Modeling and function of the p40^phoxR105Q mutant. (A) A model showing a close-up view of the Ptdlns(3)P binding cavity within the PX domain of wild-type p40^phox, and the R105Q p40^phox mutant (). The protein is shown in ribbon representation and colored in cyan. Residues that interact with Ptdlns(3)P are shown in capped-sticks rendering and color-coded based on atom types (C, N, O, and P in yellow, blue, red, and orange, respectively). Ptdlns(3)P is also color-coded based on atom types except that carbon is colored in white. Dashed lines show hydrogen bond interactions of R58, R60, V93, and R105 with Ptdlns(3)P. The interactions between amino acid 105 and Ptdlns(3)P are lost in the R105Q mutant (), as the distance between the Q105 amide oxygen and nitrogen atoms is greater than 5 Å, which precludes any hydrogen bonding. In addition, Q105 is neutral and therefore cannot compensate for the loss of the positive charge on R105, which contributes to stabilize interactions with Ptdlns(3)P. Images were created with program PyMOL (DeLano Scientific LLC). (B) Representative image collected during confocal videomicroscopy of nonstimulated (NS) PLB-985 granulocytes and during phagocytosis of SOZ, where cells are expressing the wild-type YFP-p40^phox PX domain (YFP-PX_40WT) or with R105Q mutation (YFP-PX_{40 R105Q}), as indicated. Representative of cells filmed in 3 experiments. (C-D) Results using granulocyte-differentiated PLB-p40 KD cells (1 or blue), PLB-p40 KD cells expressing p40^phoxR105Q (2 or red), PLB-p40 KD cells expressing wild-type p40^phoxwt (3 or green), and wild-type PLB-985 cells (4 or black). (C) Immunoblots probed with antibodies directed at the indicated proteins. Representative of 3 experiments. (D) PLB-985 granulocyte NADPH oxidase activity in response to PMA (200 ng/mL), fMLF (10 μM), or SOZ (2 mg/mL) as indicated, as monitored by isoluminol for extracellular activity (PMA and fMLF) or luminol for intracellular activity (SOZ). RLU indicates relative light units. (Top row) Representative kinetic data; (bottom row) NADPH oxidase activity as the mean ± SD (n = 3) relative to control wild-type PLB-985 granulocytes. Dashed blue line shows signal from PMA- or fMLF-stimulated PLB-p40 KD cells in the presence of SOD, or in the absence of stimulus for the SOZ data. *P < .05, PLB-p40 KD cells expressing p40^phoxR105Q versus PLB KD cells expressing wild-type p40^phox.