Fig. 1: Biochemical characterization of the interaction between HOIP and STK4.
From: STK4 inhibits the E3 activity of HOIP by phosphorylating its allosteric ubiquitin-binding site
a Schematic diagram showing the domain organizations of HOIP, HOIL-1L, Sharpin and STK4. In the drawing, the inter-molecular interactions of HOIP, HOIL-1L and Sharpin are indicated by black two-way arrows, while the HOIP/STK4 interaction characterized in this study is highlighted by red two-way arrows. b The summarized mapping results of the binding regions between HOIP and STK4 based on our SEC-based analyses. c SEC-based analyses of the interaction between 20 μM HOIP RING2-LDD and 30 μM STK4 KD. The “SUM” stands for the theoretical sum of the SEC profiles of STK4 (residues 1–326) K59R and HOIP (residues 854–1072). This assay was performed using a Superdex 75 Increase 10/300 GL column (GE Healthcare). d ITC-based measurement of the binding affinity of HOIP RING2-LDD and STK4 KD. The Kd error is the fitted error obtained from the data analysis software when using the one-site binding model to fit the ITC data; DP, differential power, measured by the ITC machine; ΔH, heat change measured by the ITC machine. e Overlay plot of the sedimentation velocity data of HOIP RING2-LDD (blue), STK4 KD (red), and the mixture of HOIP RING2-LDD with STK4 KD in a molar ratio of 1:1 (green) or 1:5 (black); c(s) continuous sedimentation coefficient distribution; MW molecular weight. The relevant experiments depicted in this figure have been replicated three times.
