Extended Data Fig. 1: PIP3-dependent membrane binding of Syk-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 Syk-cSH2, which is initially placed at least 10 Å away from the membrane surface. 1,2-dihexanoyl derivatives of phosphatidylcholine (PC) and phosphatidylserine (PS) are shown in gray and purple lines, respectively, and PIP3 (6 in each leaflet) in space-filling representation (carbon, oxygen, and phosphorous atoms in yellow, red and tan, respectively). The hydrophobic core of the membrane is filled with an organic solvent (DCLE), shown in transparent surface representation. Bulk water molecules and ions are not shown for clarity. b. Ensemble-averaged residue profile of the Syk-cSH2, calculated during last the 50 ns of HMMM simulations. The gray bars denote the standard deviation in the residue distances. The pY-interacting residue (R195) is highlighted in black star. The representative PIP3-interacting conformations captured at the end of membrane binding simulations are shown at the bottom panel. c. Probability histogram of the number of PIP3 within 5 Å of Syk-cSH2, calculated over last 50 ns of all the HMMM membrane binding simulations, highlights that the protein can simultaneously interact with multiple PIP3 interacting which might help in its effective recruitment. d. Histograms of the protein residues in contact (within 3.5 Å) with the 3’, 4’ and 5’ phosphate moieties (P3 (red), P4 (green), and P5 (blue)) of the PIP3 headgroup are calculated over last 50 ns of all the HMMM membrane binding simulations. Notice that K220 and K222 make contact primarily with P3 whereas H163 and K165 interact with both P3 and P4. Many other residues primarily interact with P5. e. All the residues that form contacts with PIP3 for more than 10% of the time are highlighted and those that make the closest contact with PIP3 are labeled. All the basic residues are colored in blue and polar residues in green. f. Enlarged view of interaction between K172 of the Syk-cSH2 domain and the P4 and P5 moieties of PIP3. g. A schematic representation of predicted hydrogen bonds in this binding mode. H-bonds are shown as arrows. Notice that K172 interacts with both P4 and P5, but not with P3. It is thus expected to be involved in non-specific interaction with non-3’ P-containing PtdInsPs, such as PI(4,5)P2. h. Determination of Kd for binding of Syk-cSH2 WT (blue) and mutants (K220A/K222A (red), K172A (green), and R195A (orange) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/PIP3 (77:20:3) large unilamellar vesicles (LUVs). The protein concentration was varied from 0 to 500 nM. Each data is average ± SD from 3 independent measurements.
