1.C.1 The Channel-forming Colicin (Colicin) Family
Colicins are plasmid-encoded bacteriocins which are produced by E. coli and other enteric bacteria. They exert a lethal effect on other bacteria including E. coli strains that lack the Col plasmid. The proteins bind to a cell surface receptor and are transported into the periplasm via an energy-dependent process involving a TonB- or a TolA-dependent heterooligomeric protein complex (TC #10.6). Some colicins kill their target cell by inserting into the cytoplasmic membrane where they form voltage-sensitive (trans-negative) channels that depolarize and deenergize the cell, and thereby kill it. Among these channel-forming colicins are the well-characterized homologous colicins E1, Ia, Ib, A, B, N and K. These channels transport about 107 ions per channel-second in 1 M NaCl. This ion flux rate can depolarize E. coli since its H pump rate is approximately 106 H+/cell-sec.
Colicins E1, Ia and A are 500-700 amino acyl residues in length and exhibit three domains: an N-terminal translocation domain, a central receptor domain and a C-terminal channel (C) domain. The C domain is generally about 150-180 residues long. That of colicin A has been crystallized and its structure determined to 2.4 Å resolution. It consists of 10 α-helices, two of which (helices 8 and 9) are strongly hydrophobic and insert into the membrane. These two helices comprise a helical hair pin which is the primary attachment site for insertion into the membrane. It is also the site of interaction with the colicin A immunity protein (Nardi et al., 2001). The colicin A channel has been reported to be selective for protons over other cations (and anions) by many orders of magnitude (Slatin et al., 2008).
The 3-D structures of the soluble colicin A (TC #1.C.1.3.1), B (TC #1.C.1.3.2), N (TC #1.C.1.3.3) and E1 (TC #1.C.1.2.2) have been reported at ~2.5 Å resolution (Hilsenbeck et al., 2004; Wormald et al., 1990; Zakharov and Cramer, 2002). Colicin B recognizes the FepA outer membrane receptor (TC #1.B.14.1.1) and gains access to the cytoplasmic membrane via a TonB-dependent mechanism. Unlike colicin Ia, colicin B does not have clearly delineated receptor-binding and translocation domains, but the C-terminal pore-forming domain is distinct and connected to the N-terminal domain by a long (74 Å) helix. The pore-forming domain consists of 10 α-helices arranged in a bundle like other colicins (Hilsenbeck et al., 2004). In fact, only this C-terminal domain is well conserved with the other colicins. The structures of colicin A, B, N, Ia and E1 are similar (White et al., 2006; Zakharov et al., 2002). These structures suggest that a key to their pore-forming ability are the two distinct hydrophobic helices that create a membranespanning hairpin upon bilayer association (Malenbaum et al., 1998; Shin et al., 1993; Song and Cramer, 1991). This feature of the colicin structure is also seen in the mammalian intracellular apoptotic regulators Bcl-2, Bcl-XL, Bax, and Bid, proteins known to create pores upon association with the mitochondrial or endoplasmic reticulum membranes.
Colicin E1 has been well studied with respect to its insertion into the cytoplasmic membrane of E. coli. Initially, the receptor-binding domain interacts with the vitamin B12 receptor of target E. coli cells. Following recognition, the translocation domain associates with the trimeric tolA gene product, which permits the translocation of the unfolded colicin E1 across the outer membrane and into the periplasm. From 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 a precursor of the open ion channel. The channel opens upon imposition of a trans-negative membrane potential, and the newly created pore allows escape of Na+, K+ and H+. As the bacterial cell tries to equilibrate the ion concentration using the Na+/K+-ATPase, cellular ATP reserves are rapidly depleted and cannot be replenished sufficiently so that cell death ensues (White et al., 2006). The channel allows the passage of monovalent ions, resulting in the dissipation of the cationic gradients (H+, K+ and Na+) of the target cell, causing depolarization of the cytoplasmic membrane.
If the cell is energized, the colicin A opens. According to one model, helices C5 and C6 insert into the membrane, and these two helices together with C8 and C9 comprise the channel. However, the channel is estimated to be 6-9 Å in diameter, and a channel as large as 9 Å may require the participation of six or more helices. Some experimental evidence suggests that a large portion of the C-domain is translocated across the bilayer of the membrane when the channel opens. Thus, channel opening and closing in response to the membrane potential is believed to involve a major rearrangement of the protein with several of the amphipathic helices localized to the outer surface of the membrane in the closed state but inserted in the bilayer in the open state.
Gating of colicin channels involves structural rearrangements rather than transfer of ~70 residues of the pore-forming domain across the membrane. Translocation does not depend on multimerization of the channel forming domain. When hydrophilic proteins are inserted into the translocation segment of colicin A, they are translocated to the trans side of the bilayer where they are functional. Therefore, colicin channels have a general protein translocation function (Slatin et al., 2002).
The channel-forming domain of colicin Ia has recently been shown to be homologous (46%) to an otherwise dissimilar protein, Pyocin S5 of Pseudomonas aeruginosa. This fact thus suggests (1) that colicin-like pore formers may be widespread, and (2) that domain shuffling has occurred during the evolution of these proteins (Parret and DeMot, 2000). Each pyocins contains an N-terminal receptor-binding domain, a translocation domain that transports the pyocin across the membrane, and a C-terminal DNase (or other catalytic functional domain) that can kill the cell (Ghequire and Öztürk 2018).
Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. The small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltaage-gated channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells (Mosbahi et al., 2002). A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Thus, endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein. E9 is homologous to colicin B (TC #1.C.1.3.2) in a 160 aa region (residues 130-290) in both proteins.
Colicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusual simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. In 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface (Baboolal et al., 2008). Colicin N binding to OmpF displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.
The channel formed by colicin A in planar lipid bilayers has an outsized selectivity for protons compared to any other ion, even though it allows large ions, such as tetraethylammonium, to permeate readily. Manipulations that interfere with ionic conduction, such as replacing some of the water in the pore with a nonelectrolyte, reduce the proton current along with the ionic current. The 10-helix channel-forming domains of colicins Ia and E1 are structurally homologous to that of colicin A but do not select so remarkably for protons. Slatin et al. (2010) found that selectivity is localized to the five C-terminal helices of colicin A.
The generalized reaction catalyzed by the channel-forming colicins is:
Ions (in) ions (out)