Figure 8

In vitro characterization of the putative EutQ:EutM interaction.

(A) Crystal structure of the EutM hexamer from E. coli (PDB ID: 3MPW²⁸). The predicted cytosol exposed helix is highlighted in red; residues predicted to be involved in electrostatic interactions with EutQ are shown as orange sticks. The arrow indicates the predicted orientation of the luminal and exterior faces of the EutM hexamer in the context of a Eut BMC shell. (B) Identification of EutM mutants which abolish interaction with EutQ. Green boxes indicate co-elution of EutM mutants with EutQ in pull-downs, while red boxes indicate no co-elution. (C) Representative SDS-PAGE analysis of a mutation that abolishes interaction between EutM and EutQ (for clarity only the D57A mutant is shown). Lanes 1 and 2 depict a control pull-down between wild-type EutM and EutQ CTH6, while lanes 3 and 4 demonstrate the loss of EutQ CTH6 binding by the EutM D57A mutant. Lanes 2 and 4 are the high imidazole (250 mM) elutions, while lanes 1 and 3 are elutions at low (50 mM) imidazole concentrations. (D) Cartoon representation of a EutM monomer with the residues shown to be involved in EutQ:EutM interactions highlighted as orange sticks.