articles 468 nature structural biology • volume 9 number 6 • june 2002 Similar to all GTPases, Rho proteins function as binary switches cycling between inactive GDP-bound and active GTP-bound states. Dbl-family proteins are guanine nucleotide exchange fac- tors (GEFs) that initiate Rho-mediated signaling pathways by catalyzing the release of bound GDP and the loading of GTP within cognate GTPase substrates 1 . Biochemical studies have confirmed that the functional portion of Dbl proteins consists of a Dbl homology (DH) domain and pleckstrin homology (PH) domain linked in tandem 2 . Given that Rho GTPases regulate essential cellular processes related to actin cytoskeletal organiza- tion and cell cycle progression, it is not surprising that many Dbl proteins possess transforming or invasion-inducing potential 3–5 . Recent structural studies have revealed a conserved mechanism of nucleotide exchange in Rho GTPases catalyzed by Dbl pro- teins 6 . Crystal structures of the DH/PH fragments of the Dbl members Tiam1 and Dbs bound to Rac1 and Cdc42, respectively (Tiam1–Rac1 and Dbs–Cdc42) 7,8 , display conserved DH domain helices similarly engaging and re-shaping the switch regions of their cognate GTPase to conformations that disrupt both magne- sium and nucleotide binding. Although the DH domain catalyzes nucleotide exchange in the substrate GTPase, the conserved PH domain of Dbl proteins has been implicated in membrane local- ization and allosteric modulation of exchange activity via phosphoinositide binding 9,10 . Additionally, in the Dbs–Cdc42 structure, the PH domain directly contacts Cdc42, and these interactions have been shown to be critical for effective catalysis 8 . Although the Tiam1–Rac1 and Dbs–Cdc42 structures have explained the mechanism of nucleotide exchange in Rho GTPases, the structural basis of specificity by these GEFs for sub- strate GTPases remains unknown. Dbl proteins possess a wide assortment of specificities toward Rho-family members. For instance, Tiam1 acts specifically on Rac1, whereas intersectin (ITSN) is specific for Cdc42 (refs 11,12). In contrast, Dbs and Dbl catalyze exchange within both Cdc42 and RhoA 13,14 , whereas GEFs Vav and Vav2 activate Rac1, Cdc42 and RhoA 15,16 . Rho GTPases are similar in sequence and tertiary structure, yet they trigger divergent downstream consequences exemplified by diverse changes in actin organization 17 . For instance, Rac1 specifically induces formation of lamellipodia and RhoA activa- tion leads to actin stress fiber assembly, whereas Cdc42 stimu- lates actin microspike and filopodia induction 18 . Given the distinct functions of Rho GTPases, it is critical that Dbl proteins select appropriate GTPase substrates. To investigate the mechanism of the selective activation of Rho GTPases by Dbl exchange factors, we have determined the three- dimensional structures of the DH and PH domains of ITSN and Dbs in complex with Cdc42 and RhoA, respectively. Based on these structures, we have re-designed ITSN to catalyze exchange in both Rac1 and RhoA, and have determined the functional determinants that permit Dbs to activate RhoA and Cdc42, yet not engage Rac1. Together, these findings allow a detailed analy- sis of how Dbl-family exchange factors discriminate between Cdc42, Rac1 and RhoA. GEF–GTPase crystal structures The DH/PH fragment of ITSN was crystallized in complex with Cdc42, and the 2.3 Å MAD structure was determined from crys- tals of selenomethionyl (SeMet)-substituted ITSN (Fig. 1a). In addition, the crystal structure of the DH/PH portion of Dbs bound to RhoA was determined (Fig. 1b). The overall architec- tures of ITSN–Cdc42 (Fig. 1c) and Dbs–RhoA (Fig. 1d) are simi- lar to the described Tiam1–Rac1 (ref. 7) and Dbs–Cdc42 (ref. 8) complexes. The DH domains are elongated helical bundles, with defined helices named in accordance with earlier nomenclature 8 . Conserved positions within the DH domains of ITSN, Dbs and Tiam1 bind and reorient the switch regions of each GTPase sub- strate to promote ejection of nucleotide in essentially identical fashion. The PH domains form antiparallel β-sandwiches that consist of seven β-strands capped by a C-terminal helix, which is similar to other PH domains 19 . However, the terminal helix (α6) Structural basis for the selective activation of Rho GTPases by Dbl exchange factors Jason T. Snyder 1 , David K. Worthylake 2,3 , Kent L. Rossman 1,3 , Laurie Betts 2 , Wendy M. Pruitt 2 , David P. Siderovski 2 , Channing J. Der 2 and John Sondek 1,2 Published online: 13 May 2002, DOI: 10.1038/nsb796 Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo. 1 Department of Biochemistry and Biophysics, Program in Molecular and Cellular Biophysics and 2 Department of Pharmacology, University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA. 3 These authors contributed equally to this work. Correspondence should be addressed to J.S. email: sondek@med.unc.edu © 2002 Nature Publishing Group http://structbio.nature.com