The C-terminal half of the colicin A pore-forming domain is active in vivo and in vitro.
The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of Escherichia coli from the periplasmic side and forms a functional channel. The soluble structure of pfColA consists of a ten-helix bundle containing a hydrophobic helical hairpin. Here, we generated a series of mutants in which an increasing number of sp-pfColA alpha-helices was deleted. These peptides were tested for their ability to form ion channels in vivo and in vitro. We found that the shortest sp-pfColA mutant protein that killed Escherichia coli was composed of the five last alpha-helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six alpha-helices. The peptide composed of the last five alpha-helices of pfColA generated a voltage-independent conductance in planar lipid bilayer with properties very different from that of intact pfColA. Thus, helices 1 to 4 are unnecessary for channel formation, while helix 5, or some part of it, is important but not absolutely necessary. Voltage-dependence of colicin is evidently controlled by the first four alpha-helices of pfColA.
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Laboratoire d'Ingéniérie des Systèmes Macromoléculaires Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, Marseille cedex 20, 13402, France.
Slatin
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Baty
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Duché
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D000595:Amino Acid Sequence; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004553:Electric Conductivity; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D002712:Chlorides; D003087:Colicins; D008051:Lipid Bilayers; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011188:Potassium