A 136‐amino‐acid‐residue COOH‐terminal fragment of colicin A is endowed with ionophoric activity

Abstract

DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a 'STOP-Shine-Dalgarno-sequence-ATG' cassette encoding a termination codon, a ShineDalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137-amino-acid-residue pore-forming protein designated DA70C comprising the final 136-amino-acid-residue COOH-terminal of colicin A preceded by an NH,-terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium. Its purification involved, as a final step, a partition in Triton X-114 thus demonstrating that hydrophobic regions are exposed in this protein. The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed. The results indicate that helices 1 -3 of the 204-amino-acid-residue colicin pore-forming domain (containing 10 a-heIices) are not involved in ion conduction through the channel. However, they are important in maintaining the stability of the soluble state of the COOH-terminal domain.Colicin A is a bacterial toxin produced by Escherichia coli and closely related bacteria. It belongs to the largest group of colicins comprising those which can form voltage-dependent channels in membranes, thereby destroying the cell's energy potential [l].The channel forming domain is contained in the carboxy-terminal region of the molecule [2]. A 204-aminoacid-residue COOH-terminal fragment obtained by digestion with thermolysin comprising amino acids 389 -592 from colicin A has pore-forming properties similar to those of the entire molecule [3]. This fragment has the unusual property to be both soluble in aqueous medium and able spontaneously to insert into lipid monolayers and bilayers [4]. The threedimensional structure at 25-fm resolution has been recently determined [4]. The protein consists of ten a-helices organized in a three-layer structure. Two of these helices are entirely hydrophilic and completely buried within the structure of the 204-residue COOH-terminal fragment. The colicin El COOHterminal domain is significantly similar to colicin A [1,5 -71. The minimum size of this domain that is competent for channel function is under debate [8]. However, it is clear that a colicin El COOH-terminal fragment of 152 residues is competent for channel function [9]. This would mean that helices 1 and 2, recently identified in the three-dimensional structure of colicin A [4], are not required for channel formation. It has been reported that the a-helical content of the lipid-associated COOH-terminal fragment of colicin A is similar to that of the soluble form [5]. This suggests that about the same helices as that identified in the soluble form [4] exist in the membraneinserted form of this fragment.We have previously demonstrated that DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a 'cassette' encoding a termination codon, a Shine-Dalg...

Colicin N Binds to the Periphery of Its Receptor and Translocator, Outer Membrane Protein F

“…The evidence here is that the unfolded C-terminal domain inserts in clefts at the periphery of OmpF with direct binding by its first helix. The remaining helices are sufficient to span the periplasm and form a functional toxic pore (Baty et al., 1990; Figure 5). It is not clear where the N-terminal translocation domain fits in the current proposal.…”

Colicin Biology

“…For both colicins E1 and A, several helices worth of protein can be completely eliminated from the N terminus of the domain without preventing channel formation (16,124,408,478). There remains some uncertainty about the precise location of the upstream edge of the channel, part of which results from the fact that some of the shorter fragments tested form channels with altered properties compared to the native protein and part of which may reflect true differences among the colicins.…”

Colicin Pore-Forming Domains Bind toEscherichia ColiTrimeric Porins

“…In the pore-forming regions of the colicins tested, the closest match is in the N-terminal region, which links to the receptorbinding domain (colicin N 184- . This section of the P-domain is not required for pore formation (34) and in several published models of poreforming colicin translocation it is shown adjacent to the porin (28,35). SDS-PAGE Artifact or Useful Biological Data?…”