Membrane Topology of the Colicin A Pore-forming Domain Analyzed by Disulfide Bond Engineering

Denis Duche, Institut de Biologie Structurale et Microbiologie du CNRS
Jacques Izard, Institut de Biologie Structurale et Microbiologie du CNRS
Juan M. Gonzalez-Manas, Institut de Biologie Structurale et Microbiologie du CNRS
Michael W. Parker, University of the Basque Country
Marcel Crest, St. Vincent’s Institute of Medical Research
Martine Chartier, Laboratoire de Neurobiologie
Daniel Baty, Laboratoire de Neurobiologie

Document Type Article

Vol. 271, No. 26, Issue of June 28, pp. 15401–15406, 1996 Printed in U.S.A.

Abstract

Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A. The disulfide bonds connected a-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the poreforming domain. The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope. However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds. In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that connects the a-helices 2 and 10 prevented the colicin A membrane insertion, whereas the other double-cysteine mutants inserted into lipid vesicles. The disulfide bonds that connect either the a-helices 1 and 2 or 3 and 10 were unable to prevent the formation of a conducting channel in presence of membrane potential. These results indicate that a-helices 1, 2, 3, and 10 remain at the membrane surface after application of a membrane potential.