Figure 2. MYM-type zinc fingers in ZNF261 and ZNF198 are involved in SUMO-binding.

A: GST and GST-SUMO-2(x3) were immobilized on glutathione coated plates and incubated with ³⁵S-labeled full-length or N- or C-terminus truncation fragments of ZNF261. Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. B: Pyruvate kinase or pyruvate kinase fused to the MYM-type zinc fingers from ZNF261 or ZNF198 were incubated with immobilized GST (white), GST-SUMO-2 (grey), and GST-SUMO-2(x3) (black). Unbound proteins were removed by washing and bound proteins were determined by liquid scintillation detection of eluted proteins. Plotted values represent the mean +/− S.D. from three independent experiments. C: ZNF261 was run at three concentrations in the analytical ultracentrifuge (10, 20, and 40 µM). Representative sedimentation data from the 20 µM ZNF261(1-495) data set is shown in the left panel. The data was globally fit to a single species model and the residuals between the calculated and experimental absorbance are shown below. The residuals appear randomly scattered around zero indicating that a single species model describes the data. ZNF261(1-495) (20 µM) was run with three concentrations of SUMO-2(x2) (20, 40, and 80 µM) in the analytical ultracentrifuge. Representative sedimentation data of the 20 µM ZNF261 and 80 µM SUMO-2(x2) data set is shown in the right panel. Data were globally fit to an A+B → AB model and the residuals between the calculated and experimental absorbance are shown below. The global reduced chi-squared value was 3.13.