Precise Timing of ATPase Activation Drives Targeting of Tail-Anchored Proteins

2942-Pos Board B97 Force Triggered Dissociation of the Highly Avid E9:Im9 Complex David Brockwell1, Oliver Farrance1, Renata Kaminska2, Sasha Derrington1, Colin Kleanthous2, Sheena Radford1. University of Leeds, Leeds, United Kingdom, University of Oxford, Oxford, United Kingdom. Colicin E9 is a nuclease antibiotic produced by E.coli to target and kill competing bacteria in times of stress. Immunity protein 9 (Im9) is expressed cotranslationally and binds strongly to E9, inactivating the nuclease to protect the producing cell from the cytotoxic effects of E9. The affinity between E9:Im9 is highly avid with an off-rate of the order of days. However, upon binding to an outer membrane protein of a competing cell, a complex set of protein:protein interactions coupled to protein remodelling results in rapid Im9 release allowing E9 translocation into the cytoplasm and cell death on a timescale of minutes. In order to investigate the molecular basis for such an extreme switch in complex avidity we have used an atomic force microscope to perform dynamic force spectroscopy (DFS) measurements on E9:Im9. We observe a forceinduced switching mechanism from the long lived complex (koff ~ 1x10 5 s ) in the absence of force to a much shorter lived state (koff ~ 5 s ) under the application of small biologically relevant forces. The decrease in complex lifetime under force can be abrogated by introduction of disulfide cross crosslinks. This suggests that protein dynamics lie at the heart of the bipartite function of the complex switching from a highly stable complex protective to host, to one where rapid dissociation facilitates competitor death.