Interaction of Nuclease Colicins with Membranes: Insertion Depth Correlates with Bilayer Perturbation

Abstract

BackgroundProtein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases) or RNA (RNases) hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7) and one ribosomal RNase domain (E3) using a biomembrane model system.Principal ResultsWe demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed.Conclusions/SignificanceOur results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation.

Immunity protein release from a cell‐bound nuclease colicin complex requires global conformational rearrangement

“…Where necessary, the immunity protein was removed from the colicin complex as previously described (Vankemmelbeke et al. ). The free colicin proteins were then refolded by dialysis against PBS containing 1 mmol/L DTT and stored.…”

“…ColE9/Im9 complexes were purified by metal chelate chromatography with phosphate-buffered saline pH 7.4 (PBS) elution buffer containing 500 mmol/L NaCl and 1 mol/L imidazole. Where necessary, the immunity protein was removed from the colicin complex as previously described (Vankemmelbeke et al 2012). The free colicin proteins were then refolded by dialysis against PBS containing 1 mmol/L DTT and stored.…”

“…We suggest that a conformational rearrangement requiring a cellular energy input and initiated via the recruitment of the Tol system by the colicin IUTD is propagated along the STD (IL1 and IL3), R domain (BH29), and finally DNase domain (KM1) culminating in the release of the immunity protein. The proximity of the OM surface may also contribute to this process, providing electrostatic attraction of the positively charged DNase and repulsion of the negatively charged immunity protein (Walker et al 2007;Vankemmelbeke et al 2012).…”

The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm

“…Docking onto the inner membrane was proposed to drive the unfolding of the catalytic domain (28), thereby exposing it as a substrate for FtsH. The central domains of colicin D and klebicins C and D as well as the catalytic domains of colicin D and klebicin D do not exhibit an excess of positive charges, facilitating their electrostatically driven interaction with the inner membrane.…”

“…Although such a chargedependent interaction has been proposed as essential to initiate the late import steps, this was not found to be relevant for colicin D (9,28).…”