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  • The EMBO Journal (1998) 17, 3651 - 3659
  • doi:10.1093/emboj/17.13.3651

A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the bold beta-phosphate to destabilize GDP on ARF1

Sophie Béraud-Dufour1,3, Sylviane Robineau1,3, Pierre Chardin1, Sonia Paris1, Marc Chabre1, Jacqueline Cherfils2 and Bruno Antonny1

  1. CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560 Valbonne, France
  2. CNRS, Laboratoire d'Enzymologie et Biochimie Structurales, 91198 Gif-sur-Yvette, France
  3. S.Béraud-Dufour and S.Robineau contributed equally to this work

Correspondence to:

Bruno Antonny, E-mail: antonny@ipmc.cnrs.fr

Received 27 March 1998; Accepted 29 April 1998; Revised 28 April 1998

The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.

  • Keywords:

    • ARF,
    • ARNO,
    • G protein,
    • guanine nucleotide exchange factor,
    • Sec7 domain