Mutagenesis of the Cooh-Terminal Channel Domain of Colicin E1 Affecting the Ion Selectivity of the Channel

Abstract

Colicin El was altered by oligonucleotide-directed mutagenesis at the site of three charged residues, Asp-509, Lys-510, and Lys-512 on the COOH-side of the 35 residue hydrophobic segment in the channel-forming domain, and at the site of a neutral residue, Thr-501, in this segment. A mutation in this segment at Gly-502 was also obtained by in vivo mutagenesis. Asp-509 and Lys-512 were changed to amber and ochre stop codons, respectively, while Lys-510 was mutated to a Met codon. Proteins truncated after residue 508 or 511, and missing the last 14 or 11 residues, were obtained from a non-suppressing cell strain harboring the mutant plasmid, while full length colicin molecules with single residue changes at Asp-509 to Leu, Ser, and Gin, and Lys-512 to Tyr were obtained using appropriate suppressor strains. The truncated colicins displayed (i) low cytotoxicity, ~1% of intact wild type colicin, (ii) ten-fold less in vitro channel activity with liposomes, and (iii) smaller labeling of the colicin in liposomes by a phospholipid photoaffinity probe, showing that one or more of the residues following Asn-511 is necessary for both in vivo and in vitro activity and insertion into the bilayer. Substitution of a neutral residue for either of the positively charged Lys residues in point mutants Lys-510 → Met or Lys-512 → Tyr did not significantly affect in vivo or in vitro activity. However, the anion selectivity of the channel was significantly decreased by the latter mutations, indicating that Lys-5l0 and Lys-5l2 affect channel selectivity, probably at the surface of the channel.