks from each of the zinc fingers. The chemical shift change of the backbone amide group (Δδ_HN−N) is computed with Δδ_HN−N = √ _(δ_H ²+ (δ_N/10)²), where δ_H and δ_N are the changes in ¹H and ¹⁵N chemical shift, respectively. If changes in the spectra are observed and the signals are in fast exchange, the dissociation constant (K_D) is calculated by fitting the NMR titration data to the formula: $\Delta {\delta }_{\text{HN-N}}=\frac{{\delta }_b-{\delta }_f}{2\left[P_0\right]}\left(\left[P_0\right]+\left[L_0\right]+K_D-\sqrt{{\left(\left[P_0\right]+\left[L_0\right]+K_D\right)}^2-4\left[P_0\right]\left[L_0\right]}\right)$ where y is Δδ, δ_b-δ_f is the difference in chemical shift between bound and free state (= Δδ_max), P₀ is the total protein concentration and L₀ is the total ligand concentration. A simple two-state binding model is assumed. Protein concentration is treated as a constant (measured before starting the titration). For our experiments P₀ was 150 μM. (D) The direct interaction of myosin VI CBD with TAX1BP1 was measured as changes in thermophoretic mobility of 75 nM fluorescently labeled myosin VI CBD in the presence of various TAX1BP1 concentrations. The binding affinity of the complex was estimated by a fit to a Hill function, as a K _D of about 5–10 μM. The experiment was repeated in the presence of 1 mM ubiquitin. Even the presence of a large excess of free ubiquitin does not change the binding affinity between myosin VI and TAX1BP1. Experiments were performed in triplicate (no ubiquitin) or duplicate (1mM ubiquitin) and error bars represent s.d. (E) Pull-down assay of myosin VI CBD and TAX1BP1 in the presence of ubiquitin. 15 μM TAX1BP1 was incubated for 30 min with increasing amounts of ubiquitin (0–1 mM) before adding 10 μM GST-myosin VI CBD. After pull-down with Glutathione-Sepharose beads, the amount of TAX1BP1 binding to GST-myosin VI CBD was visualised by SDS-PAGE. Upper gel shows the total input and lower panel the pull-down.