Ion-channel-forming colicins

Stroud, Robert M.; Reiling, K. Kinkead; Wiener, Michael C.; Freymann, Douglas

doi:10.1016/s0959-440x(98)80132-2

Cited by 62 publications

(46 citation statements)

References 39 publications

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“…At pH 4.0, higher salt concentrations seem to be needed to diminish the DOPG-induced effect on max , presumably reflecting the increase in overall positive charge of the E9 DNase at this pH. 2 Finally, binding of the immunity protein, Im9, abolishes protein-lipid complex formation. We speculate that this is probably because of decreasing the electrostatic interaction with anionic phospholipids upon binding of the acidic immunity protein (pI ϭ 4.5) to the exosite of the basic E9 DNase domain (pI ϭ 9.5).…”

Section: Discussionmentioning

confidence: 98%

“…However, it has been shown previously that binding of a stoichiometric amount of Zn 2ϩ causes a considerable increase in the conformational stability of the E9 DNase (19). This is manifest by an increase of 22°C (from 37 to 59°C) in the melting temperature of the protein at pH 7.5, a considerable decrease in the susceptibility of the protein to proteolysis, and a decrease in affinity of 2 the protein for the hydrophobic dye ANS (19). In view of these differences, we performed a similar experiment to that described in Fig.…”

Section: E9 Dnase Specifically Interacts With Negatively Chargedmentioning

confidence: 95%

“…These data are consistent with the E9 DNase forming non-voltage gated channels in planar lipid bilayers both at pH 4.0 and pH 7.5 and indicates that the protein does not have to be in its native conformation to interact with membranes. 2 Effect of Ligand Binding and Disulfide Bond Formation on the Ability of the E9 DNase to Interact with Lipids-The experiments described above were performed on protein preparations of the E9 DNase in the absence of bound metal. However, it has been shown previously that binding of a stoichiometric amount of Zn 2ϩ causes a considerable increase in the conformational stability of the E9 DNase (19).…”

Section: E9 Dnase Specifically Interacts With Negatively Chargedmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Mosbahi

Walker

Lea

et al. 2004

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 98%

Section: E9 Dnase Specifically Interacts With Negatively Chargedmentioning

confidence: 95%

Section: E9 Dnase Specifically Interacts With Negatively Chargedmentioning

confidence: 99%

mentioning

confidence: 99%

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

Mosbahi

Walker

Lea

et al. 2004

Journal of Biological Chemistry

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“…The lethal action of colicins against their target cells is manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1)(2)(3)(4)(5)(6)(7). In this context, the bacterial machinery responsible for colicin biological activity features important mechanisms that are fundamental to various biological processes.…”

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confidence: 99%

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

Musse

Merrill

2003

Journal of Biological Chemistry

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The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, due to nutrient depletion or overcrowding, and these proteins often target-sensitive bacterial strains (1). The lethal action of colicins against their target cells is manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1-7). In this context, the bacterial machinery responsible for colicin biological activity features important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes.Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that possesses the naturally occurring colE1 plasmid; the holoprotein consists of three functional segments, the translocation, receptor-binding, and channel-forming domains. Initially, the receptor-binding domain (8) interacts with the vitamin B 12 receptor of target cells. Following recognition, the translocation domain associates with the tolA gene product, which permits the translocation of colicin E1 across the outer membrane and into the periplasm (9). In the periplasm, the channel domain undergoes a conformational change to an insertion-competent state and then inserts spontaneously into the cytoplasmic membrane of the host cell, forming an ion channel. The channel allows the passage of monovalent ions, resulting in the dissipation of the cationic gradients (H ϩ , K ϩ , and Na ϩ ) of the target cell, causing depolarization of the cytoplasmic membrane. In an effort to compensate for the membrane depolarization effected by the colicin E1 channel, Na ϩ /K ϩ -ATPase activity is increased in the host cell, resulting in the consumption of ATP reserves, without concomitant replenishment (4). The final outcome is host cell death.In addition to structural similarities between colicin E1 and several membrane-targeting proteins, an intriguing characteristic shared by colicin E1 and many membrane-active proteins is the observed requirement of an acidic environment for activation of the soluble structure in order to bind and insert into target cell membranes. The important effect of an acidic environment in conferring an optimum activity in vitro, and perhaps in vivo, for colicin E1 has been attributed to an onset of acid-induced protein unfolding events, resulting in an increased structural mobility/flexibility that may potentiate the complex sequence of structural changes of the channel peptide observed at the surface of a membrane (10 -12).An important contribution to our understanding of the mechanism of colicin E1 low pH activation was conducted by Merrill et al. (13) in which a precise pH-sensitive region with...

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Colicin

2004

Encyclopedic Dictionary of Genetics, Genomics and Proteomics

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Ion-channel-forming colicins

Cited by 62 publications

References 39 publications

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

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

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

Colicin

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