Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

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 ( 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 max , from 354 to 342 nm. Changes in 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 preempts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.Unraveling the interactions and mechanisms that enable proteins to cross biological membranes is of considerable interest, as the ability to target specific exogenous enzymes to the cytosol is likely to facilitate the design and discovery of novel chemotherapeutic agents. Many protein toxins have evolved to deliver a cytotoxic domain or subunit to the cytoplasm of susceptible cells, and so they provide an invaluable tool for studying protein translocation from the extracellular environment to their cellular targets, often located in the cytoplasm (1). 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. Cell death occurs as a consequence of ion channel formation across the cytoplasmic membrane, inducing depolarization of the membrane. Association of the cytotoxic domain with the membrane is thought to lead to destabilization and unfolding of the protein, yielding a "molten...

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria

“…This lipid-induced destabilization was also observed in thermal unfolding experiments followed by CD in the far-UV region (12). Such a loss of cooperativity upon diC8PI binding is characteristic of membrane-bound proteins lacking tertiary structure interactions (17,18). In line with these results, limited proteolysis experiments in the presence of diC8PI micelles or anionic SUVs increased the susceptibility of PimA to proteolysis (12).…”

“…Such a loss of cooperativity upon binding of PI or DOPG liposomes is characteristic of membrane-bound proteins in molten globule-like (MG-like) states, i.e. displaying native-like secondary structures but lacking a stable tertiary structure (17,18,21).…”

FtsH-dependent Processing of RNase Colicins D and E3 Means That Only the Cytotoxic Domains Are Imported into the Cytoplasm

“…The presence of an unstructured terminal tail of membrane substrates that penetrate into the cytoplasm appears to be important in initiating FtsH-de- pendent proteolysis (48). It is conceivable that DNase colicins have a similar access to FtsH, because their nuclease domain exhibits an endogenous channel-forming activity, possibly allowing its self-propulsion into the cytoplasm (14,15). An electrostatically mediated interaction of the nuclease domain of E type colicins with the inner membrane has been reported (16).…”

“…The DNase domains of colicins E9 and E2 exhibit nonvoltage-gated channel forming activity in planar lipid bilayers, which involves changes in their conformation (14). Such channels, unlike those formed by pore-forming colicins, are not directly responsible for cell killing but may allow "self-propulsion" of the toxic domains into cytoplasm, driven by an electrostatic association of the DNase domain of colicin E9 with the inner membrane (15). Although such an association between the RNase colicin E3 and anionic phospholipid surfaces has been reported, no RNase colicin has been found to exhibit any channel forming activity (16).…”

Colicin Biology

“…Dover et al suggested that the poreforming domain of colicin N might translocate through OmpF, following the same route as the N-terminal domain (161). Alternatively, the C-terminal domain might translocate through the OM bilayer by conformational changes induced by its interaction with the phospholipids (468,469,470) or at the interface between the porin trimer and the LPS leaflet. It has also been proposed that for group A colicins, interactions between the colicin N-terminal domain and the Tol machinery would displace Tol-Tol interactions (such as the TolB-Pal complex) (417) and then induce an OM defect and the subsequent entry of the cytotoxic domain (147).…”