UNCOUPLED STEPS OF THE COLICIN-A PORE FORMATION DEMONSTRATED BY DISULFIDE BOND ENGINEERING

Four disulfide bonds were engineered into the pore-forming domain of c olicin A to probe the conformational changes associated with its membr ane insertion and channel formation. The soluble pore-forming domain c onsists of 10 alpha-helices with two outer layers (helices 1, 2, and 3 -7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has be en hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichi a coli cells. Activity can be restored by addition of a reducing agent . In vitro studies with brominated lipid vesicles and planar lipid bil ayers show that the disulfide bond which connects the helices 1 to 9 p revents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bri dges prevented the formation of a conducting channel in the presence o f a membrane potential. This novel approach indicates that membrane in sertion and channel formation are two separate steps. Moreover, the ef fects of the distance constraints introduced by the different disulfid e bonds on colicin A activity indicate that the helical pair 1 and 2 m oves away from the other helices upon membrane insertion, Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast , induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results ar e discussed in light of several proposed models of the membrane-bound colicin and channel structures.