The mammalian growth factor receptor-binding protein Grb2 is an adaptor that mediates activation of guanine nucleotide exchange on Ras. Grb2 binds to the receptor through its SH2 domain and to the carboxyl-terminal domain of Son of sevenless through its two SH3 domains. It is thus a key element in the signal transduction pathway. The crystal structure of Grb2 was determined to 3.1 angstrom resolution. The asymmetric unit is composed of an embedded dimer. The interlaced junctions between the SH2 and SH3 domains bring the two adjacent faces of the SH3 domains in van der Waals contact but leave room for the binding of proline-rich peptides.

Src homology 3 (SH3) domains are found in numerous cytoplasmic proteins involved in intracellular signal transduction. We used 2‐D 1H NMR to determine the structure of the SH3 domain of the guanosine triphosphatase‐activating protein (GAP), an essential component of the Ras signaling pathway. The structure of the GAP SH3 domain (275‐350) was found to be a compact beta‐barrel made of six antiparallel beta‐strands arranged in two roughly perpendicular beta‐sheets with the acidic residues located at the surface of the protein. The Trp317, Trp319, Thr321 and Leu323 residues belonging to the sequence (317‐326), which was shown to be essential for Ras signaling, formed two nearby lipophilic bulges followed by a hydrophilic domain (Arg324‐Asp326). These structural data could be used to characterize the still unidentified downstream components of GAP, which are involved in Ras signaling, and to rationally design inhibitors of this pathway.

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We report here the purification and the crystallization of the modular protein Grb2. The protein was expressed as a fusion with glutathione‐S‐transferase and purified by affinity chromatography on glutathione agarose. It was apparent from reverse phase chromatography that the purified protein was conformationally unstable. Instability was overcome by the addition of 100 mM arginine to the buffers. Because Grb2 appeared to be extremely sensitive to oxidation, crystallization experiments were performed with a dialysis button technique involving daily addition of fresh DTT to the reservoirs. The presence of 8 to 14% glycerol was necessary to obtain mono‐crystals. These results are discussed in relation with the modular nature of Grb2. © 1996 Wiley‐Liss, Inc.

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