Structural and mechanistic basis of immunity toward endonuclease colicins

The crystal structure of the cytotoxic endonuclease domain from the bacteri al toxin colicin E9 in complex with its cognate immunity protein Im9 reveal s that the inhibitor does not bind at the active site, the core of which co mprises the HNH motif found in intron-encoded homing endonucleases, but rat her at an adjacent position leaving the active site exposed yet unable to b ind DNA because of steric and electrostatic clashes with incoming substrate . Although its mode of action is unorthodox, Im9 is a remarkably effective inhibitor since it folds within milliseconds and then associates with its t arget endonuclease at the rate of diffusion to form an inactive complex wit h sub-femtomolar binding affinity. This hyperefficient mechanism of inhibit ion could be well suited to other toxic enzyme systems, particularly where the substrate is a polymer extending beyond the boundaries of the active si te.