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

Abstract

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...

Topography of the Hydrophilic Helices of Membrane-Inserted Diphtheria Toxin T Domain: TH1−TH3 as a Hydrophilic Tether

“…Low pH-induced breakdown of favorable Coulombic interactions between spacially close residue pairs that have opposite charge at neutral pH (Arg210-Glu362, Lys216-Glu259, Lys229-Glu249, and Glu292-Lys299) may also contribute to unfolding. Finally, changes in hydrogen-bonding between side chains and backbone groups, analogous to changes that have been observed for residues in colicin E1, may also play an important role in this process (48). Figure 6 (top) schematically summarizes the low pH-induced changes in the conformation of the TH 1-3 region in solution.…”

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

“…In that case, 3 Asp residues distributed within two helices triggered disruption of the helical structure upon acidification (27). Hence, acidic residues are used by several toxins as low pH sensors to induce a destabilizing effect on specific target and key helices whose collapse will enable structural rearrangements upon low pH exposure.…”

“…Hence, acidic residues are used by several toxins as low pH sensors to induce a destabilizing effect on specific target and key helices whose collapse will enable structural rearrangements upon low pH exposure. These acidic residues (Asp until now) can control the integrity of these helices because they belong to the helix residues (27) or because they are the N-cap residue (this study).…”

“…In several toxins, such as botulinum toxins, diphtheria toxin, and colicins whose conformation changes in response to low pH, acidic residues were identified as low pH sensors triggering structural modifications (25)(26)(27). We therefore monitored the pH dependence of membrane insertion for all Asp-358/Glu-359 mutants using Trp fluorescence quenching by brominated liposomes.…”

Subunit–subunit interactions are critical for proton sensitivity of ROMK: Evidence in support of an intermolecular gating mechanism

“…For most ion pairs exposed to solvent, changes in the pH of the surrounding solution can lead to either making or breaking of these salt bridges (22). Acid pH breaks a critical salt bridge between transmembrane domains and activates the bacterial colicin ion channel (23), and mutation of a salt bridge between subunits of the AMPA receptor decreases dimer stability and accelerates deactivation (24). Thus, we reasoned that if subunit-subunit interactions are important to pH sensing of ROMK, then disruption of this R-E ion pair should alter channel pH 0.5 .…”