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J. Biol. Chem., Vol. 281, Issue 17, 11805-11814, April 28, 2006

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Dual Specificity of the Interfacial Inhibitor Brefeldin A for Arf Proteins and Sec7 Domains^*

Jean-Christophe Zeeh¹², Mahel Zeghouf², Cedric Grauffel³, Bernard Guibert, Elyette Martin⁴, Annick Dejaegere⁵, and Jacqueline Cherfils⁶

From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif sur Yvette, France and the Biocomputing Group, Structural Biology and Genomics Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP 10142, F-67404 Illkirch Cedex, France

Guanine nucleotide exchange factors (GEFs), which activate small GTP-binding proteins (SMG) by stimulating their GDP/GTP exchange, are emerging as candidate targets for the inhibition of cellular pathways involved in diseases. However, their specific inhibition by competitive inhibitors is challenging, because GEF and SMG families comprise highly similar members. Nature shows us an alternative strategy called interfacial inhibition, exemplified by Brefeldin A (BFA). BFA inhibits the activation of Arf1 by its GEFs in vivo by stabilizing an abortive complex between Arf-GDP and the catalytic Sec7 domain of some of its GEFs. Here we characterize the specificity of BFA toward wild-type (ARNO and BIG1) and mutant Sec7 domains and toward class I, II, and III Arfs. We find that BFA sensitivity of the exchange reaction depends on the nature of both the Sec7 domain and the Arf protein. A single Phe/Tyr substitution is sufficient to achieve BFA sensitivity of the Sec7 domain, which is supported by our characterization of brefeldin C (BFC), a BFA analog that cannot interact with the Tyr residue, and by free energy computations. We further show that Arf1 and Arf5, but not Arf6, are BFA-sensitive, despite their having every BFA-interacting residue in common. Analysis of Arf6 mutants points to the dynamics of the interswitch, which is involved in membrane-to-nucleotide signal propagation, as contributing to, although not sufficient for, BFA sensitivity. Altogether, our results reveal the Tyr/Phe substitution as a novel tool for monitoring BFA sensitivity of cellular ArfGEFs and document the exquisite and dual specificity that can be achieved by an interfacial inhibitor.

Received for publication, January 6, 2006 , and in revised form, February 7, 2006.

^* This work was supported by grants from the Association pour la Recherche contre le Cancer and the French Research Ministry (to J. C.). All authors are members of the CNRS research network GDR2823. 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.

¹ Supported by a grant from the French Ministry.

² These authors contributed equally to this work.

³ Supported by a grant from CNRS/Région Alsace.

⁴ Supported by a grant from the Ligue contre le Cancer.

⁵ Supported by CNRS, INSERM, and the University Louis Pasteur de Strasbourg.

⁶ To whom correspondence should be addressed. Tel.: 33-1-69-82-34-92; Fax: 33-1-69-82-31-29; E-mail: cherfils{at}lebs.cnrs-gif.fr.

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