Figure 5. Mutation of Eno C-terminal lysine 434 reduces Eno-eRNA/eDNA interaction.

(A) Bovine serum albumin (BSA), wt pneumococcal Eno (Eno) and Eno mutant (Eno^K434G) were spotted on the nitrocellulose membrane. The binding of proteins to eRNA, eDNA and Plg was tested by dot blot. Commassie brilliant blue (CBB) staining was used as a loading control. (B) The binding of pneumococcal Eno^wt (●), Eno^K434G (▲) or BSA (○) to eRNA was determined using the solid-phase binding assay. Immobilized proteins were incubated with different concentration of biotinylated RNA. Eno/eRNA complexes were detected by chemiluminescence. (C) The binding of pneumococcal Eno^wt (●), Eno^K434G (▲) and BSA (○) to eDNA was determined using the solid-phase binding assay. The immobilized proteins were incubated with different concentration of biotinylated eDNA. Eno/eDNA complexes were detected by chemiluminescence. Data represent mean values ± SEM; n = 3; ^###p ≤ 0.001; ***p ≤ 0.001 vs wt Eno (●). (D) The binding of Eno^wt (●), Eno^K434G (▲) or BSA (○) to Plg was determined using the solid-phase binding assay. Immobilized proteins were incubated with different concentration of biotinylated Plg. Eno/Plg complexes were detected by chemiluminescence. Data represent mean values ± SEM; n = 3; ^###p ≤ 0.001; ***p ≤ 0.001 vs wt Eno (●).