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J. Biol. Chem., Vol. 282, Issue 33, 24239-24245, August 17, 2007

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Concerted Protonation of Key Histidines Triggers Membrane Interaction of the Diphtheria Toxin T Domain^*

Aurélie Perier¹, Anne Chassaing¹, Stéphanie Raffestin, Sylvain Pichard, Michel Masella, André Ménez, Vincent Forge, Alexandre Chenal²³, and Daniel Gillet²⁴

From the Commissariat à l'Energie Atomique, Institut de Biologie et Technologies de Saclay, Service d'Ingénierie Moléculaire des Protéines, Gif-sur-Yvette F-91191, France and Institut de Recherche en Technologies et Sciences pour le Vivant, Laboratoire de Chimie Biologie des Métaux, Grenoble F-38054, France

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His²²³-His²⁵⁷ and His²⁵¹ are the most sensitive triggers for the formation of the molten globule state in solution, whereas His³²²-His³²³ and His²⁵¹ are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.

Received for publication, April 24, 2007 , and in revised form, June 13, 2007.

^* This work was supported by the Commissariat à l'Energie Atomique (Signalization and Membrane Transport Program of the Life Science Division) and by Action Concertées Incitative-microbiology program of Fonds National de la Science Grant MIC 0325. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2.

¹ These authors contributed equally to this work.

² These authors contributed equally to the elaboration and direction of this work.

³ To whom correspondence may be addressed: Unité de Biochimie des Interactions Macromoléculaires, URA CNRS 2185, Dépt. de Biologie Structurale et Chimie, Institut Pasteur, 25-28 Rue du Dr. Roux, 75724 Paris cedex 15, France. Tel.: 33-1-44-38-92-12; Fax: 33-1-40-61-30-42; E-mail: chenal{at}pasteur.fr. ⁴ To whom correspondence may be addressed. E-mail: daniel.gillet{at}cea.fr.

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