Fig. 5: Binding pocket mutants show decreased bactericidal activity and binding.

A Zone of inhibition assays demonstrating sensitivity of Cir mutants to WT MccV. Cir mutations C435A, R436E, R490E, R490A, and R436E/R490E exhibit losses in sensitivity to WT MccV secreted from E. coli. The third column shows that the toxic activity of MccV is attenuated when residue D85 is mutated. B MccV binds Cir with high affinity. Purified MccV was biotinylated and loaded onto streptavidin-coated biolayer interferometry (BLI) sensors at a concentration of 100 nM, then probed with a dilution series (5 μM ->78 nM in twofold steps) of wildtype (WT) Cir to determine affinity. C Mutant binding assays. 100 nM WT or mutant biotinylated MccV was loaded onto BLI sensors and probed with dilutions of WT or mutant Cir to compare binding against the WT partners. In each mutant assay, 5 μM WT Cir or 100 nM WT MccV was included as a positive control (red trace). For all BLI experiments, buffer alone (PBS-T) was added as a negative control. Representative BLI traces are shown from n = 3 experiments. D Table showing affinity and Cir/MccV-dependent sensitivity for mutants with relevant phenotypes. Note that R490A failed to properly fit to the binding model due to poor dose response.