Figure 3. αCat homodimers prefer PIP 3... | Rockefeller University Press

Figure 3.

$Figure 3. αCat homodimers prefer PIP3 phosphoinositides. (A) Phosphoinositide (PtdInsP) selectivity of WT, full-length (FL) αCat dimer. SPR sensorgrams for seven different POPC/POPS/PtdInsP (77:20:3) vesicles are shown (from top to bottom): PIP3 >> PIP2 > phosphatidylinositol-3,4-bisphosphate (PI34P2) ≈ phosphatidylinositol-5-monophosphate (PI5P) > phosphatidylinositol-4-monophosphate (PI4P) > phosphatidylinositol-3,5-bisphosphate (PI35P2) ≈ phosphatidylinositol-3-monophosphate (PI3P). 200 nM αCat was used for all measurements. Data are representative of three experiments. (B) Binding of αCat homodimer versus monomer (200 nM each) to POPC/POPS/PIP3 (77:20:3) vesicles. Data are representative of three experiments. (C) Chromatographic separation of monomer (M) and dimer (D) fractions of recombinant αCat proteins. (D) αCat schematic with KKR-basic patch overlaid on crystal structure of αCat dimer. Subunit A in gray; subunit B in green/blue; basic patch in red. (E) Circular dichroism (CD) spectra thermal denaturation analysis of purified recombinant protein; FLαCat (red trace) or FLαCatKKR<3A (blue trace) proteins. No global differences between WT and mutant protein were detected. Wavelength scan: n = 4; temperature scan: n = 2. (F) SDS-PAGE (top) and BN-PAGE (bottom) analysis of purified proteins (10 µg). (G) Membrane binding isotherms for FL αCat dimer (open symbols) and FLKKR<3A mutant dimer (closed symbols). n = 3; data are mean ± SD. Lines represent theoretical curves constructed from apparent dissociation constant (Kd) and the maximal Req value (Rmax) determined from by nonlinear least squares analysis of the binding isotherm using the equation Req = Rmax/(1 + Kd/Po). Kd = 30 ± 3 nM for WT and 73 ± 15 nM for the mutant. (H) PIP3 versus PIP2 selectivity of WT FLαCat dimer (cyan) and FLαCatKKR<3A mutant dimer (orange). Data are representative of three experiments. Notice that although the mutation decreased the overall membrane affinity of the αCat dimer, it did not affect the PtdInsP selectivity of the protein, as indicated by essentially the same (RU for PIP3)/(RU for PIP2) ratio for WT and the mutant. Each SPR measurement was performed in 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl and 1 mM TCEP using L1 chip coated with POPC/POPS/PtdInsP (77:20:3) vesicles as the active surface. 200 nM αCat was used for both WT and mutant proteins. POPC vesicles were used to coat the control surface for most experiments. For A, POPC/POPS (80:20) vesicles were used for the control surface to eliminate the potential contribution of POPS to PtdInsP selectivity.$

αCat homodimers prefer PIP₃ phosphoinositides. (A) Phosphoinositide (PtdInsP) selectivity of WT, full-length (FL) αCat dimer. SPR sensorgrams for seven different POPC/POPS/PtdInsP (77:20:3) vesicles are shown (from top to bottom): PIP₃ >> PIP₂ > phosphatidylinositol-3,4-bisphosphate (PI34P₂) ≈ phosphatidylinositol-5-monophosphate (PI5P) > phosphatidylinositol-4-monophosphate (PI4P) > phosphatidylinositol-3,5-bisphosphate (PI35P₂) ≈ phosphatidylinositol-3-monophosphate (PI3P). 200 nM αCat was used for all measurements. Data are representative of three experiments. (B) Binding of αCat homodimer versus monomer (200 nM each) to POPC/POPS/PIP₃ (77:20:3) vesicles. Data are representative of three experiments. (C) Chromatographic separation of monomer (M) and dimer (D) fractions of recombinant αCat proteins. (D) αCat schematic with KKR-basic patch overlaid on crystal structure of αCat dimer. Subunit A in gray; subunit B in green/blue; basic patch in red. (E) Circular dichroism (CD) spectra thermal denaturation analysis of purified recombinant protein; FLαCat (red trace) or FLαCat^KKR<3A (blue trace) proteins. No global differences between WT and mutant protein were detected. Wavelength scan: n = 4; temperature scan: n = 2. (F) SDS-PAGE (top) and BN-PAGE (bottom) analysis of purified proteins (10 µg). (G) Membrane binding isotherms for FL αCat dimer (open symbols) and FL^KKR<3A mutant dimer (closed symbols). n = 3; data are mean ± SD. Lines represent theoretical curves constructed from apparent dissociation constant (K_d) and the maximal R_eq value (R_max) determined from by nonlinear least squares analysis of the binding isotherm using the equation R_eq = R_max/(1 + K_d/P_o). K_d = 30 ± 3 nM for WT and 73 ± 15 nM for the mutant. (H) PIP₃ versus PIP₂ selectivity of WT FLαCat dimer (cyan) and FLαCat^KKR<3A mutant dimer (orange). Data are representative of three experiments. Notice that although the mutation decreased the overall membrane affinity of the αCat dimer, it did not affect the PtdInsP selectivity of the protein, as indicated by essentially the same (RU for PIP₃)/(RU for PIP₂) ratio for WT and the mutant. Each SPR measurement was performed in 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl and 1 mM TCEP using L1 chip coated with POPC/POPS/PtdInsP (77:20:3) vesicles as the active surface. 200 nM αCat was used for both WT and mutant proteins. POPC vesicles were used to coat the control surface for most experiments. For A, POPC/POPS (80:20) vesicles were used for the control surface to eliminate the potential contribution of POPS to PtdInsP selectivity.