Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids: IMPLICATIONS FOR COLICIN TRANSLOCATION INTO BACTERIA

Khédidja Mosbahi,Daniel Walker,Edward Lea,Geoffrey R Moore,Richard James,Colin Kleanthous

doi:10.1074/jbc.m400402200

Abstract

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (lambda(max)) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in lambda(max), from 354 to 342 nm. Changes in lambda(max) were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.

Highlights

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers
Our current work focuses on the mechanism by which the cytotoxic DNase domain of the microbial toxin colicin E9 is able to translocate into the cytoplasm of susceptible cells to reach its cellular target, the bacterial chromosome
We found that the addition of negatively charged DOPG phospholipid vesicles to the E9 DNase at pH 7.5 gave rise to a significant red-shift in the ␭max of the fluorescence of the protein that was not observed with neutral DOPC vesicles

Summary

Introduction

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers The transition from the water-soluble to membrane-bound state has perhaps been most intensely studied in the pore-forming colicins [2, 3] This family of bacterial toxins, like all colicins, share a common three-domain structure, with receptor-binding and translocation domains that facilitate binding to the cell surface and mediate delivery of the channel-forming cytotoxic domain to the inner membrane. Colicin E9 DNase Interactions with Phospholipids channels formed by the E9 DNase domain do not in themselves cause cell death, because a mutant protein lacking DNase activity is still able to induce channel formation in planar lipid bilayers but is not cytotoxic [10]. This possibility was inferred from the observation that the introduction of a specific disulphide bond in the E9 DNase domain had little effect upon the endonuclease activity of the DNase domain but abolished both channel activity and colicin cytotoxicity

Methods

Results

Conclusion