X-ray reflectivity was used to study the interaction of the PX domain of p40phox protein (p40phox-PX) with a Langmuir monolayer of a mixture of SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SOPS (1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine), and DPPtdIns(3)P (1,2-dipalmitoylphosphatidylinositol 3-phosphate) lipids supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of the p40phox-PX domain and a slab model that represents the lipid layer, yielding an electron density profile of the lipid layer and bound PX domains. This analysis determines the angular orientation and penetration depth of the p40phox-PX domain bound to the SOPC/SOPS/DPPtdIns(3)P monolayer. The best fit orientation is characterized by the following angles: θ = 30 ± 10° and φ= 140 ± 30°. These angles describe rotations, about axes in a coordinate system fixed to the domain, that are required to orient the domain with respect to the lipid layer at the interface. The protein penetrated into the lipid layer by 9 ± 2 Å, indicating that the protein penetrated into the headgroup region, but not deeply into the hydrocarbon region of the monolayer. In this analysis, polar Tyr94 and hydrophobic Val95 penetrated deepest into the lipid monolayer. The backbone of these residues was ∼5 Å above the headgroup-buffer interface, i.e., at the level of the SOPC/SOPS lipid phosphates. Positively charged Lys92 and Lys 98 were also near the SOPC/SOPS lipid phosphates. This position of the protein allows for a favorable electrostatic contribution to binding.
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