Pore Formation

From Proteopedia

Many Colicins produced by E. coli cells kill other E. coli cells through the formation of a pore in the membrane of the cell. This kills the cell that is targeted by disrupting the ionic gradient of the cell^[1]. The colicins that target their cells in this way are known as lysis proteins, as they lyse the target cell^[2]. Colicins are alpha-pore forming toxins, where they form a channel of alpha-helices. The pores formed leak ions, which depolarises the membrane of the targeted cell, and causing cell death^[3]. The activity of these channel-forming colicins is largest in vitro at an acidic ambient pH^[4]. The colicins that have this pore-forming domain are Colicin A, Colicin Ia, Colicin E1, Colicin N and Colicin B.

Production and formation

Colicin lysis proteins are present in a colicin operon, alongside a regulatory gene called cal. The cal gene product has a self-regulatory role, whereby it exerts a positive effect on its own synthesis. As it is within the same operon as the lysis protein and the rest of the colicin, it also up-regulates synthesis of these^[5].

Structure

The pore-forming domains of colicins are globular and formed of 10 alpha helices. Two hydrophobic helices form the core of the channel, making a hydrophobic helical hairpin that inserts into the membrane during the early stage of pore-formation. These pore forming regions are initially folded up on the surface of the soluble precursors, which unfold at the membrane.

Structures have been determined for the pore-forming domains for Colicin A and Colicin E1, along with complete structures for Colicin Ia, Colicin B and Colicin N.

The structure of these bacterial toxins challenge the notions that protein sequence determines the unique 3D structure, and that membrane and soluble proteins are very distinct. Colicins are able to transform from soluble monomeric proteins to oligomers that form transmembrane channels.

The structure of the pore state is still unclear. Discovery of the structure is problematic, as the helices are too short to span a standard bilayer. The current theoretical model is that of the toroidal lipidic pore. In this, the protein exerts a detergent-like action, inducing a high curvature in the bilayer. These pores are characterised by their sensitivity to lipids that alter the curvature of the membrane. This curved region of the membrane could then be lined by helices that are shorter than could normally cross the bilayer^[6].

Membrane insertion

3 stages are involved in the insertion of the pore-forming region of colicins into the membrane of the target cell: Binding, Unfolding and Insertion.

After binding to the membrane, the pore-forming domain unfolds and releases the hydrophobic helices to initiate insertion. This unfolding is shown to be linked to a low pH. 3 conserved aspartate residues in the domain form a hydrogen-bonding domain which is disrupted by the low pH, as is a critical salt bridge required for stability of the folded protein. This causes local unfolding of a helix. The helices then loosely associate on the surface of the membrane, as the hydrophobic hairpin inserts into the bilayer^[7].

Pore-forming Colicins

Colicin A
Colicin E1
Colicin N
Colicin S4
Colicin K
Colicin U
Colicin 5
Colicin 6
Colicin 7
Colicin 8
Colicin 9
Colicin 10
Colicin Ia
Colicin Ib
Colicin B
Colicin V
Colicin Y

3D structures of colicin

Colicin

References

1. ↑ Cutler SA, Lonergan SM, Cornick N, Johnson AK, Stahl CH. Dietary inclusion of colicin e1 is effective in preventing postweaning diarrhea caused by F18-positive Escherichia coli in pigs. Antimicrob Agents Chemother. 2007 Nov;51(11):3830-5. Epub 2007 Aug 27. PMID:17724148 doi:10.1128/AAC.00360-07
2. ↑ Cavard D. Role of the colicin A lysis protein in the expression of the colicin A operon. Microbiology. 1997 Jul;143 ( Pt 7):2295-303. PMID:9245818
3. ↑ Tilley SJ, Saibil HR. The mechanism of pore formation by bacterial toxins. Curr Opin Struct Biol. 2006 Apr;16(2):230-6. Epub 2006 Mar 24. PMID:16563740 doi:10.1016/j.sbi.2006.03.008
4. ↑ Sobko AA, Rokitskaya TI, Kotova EA. Histidine 440 controls the opening of colicin E1 channels in a lipid-dependent manner. Biochim Biophys Acta. 2009 Sep;1788(9):1962-6. Epub 2009 Jun 26. PMID:19560438 doi:10.1016/j.bbamem.2009.06.017
5. ↑ Cavard D. Role of the colicin A lysis protein in the expression of the colicin A operon. Microbiology. 1997 Jul;143 ( Pt 7):2295-303. PMID:9245818
6. ↑ Tilley SJ, Saibil HR. The mechanism of pore formation by bacterial toxins. Curr Opin Struct Biol. 2006 Apr;16(2):230-6. Epub 2006 Mar 24. PMID:16563740 doi:10.1016/j.sbi.2006.03.008
7. ↑ Tilley SJ, Saibil HR. The mechanism of pore formation by bacterial toxins. Curr Opin Struct Biol. 2006 Apr;16(2):230-6. Epub 2006 Mar 24. PMID:16563740 doi:10.1016/j.sbi.2006.03.008