Investigating Protein-Protein Interaction Networks with Force Spectroscopy

Abstract

Colicins are antimicrobial proteins produced by bacteria. Their highly active modes of killing make colicins of interest as a potential new class of antibiotics. However, the precise mechanism of cytotoxicity is not understood thus limiting their translation to biotechnological applications.It is thought that colicin intoxication occurs via a series of protein-protein interactions (PPIs) that span the periplasm (Housden et al. Science, 2013). Mechanical force may play a role in this process by an inside-out energy transduction mechanism. Periplasmic proteins that are subverted by colicins have either a known role in applying force during their normal function in-vivo or are highly homologous to proteins that do.In this work we study the colicin E9 system and the PPIs that remodel during its translocation across the outer and inner membranes of E. coli. Through a series of in-vitro single molecule force spectroscopy experiments performed using an atomic force microscope, we identify a mechanism by which dissociation of the highly avid colicin E9-immunity protein interaction (fM affinity, half-life days) can take place via the application of small forces (<20 pN) during colicin translocation (Farrance et al. PLoS Biol, 2013). Such a mechanism could account for the disparity between measured lifetimes of colicin-immunity protein interactions and the timescale on which colicins kill (days vs. minutes) for which immunity protein release is a prerequisite.Further and ongoing pulling experiments on other PPIs formed during colicin E9 translocation involving periplasmic proteins from the Tol complex also show interesting behaviour under applied forces. These results suggest that the PPI network formed during E9 translocation would be able to support and transduce significant forces and these may be crucial in understanding the mechanism by which colicins gain access to the cellular interior and ultimately kill their target.