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J. Biol. Chem., Vol. 279, Issue 21, 22145-22151, May 21, 2004

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Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

IMPLICATIONS FOR COLICIN TRANSLOCATION INTO BACTERIA^*

Khédidja Mosbahi, Daniel Walker, Edward Lea, Geoffrey R. Moore||, Richard James||, and Colin Kleanthous**

From the Department of Biology, University of York, York YO10 5YW, United Kingdom, Schools of Biological Sciences and ^||Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom, and ^||Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (_max) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in _max, from 354 to 342 nm. Changes in _max were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.

Received for publication, January 14, 2004 , and in revised form, March 10, 2004.

^* This work was funded by the Biotechnology and Biological Sciences Research Council of the United Kingdom. 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.

^** To whom correspondence should be addressed. Tel.: 44-1904-328820; E-mail: ck11{at}york.ac.uk.

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