Novel intermediate of Rac GTPase activation by guanine nucleotide exchange factor

The biochemical role of guanine nucleotide exchange factors (GEFs) in catalyzing small GTPase GDP-GTP exchange is thought to be twofold: stimulation of GDP dissociation and stabilization of a nucleotide-free GTPase intermediate. Here we report that TrioN, a Dbl family GEF, activates Rac1 by facilitating GTP binding to, as well as stimulating GDP dissociation from, Rac1. The TrioN-catalyzed GDP dissociation is dependent upon the structural nature and the concentration of free nucleotide, and nucleotide binding serves as the rate-limiting step of the GEF reaction. The TrioN-stimulated nucleotide exchange may undergo a novel two nucleotide-one G-protein intermediate involving two cryptic subsites on Rac1 induced by the GEF, with one subsite contributing to the recognition of the β/γ phosphates of the incoming GTP and another to the binding of the guanine base of the leaving GDP. We propose that the Rac GEF reaction may proceed by competitive displacement of bound GDP by GTP through a transient intermediate of GEF-[GTP-Rac-GDP].