Untitled

Abstract

The crystal structure of the cytotoxic endonuclease domain from the bacterial toxin colicin E9 in complex with its cognate immunity protein Im9 reveals that the inhibitor does not bind at the active site, the core of which comprises the HNH motif found in intron-encoded homing endonucleases, but rather at an adjacent position leaving the active site exposed yet unable to bind 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 target endonuclease at the rate of diffusion to form an inactive complex with sub-femtomolar binding affinity. This hyperefficient mechanism of inhibition could be well suited to other toxic enzyme systems, particularly where the substrate is a polymer extending beyond the boundaries of the active site.

Homing in on the Role of Transition Metals in the HNH Motif of Colicin Endonucleases

“…From these studies it was argued that inactivation of the colicin occurs through steric and electrostatic occlusion of substrate DNA, consistent with solution data demonstrating the inability of the complex to bind dsDNA, even though the active site cleft of the DNase is wide enough to accommodate substrate (17). The two structures also revealed that colicin DNases are metalloproteins, containing single, tetrahedrally coordinated transition metals within their active sites, located more than 10 Å away from the proteinprotein interface.…”

“…Thus, the immunity protein takes the form of a membrane protein that prevents the insertion of an incoming colicin into the inner membrane (16). By contrast, nuclease colicins can kill cells the moment their synthesis is complete and so have evolved a highly efficient immunity system to combat suicide (17,18). The 9.5-kDa immunity protein Im9, 1 for example, folds into its distorted fourhelical bundle structure with a rate constant of 2200 s Ϫ1 ; thus, folded immunity protein appears in Ͻ1 ms once SOS induction of the colicin operon is activated (17).…”

“…Recent crystal structures of the 15-kDa DNase domains of colicins E9 and E7 in complex with their cognate immunity proteins Im9 and Im7, respectively, revealed that the immunity protein does not bind within the active site cleft of the DNase but adjacent to it (17,22). From these studies it was argued that inactivation of the colicin occurs through steric and electrostatic occlusion of substrate DNA, consistent with solution data demonstrating the inability of the complex to bind dsDNA, even though the active site cleft of the DNase is wide enough to accommodate substrate (17).…”

“…By contrast, nuclease colicins can kill cells the moment their synthesis is complete and so have evolved a highly efficient immunity system to combat suicide (17,18). The 9.5-kDa immunity protein Im9, 1 for example, folds into its distorted fourhelical bundle structure with a rate constant of 2200 s Ϫ1 ; thus, folded immunity protein appears in Ͻ1 ms once SOS induction of the colicin operon is activated (17). The folded Im9 then associates with the colicin E9 DNase at the rate of diffusion to form an inactive complex with an equilibrium dissociation constant of 9.3 ϫ 10 Ϫ17 M, one of the highest affinity proteinprotein interactions yet reported (19).…”

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

“…The immunity protein serves to protect the producing cell from the lethal effects of the toxin but must be jettisoned before translocation into the target cell. Unusually for an enzymeinhibitor complex, the immunity protein does not bind directly to the active site of the E9 DNase, but rather to an adjacent exosite (14,15). The catalytic center of the E9 DNase domain contains the HNH motif, which is the site for both DNA and metal binding (16).…”