A force-activated trip switch triggers rapid dissociation of a colicin from its immunity protein.

Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (K(d) ≈ fM, lifetime ≈ 4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (<20 pN) increases the rate of complex dissociation 10(6)-fold, to a timescale (lifetime ≈ 10 ms) compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21-30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods.

The effect of protein complexation on the mechanical stability of Im9.

Force mode microscopy can be used to examine the effect of mechanical manipulation on the noncovalent interactions that stabilize proteins and their complexes. Here we describe the effect of complexation by the high affinity protein ligand E9 on the mechanical resistance of the simple four-helical protein, Im9. When concatenated into a construct of alternating I27 domains, Im9 unfolded below the thermal noise limit of the instrument ( approximately 20 pN). Complexation of E9 had little effect on the mechanical resistance of Im9 (unfolding force approximately 30 pN) despite the high avidity of this complex (K(d) approximately 10 fM).