Effector Recognition by the Small GTP-binding Proteins Ras and Ral

doi:10.1074/jbc.274.25.17763

Effector Recognition by the Small GTP-binding Proteins Ras and Ral*

From the ^‡Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany, the ^‖Institut Curie, INSERM 248, 26 rue d’Ulm, F-75248 Paris, Cedex 05, France, and the ^‡Protein Design Group, Centro Nationale de Biotecnologı́a, Campus de la Universidad Autónoma, Cantoblanco, Madrid M-28049, Spain

Abstract

The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral·GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.

Footnotes

↵* This work was partially supported by Institut Curie, Ligue contre le Cancer, Association pour la Recherche sur le Cancer, and Grant BIO4-CT96-1110 of the European Community.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ These authors contributed equally.
↵¶ Supported by Grant BIO4-CT96-1110 of the European Community.
↵** Supported by a Ministère de l’Education Nationale de la Recherche et de la Technologie Ph.D. fellowship.
↵§§ To whom correspondence should be addressed. Tel.: 49-231-133-2105; Fax: 49-231-133-2199; E-mail:robbert.cool{at}mpi-dortmund.mpg.de.
↵2 P. Bork, C. Sander, and A. Valencia, unpublished results.
↵3 I. R. Vetter, manuscript in preparation.
Abbreviations:

GAP

GTPase activating protein

GDS

guanine nucleotide dissociation stimulator

GEF

guanine nucleotide exchange factor

Gpp(NH)p

5′-guanylylimidodiphosphate

GST

glutathione S-transferase

RBD

Ras-binding domain

PCR

polymerase chain reaction

wt

wild type

RalBD

Ral-binding domain
- Received February 5, 1999.
- Revision received March 25, 1999.
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