Intramembrane helix-helix interactions as the basis of inhibition of the colicin E1 ion channel by its immunity protein.

It had previously been hypothesized that the ability of a small number of immunity protein molecules in the cytoplasmic membrane to confer protection against the lethal effects of a channel-forming colicin involves a complex stabilized by electrostatic or polar interactions between immunity protein, the colicin channel, and specific sites on the cytoplasmic membrane surface defined by the presence of the tol gene translocation proteins. The hypothesis was tested (a) by constructing a hybrid colicin molecule, IaE1, containing the E1 channel domain, and the translocation and receptor domains of Ia, and (b) by altering charged residues in all peripheral regions of the immunity protein to neutral residues. It was concluded that the specificity of immunity protein requires neither specific translocation proteins, nor a specific arrangement of charged residues in the immunity protein. (c) In addition, by making 65 site-directed mutations, "immunity by-pass" mutants were found at five different loci, Ala474, Ser477, His440, Phe443, and Gly444, on two proposed membrane-spanning helices of the open colicin channel, one hydrophobic (A471-A488) and one amphiphilic (V441-W460). The mutants in the hydrophobic helix showed a larger bypass effect. The "bypass" phenotype could be assayed by (i) cytotoxicity and (ii) K+ efflux in imm+ cells caused by a bypass mutant but not wild-type colicin. It is concluded that the immunity protein exerts its specific effect through rapid lateral diffusion in the cytoplasmic membrane and helix-helix recognition and interaction with at least one hydrophobic and one amphiphilic trans-membrane helix of the colicin channel. Interaction with the amphiphilic helix implies that the immunity protein can react with the channel in the open state.