Activation of the Ras superfamily of small GTPases. Workshop on exchange factors.

Introduction The Center for International Meetings on Biology workshop on ‘Exchange Factors’ provided an up-to-date view of how guaninenucleotide-exchange factors (GEFs) affect the biochemical pathways that regulate the Ras superfamily of small GTPases, and how these molecules function as part of an interrelated network of regulatory proteins that mediate the biological responses of Ras-like GTPases (Fig. 1). Reflecting the complex nature of GEF functions, the meeting focused on their structure–function relationships, regulation and biological functions. Particular emphasis was placed on a subset of regulators of the Ras and Rho subfamilies that contain catalytic CDC25 homology domains or Dbl homology (DH)/pleckstrin homology (PH) cassettes, respectively (Fig. 2; Quilliam et al., 2002; Zheng, 2001). Mechanism of GEF reactions The Ras superfamily of GTPases can be subdivided into the Ras, Rho, Rab, ARF and Ran subfamilies. Whereas the GEFs that regulate each subfamily are structurally distinct, those that regulate members of any given subfamily show a high degree of sequence conservation. Recent structural studies of GEF–GTPase complexes have also revealed mechanistic features that are shared by all GEFs. A. Wittinghofer (Dortmund, Germany) summarized some of the common principles of exchange-factor-catalysed reactions. GEFs typically stimulate the intrinsic release of GDP from the GTPase by ~1 × 105-fold (Vetter & Wittinghofer, 2001), through interactions both with the switch regions (I and II) and with the phosphate-binding loop of the GTPase. On the basis of studies of Ran regulation by the exchange factor RCC1, Wittinghofer proposed that there are several transition steps during the exchange reaction of Ras proteins, with the guanine ring of GDP being dislodged first, to generate a low-affinity Ras–GDP:GEF complex. Subsequently, the phosphate moiety and an Mg2+ ion are displaced, and a high-affinity Ras–GEF complex is established. Because dislodging the bound Mg2+ ion is not sufficient for GDP dissociation, it seems that the disruption of contacts with the phosphate moiety is the key step required for rapid nucleotide release. According to the principle of micro-reversibility, the entry of GTP into the binding pocket to displace the GEF and create a high-affinity nucleotide complex would have to occur in the reverse order: that is, phosphate first, base second. Y. Zheng (Cincinnati, OH, USA) presented an alternative model for the mechanism of activation of the Rho subfamily GTPase, Rac1, by the Rho-family GEF, Trio. The fact that the Trio-catalysed GEF reaction depends on the nature and concentration of free nucleotides was taken to indicate that the binding of incoming GTP is required for GDP displacement, and that a two-nucleotide–one-G-protein intermediate, GTP–Rac1–GDP, is involved. This interpretation was one of the more extensively debated issues of the workshop.