Extended Data Fig. 7: Target validation of WC36.
From: Targeting lipid–protein interaction to treat Syk-mediated acute myeloid leukemia
a. Design of mutants that may potentially inhibit WC36 binding without affecting PIP3 binding. In the model structures of Syk-cSH2-PIP3 and Syk-cSH2-WC36 complexes (superimposed here as in Fig. 2d), the side chain of E164 forms a hydrogen bond (the red dotted line) with the N-H group of the imidazole moiety of WC36 but is remote from the PIP3 head group. We thus reasoned that the mutation of E164 might selectively suppress WC36 binding without impacting PIP3 binding. The blue dotted line indicates the location of the membrane surface. b. Membrane binding activity of EGFP-Syk-cSH2-WT (blue), -E164D (red), -E164A (purple), -E164Q (green), and -E164K (orange) was measured by the fluorescence quenching assay using POPC/POPS/PIP3/dabsyl-PE (67:20:3:10 in mol%) LUVs. The quenching of the EGF fluorescence by dabsyl-PE (△F) of was determined as a function of the total lipid concentration in the vesicles. E164D showed essentially the same vesicle affinity as WT. The protein concentration was kept at 25 nM. c. Inhibition of membrane binding activity of EGFP-Syk-cSH2-WT (blue), -E164D (red), -E164A (purple), -E164Q (green), and -E164K (orange) by WC36. 25 nM of Syk-cSH2 proteins and 50 μM POPC/POPS/PIP3/dabsyl-PE (67:20:3:10 in mol%) LUVs were employed. % inhibition ((△F / △Fmax) x 100)) was plotted as the function of the WC36 concentration where △F and △Fmax indicate fluorescence quenching in the presence of and absence of WC36, respectively. Neither E164D nor W164Q was inhibited by WC36 whereas WT was. For Expended Data Fig. 7b, d, each data represents average ± SD from 3 independent measurements. d. The structure of WC36B.
