Extended Data Fig. 4: PIP3-dependent membrane binding of PLCγ2-cSH2.
From: Targeting lipid–protein interaction to treat Syk-mediated acute myeloid leukemia
a. A representative simulation box for the HMMM membrane binding simulations of PLCγ2-cSH2, which is initially placed at least 10 Å away from the membrane surface. See Extended Data Fig. 1 for details. b. A predicted mode of binding of PLCγ2-cSH2 to a PIP3 molecule in the membrane. Left panel: a PLCγ2-cSH2 residues (C and O, N atoms are shown in white, red and blue, respectively) interacting with a PIP3 molecule (C and O, N atoms are shown in yellow, red and blue, respectively) are shown in stick presentation and labeled. Gray circles indicate PC and PS molecules. This representative structure is one of multiple low energy conformations obtained from the simulation and docking and not all of residues predicted for PIP3 binding are shown. Right panel: the structure is rotated 90° for the top of the protein surface to face the membrane. The protein is shown in surface representation to better illustrate the shape of the primary lipid binging site. c. Superimposition of PIP3 and VG370 docked into the primary lipid binding groove of PLCγ2-cSH2 in the membrane environment. In its lowest energy binding mode, VG370 binds deeper to the groove than the PIP3 headgroup, leading to tight binding and effective inhibition of PIP3 binding. d. Determination of Kd for binding of PLCγ2-cSH2 WT (circles) and K727A/K728A (triangles) to POPC/POPS/PIP3 (77:20:3) LUVs. The protein concentration was varied from 0 to 500 nM. Each data is average ± SD from 3 independent measurements. (e) Selectivity of PLCγ2-cSH2 WT (red) and K727A/K728A (blue) for POPC/POPS/PIP3 (77:20:3) (solid lines) over POPC/POPS/PI(4,5)P2 (PIP2) (77:20:3) (broken lines) vesicles determined by SPR analysis. The protein concentration was 100 nM.
