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J. Biol. Chem., Vol. 278, Issue 51, 51023-51034, December 19, 2003

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Identification and Characterization of an Amphipathic Leucine Zipper-like Motif in Escherichia coli Toxin Hemolysin E

PLAUSIBLE ROLE IN THE ASSEMBLY AND MEMBRANE DESTABILIZATION^*

Sharada Prasad Yadav, Bijoy Kundu¶, and Jimut Kanti Ghosh||

From the Molecular and Structural Biology Division and the ^¶Medicinal Chemistry Division, Central Drug Research Institute, Lucknow 226 001, India

Hemolysin E (HlyE) is a 34 kDa protein toxin, recently isolated from a pathogenic strain of Escherichia coli, which is believed to exert its toxic activity via formation of pores in the target cell membrane. With the goal of understanding the involvement of different segments of hemolysin E in the membrane interaction and assembly of the toxin, a conserved, amphipathic leucine zipper-like motif has been identified. In order to evaluate the possible structural and functional roles of this segment in HlyE, a 30-residue peptide (H-205) corresponding to the leucine zipper motif (amino acid 205-234) and two mutant peptides of the same size were synthesized and labeled by fluorescent probes at their N termini. The results show that the wild-type H-205 binds to both zwitterionic (PC/Chol) and negatively charged (PC/PG/Chol) phospholipid vesicles and also self-assemble therein. Detailed membrane-binding experiments revealed that this synthetic motif (H-205) formed large aggregates and inserted into the bilayer of only negatively charged lipid vesicles but not of zwitterionic membrane. Although both the mutants bound to zwitterionic and negatively charged lipid vesicles, neither of them inserted into the lipid bilayers nor assembled in any of these lipid vesicles. Furthermore, H-205 adopted a significant helical structure in membrane mimetic environments and induced the permeation of monovalent ions and release of entrapped calcein across the phospholipid vesicles more efficiently than the mutant peptides. The results presented here indicate that this H-205 (amino acid 205-234) segment may be an important structural element in hemolysin E, which could play a significant role in the binding and assembly of the toxin in the target cell membrane and its destabilization.

Received for publication, September 10, 2003 , and in revised form, October 1, 2003.

^* This is CDRI communication number 6454. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Junior Research Fellowship from CSIR, India.

^|| To whom correspondence should be addressed. Tel.: 2212411-18 (ext. 4282); Fax: 091-522-223405/223938/229504; E-mail: jighosh{at}yahoo.com.

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