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. 2015 Dec 9;4:e10766. doi: 10.7554/eLife.10766

Figure 4. Electrostatic interaction of oppositely charged residues underlies CyaA binding to CR3.

(A) 3D structure of CR3 was modeled by homology onto the known 3D structure of CR4. The structure of the CD11b binding site within the segment 1166-1287 of CyaA was predicted using I-TASSER. For clarity, only the CD11b subunit is shown. (B) To identify interacting residues, a flexible side chain docking of the segment 1166-1287 of CyaA to CR3 was performed using the ClusPro server. (C) Different concentrations of Dy647-labeled intact CyaA or its variants with point mutations in the CD11b binding site of the toxin were incubated with 2x105 CHO cells expressing intact CR3 and the cells were analyzed by flow cytometry. Mean fluorescence intensities of binding of intact CyaA or its variants were plotted against the toxin concentrations. Each point represents the mean value ± SD of four independent experiments. Binding of CyaA mutant variants to cells was at all measured concentrations significantly lower than binding of intact CyaA (p<0.0001; ANOVA). RFI, relative fluorescence intensity. (D) Different concentrations of intact CyaA and its mutant variants were incubated with 1x105 CHO cells expressing intact CR3 and intracellular levels of cAMP were determined by ELISA. Each point represents the mean value ± SD of two independent experiments performed in triplicate. Intoxication of cells by CyaA mutant variants was at all measured concentrations significantly lower than intoxication of cells by intact CyaA (p<0.0001; for CyaAE1232+D1234A at 20 ng/ml p<0.001; ANOVA). (E) 2x105 CHO cells expressing integrin molecules were incubated with 2 µg/ml of CyaA-biotin, the surface-bound toxin was labeled with streptavidin-PE and the cells were analyzed by flow cytometry. CyaA binding was expressed as percentage of toxin binding to CHO cells expressing the native form of CD11b/CD18. Each bar represents the mean value with SD of two independent experiments performed in duplicate. Significantly reduced binding of CyaA to mutant integrins in comparison with intact CD11b/CD18 is indicated (****, p<0.0001; ANOVA). (F) 1x105 CHO cells expressing different integrin molecules were incubated with various concentrations of CyaA and the amounts of accumulated cAMP were determined in cell lysates by ELISA. Each point represents the mean value ± SD of six independent experiments. Intoxication of cells expressing CD11bR662A+R664A+R666A/CD18 or no β2 integrin was in concentrations ranging from 5 to 20 ng/ml of CyaA significantly lower than intoxication of cells expressing intact CD11b/CD18 (p<0.0001; ANOVA).

DOI: http://dx.doi.org/10.7554/eLife.10766.012

Figure 4.

Figure 4—figure supplement 1. Expression of CD11b/CD18 and of its mutant variants on the surface of CHO cells and binding of CyaA to transfected cells.

Figure 4—figure supplement 1.

A CHO cell line stably expressing the intact CD18 subunit was transfected with plasmid constructs encoding intact CD11b or its mutant variants with the charged and hydrophilic residues within the segment 614-682 replaced with alanine residues. Cells stably expressing integrin molecules were selected using a cell sorter. The expression levels of CD11b/CD18 (green) and its mutant variants (red) on the cell surface were examined by flow cytometry after staining of 2x105 cells with the 2LPM19c mAb recognizing the I-domain of CD11b (left panels), or the OKM1 mAb recognizing the mutagenized segment 614-682 of CD11b (middle panels). The cells were also analyzed for capacity to bind CyaA: 2x105 transfected cells were incubated with 2 μg/ml of CyaA-biotin, the surface-bound toxin was labeled with streptavidin-PE and the cells were analyzed by flow cytometry (right panels). CHO cells expressing no β2 integrin (transfected with empty vectors) were processed in parallel and used as negative control (grey). Typical flow cytometry histograms from one representative binding experiment out of four performed are shown. RFI, relative fluorescence intensity. In contrast to other substitutions in the segment 614-682 of CD11b, the triple substitution E625A+N627A+R629A abolished binding of OKM1, suggesting that the epitope recognized by OKM1, or part of it, comprises these residues.
DOI: http://dx.doi.org/10.7554/eLife.10766.013
Figure 4—figure supplement 2. Staining of CHO cells expressing intact CD11b/CD18 and of its two mutant variants with the four mAbs that block the binding of CyaA.

Figure 4—figure supplement 2.

2x105 CHO cells stably expressing intact CD11b/CD18, or its mutant variants exhibiting significantly reduced capacity to bind CyaA, were stained with the anti-CD11b mAbs that block CyaA binding. The cells were analyzed by flow cytometry and antibody binding was expressed as percentage of mAb binding to CHO cells expressing the native form of CD11b/CD18. Each bar represents the mean value with SD of four independent experiments. Significant differences between mean values of mAb binding to cells expressing mutant integrin and cells expressing intact CD11b/CD18 are indicated (****, p< 0.0001; ANOVA).
DOI: http://dx.doi.org/10.7554/eLife.10766.014