Activity was measured as a function of increasing PC mole fractions for 60 min at 37☌. ( F) Effect of Y96F, Y96A, N64A, and N65D mutations on the allosteric sigmoidal constant (K half) of human cPLA 2α activity. cPLA 2α activities (nmol of arachidonic acid released/min/mg of recombinant cPLA 2α) were collected on eight separate occasions and are presented as n = 4 for Y96F, n = 4 for Y96A, n = 4 for N64A, n = 4 for N65D, and n = 8 for WT. The PC mole fraction was held constant at 0.285. Activity was measured as a function of PC molar concentration for 60 min at 37☌. Proteins were purified as described Stahelin et al. ( E) Effect of the Y96F, Y96A, N64A, and N65D mutations on the dissociation constant (K s A) of human cPLA 2α activity. ( D) Relative binding affinity of C2-domain point mutants and control protein obtained for binding isotherms shown in panel (C). ( C) FRET-binding isotherms showing the POPC–DHPC bicelle-dilution vesicle dependence of point mutant and control protein (0.5 μM) equilibrium adsorption at 50 μM Ca 2+ (see 'Materials and methods'). ( B) FRET binding isotherms showing the Ca 2+ dependence of point mutant and control protein (0.5 μM) equilibrium adsorption to POPC–DHPC bicelle-dilution vesicles (4 μM) (see 'Materials and methods'). ( A) SPR binding isotherms showing point mutant and control protein equilibrium adsorption to immobilized 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles saturating a L1 sensor chip at 5 μl/min solution flow rates (see 'Materials and methods'). Root mean square deviation = 0.7 Å after superimposition of Cα atoms. ( E) Superimposition of the chicken cPLA 2α C2-domain with bound DHPC (colored as in Figure 1B) on the human lipid-free structure (PDB: 1RLW, cyan). ( D) Fo- Fc omit electron density map for the bound DHPC molecule at the 2.5σ contour level. Ca1 and Ca4 (blue spheres) are in a similar position in the apo-form structure whereas Ca PC (magenta sphere) is unique to the DHPC-bound form. The DHPC molecule (beige stick) straddles the β1–β2 loop (CBL1, blue), β3–β4 loop (CBL2, cyan) and β5–β6 loop (CB元, red). ( B) Ribbon structure representation of the cPLA 2α C2-domain bound to 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC). The human and chicken cPLA 2α CBL sequences are 92% identical and 94.5% highly conserved (see Figure 1-figure supplement 1 for full-length sequence alignment). Conversely, residues that interact with PS in the PKCα-PS structure (magenta) are highly conserved in PKCs and Syt1, but not in cPLA 2α. Residues interacting directly with PC in our structural complex (blue or blue asterisk) are absolutely conserved among eukaryotic cPLA 2α proteins but not in PKCs and Syt1. ( A) Sequence alignment of C2-domain calcium-binding loop (CBL) regions in cPLA2α from different eukaryotes compared to human PKCs and Syt1.
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