GTP-binding (G) proteins regulate the flow of information in cellular signaling pathways by alternating between a GTPbound "active" state and a GDP-bound "inactive" state. Cdc42, a member of the Rho family of Ras-related small G-proteins, plays key roles in the regulation of cell shape, motility, and growth. Here we describe the high resolution x-ray crystal structure for Cdc42 bound to the GTP analog guanylyl ␤,␥-methylenediphosphonate (GMP-PCP) (i.e. the presumed signaling-active state) and show that it is virtually identical to the structures for the signaling-inactive, GDP-bound form of the protein, contrary to what has been reported for Ras and other G-proteins. Especially surprising was that the GMP-PCP-and GDP-bound forms of Cdc42 did not show detectable differences in their Switch I and Switch II loops. Fluorescence studies using a Cdc42 mutant in which a tryptophan residue was introduced at position 32 of Switch I also showed that there was little difference in the Switch I conformation between the GDP-and GMP-PCPbound states (i.e. <10%), which again differed from Ras where much larger changes in Trp-32 fluorescence were observed when comparing these two nucleotide-bound states (>30%). However, the binding of an effector protein induced significant changes in the Trp-32 emission specifically from GMP-PCPbound Cdc42, as well as in the phosphate resonances for GTP bound to this G-protein as indicated in NMR studies. An examination of the available structures for Cdc42 complexed to different effector proteins, versus the x-ray crystal structure for GMP-PCP-bound Cdc42, provides a possible explanation for how effectors can distinguish between the GTP-and GDPbound forms of this G-protein and ensure that the necessary conformational changes for signal propagation occur.Cdc42 is a member of the Rho family of Ras-related small G-proteins and is an essential protein found in all eukaryotic organisms, including yeast, flies, and mammals (1-3). Like Ras, the founding member of the small G-protein family (4), Cdc42 undergoes a GTP-binding/GTP-hydrolytic cycle that enables it to act as a molecular switch in cells. It is activated to undergo GDP-GTP exchange by members of the Dbl family of guanine nucleotide exchange factors (5-7). GTP-bound Cdc42 can bind and/or activate over 20 downstream effector proteins that are responsible for mediating a diversity of cellular functions, including actin cytoskeletal remodeling, cell polarity, intracellular trafficking, epidermal growth factor receptor degradation, and cell cycle progression (1)(2)(3)8). These different signals are terminated when Cdc42 is deactivated through its ability to hydrolyze GTP, a reaction that is catalyzed by GTPase-activating proteins (GAPs) 2 (9). Structural studies of a number of GTP-binding proteins, beginning with the bacterial elongation factor Ef-Tu, and including H-Ras and the ␣ subunits of various members of the family of large G-proteins, have shown that a conserved architecture exists for GTP binding and hydrolytic activity, comprising five...