Figure 4

PcrG restores the monomeric state of oligomeric ΔPcrV _(128–294) by forming a high affinity heterodimeric PcrG-ΔPcrV _(128–294) complex. A. SDS PAGE showing the interaction of PcrG with ∆PcrV_(128–294) and ∆PcrV_(1–127). When ∆PcrV_(128–294) was incubated with PcrG and subjected to Ni-NTA affinity chromatography, both ∆PcrV_(128–294) and PcrG were seen in the elution fraction. However, when ∆PcrV_(1–127) was incubated with PcrG, only PcrG was seen in the elution. This revealed that only ∆PcrV_(128–294) forms a complex with PcrG. B. The region corresponding to ∆PcrV_(128–294) was shown in deep blue colour in the homology model of PcrV. This region encompasses helix-7, C-terminal globular domain, and helix-12. C. Surface plasmon resonance sensogram of ∆PcrV_(128–294) and PcrG. D. Size exclusion chromatography profile of ∆PcrV_(128–294), PcrG-∆PcrV_(128–294), and PcrG with the corresponding SDS PAGE showing the proteins present in each of the peaks. E. The DLS profile of ∆PcrV_(128–294), PcrG-∆PcrV_(128–294) complex, and PcrG with corresponding hydrodynamic diameter. F. ∆PcrV_(128–294) forms a 1:1 heterodimeric complex with PcrG, shown by chemical crosslinking with 0.5 mM, 1 mM and 2 mM EGS-sulfonate. G. Size exclusion chromatography profile of ∆PcrV_(1–127) with corresponding SDS PAGE. M denotes the protein molecular weight marker in SDS PAGE (10, 17, 26, 34, 43, 55 kDa bands from bottom to top).