Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans
Fig 5
LigAC and LigBC interact with heparin mainly via Big 7 and Big 8.
(A) Using different LigA and LigB tandem domain constructs, we observed that Big7-8 are the major heparin binding sites on the C-terminus variable regions of LigB and LigA. (B) Binding of C4BP to different LigA and LigB tandem domain constructs is affected by heparin. Immobilized Lig protein constructs were incubated with C4BP mixed with different concentrations of heparin. C4BP bound to each Lig protein fragments in the absence of heparin was set as 100%. Each point represents the mean absorbance value at 492 nm or 630 nm +/- the SD of 3 independent experiments each performed in triplicate. Binding of heparin to LigA7-8, LigA8-13 and LigB 7–8, LigB7-12 were compared to LigA10-11 and LigB 9–10, respectively. ***p<0,001 (C) Estimation of the dissociation constant (Kd) for the interactions of LigAC and LigBC with heparin. Kd was calculated by fitting the data to the equation Y = Bmax*X/(Kd + X) using GraphPad Prism 5.0 (GraphPad Software, Inc.). Each data point represents the mean +/- SD of three trials in triplicate.