Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.

Abstract

The channel-forming activity of colicin El in artificial membranes is known to increase at low pH values and to have a maximum near pH 4 in such membrane vesicles. The present work demonstrates that this pH dependence of activity can be attributed to membrane binding. Maximal binding of colicin El and a more slowly binding channelforming carboxyl-terminal tryptic peptide occurred at acidic pH values, with the effective pK values for binding equal to 4.6 and <4.0, respectively. The binding did not require imposition of a transmembrane potential. Insertion of the tryptic peptide into the membrane was shown by retention of bound [ HIleucine-labeled peptide by vesicles after digestion with protease, as well as by retention of the peptide in salt-washed vesicles. The retention after protease treatment was also used to estimate the amount of carboxyl-terminal peptide inserted into the membrane. Approximately 12 of the 21 leucines present in the carboxyl-terminal peptide were retained after Pronase treatment at pH < 4. Reversibility of the insertion at low pH values was seen after an alkaline shift of pH to 6.0, resulting in a decrease of the protease-inaccessible fraction of the bound protein. A model is presented describing a mechanism in which protonation of one or more carboxyl residues is necessary for effective binding and insertion into the membrane by the channel-forming domain of colicin El. This model may also be relevant to the mechanism of membrane insertion by certain toxins.Colicin El is a 522-residue bactericidal protein, known to exert its lethal effect through an ability to depolarize the cytoplasmic membrane of sensitive Escherichia coli (1-3). Studies of colicin El action using artificial membranes have shown that the deenergization results from an ion channel formed by the colicin molecule, which dissipates the cellular membrane potential (ref. 4, reviewed in refs. 5-7). Colicin El requires an acidic pH in vitro to form ion channels (8), and its action is dependent upon the sign and magnitude of the membrane potential (4, 8, 9). The channel-forming activity of colicin El has been shown to reside in a Mr 20,000 carboxyl-terminal tryptic peptide (10). The tryptic peptide and colicin required a low pH for in vitro activity. The effective pK values for activity, assayed by using artificial vesicles, were 4.6 and 3.8, respectively, for the colicin and the tryptic peptide (8). This requirement of an acidic pH for colicin El action resembles the pH dependence in vitro for the actions of diphtheria toxin (11)(12)(13)(14) and tetanus toxin (15), and it suggests that an explanation of this pH effect may bear on a general molecular mechanism for insertion into membranes of other colicins (2-4, 7, 16, 17), as well as the above-mentioned channel-forming toxin molecules (11)(12)(13)(14)(15).The purpose of the present work was to examine the mechanism of binding and of insertion into artificial membranes by the channel-forming domain of colicin El, and specifically its dependence on pH and protonation of o...

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

“…The C-terminal domain of WT colicin E1 shows a strong, cooperative pH dependence for membrane binding with a pK a for association near 4.1 (WT, Fig. 2, a-d, diamonds), which agrees well with previous reports (11,(27)(28)(29)(30). In contrast to the WT protein, the binding isotherms of the double Asp-toSer-substituted and disulfide-bonded mutant channel peptides exhibited altered binding profiles with an alkaline-directed shift in pH-dependence (Fig.…”

Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli.

“…5 The rate of progression from reversible to irreversible growth inhibition in phosphate-buffered nutrient broth shows some strain to strain variation (Table IV) but in all cases is more rapid at pH 6.0 than at pH 7.4. Such a pH dependence of cytotoxicity is shared by several prokaryotic and eukaryotic toxins whose action depends on penetration across lipid bilayers as part of the target cell damage (27)(28)(29)(30)(31)(32)(33)(34). We are now seeking evidence of penetration of a portion of bound BPI, or a biologically active fragment (12) further into the bacterial envelope.…”

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase

“…acidic pH required for activity of toxins such as diphtheria (London, 1992) and the channel-forming colicins (Davidson et al, 1985;Merrill et al, 1990); and (2) the more acidic environment near the negatively charged membrane surface (van der Coot et al, 1991;Muga et al, 1993).…”