Binding of Rac1, Rnd1, and RhoD to a Novel Rho GTPase Interaction Motif Destabilizes Dimerization of the Plexin-B1 Effector Domain

Abstract

Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, ␤-strands 3 and 4 and a short ␣-helical segment of the plexin domain. The 2.0 Å resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel ␤-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.Members of the plexin family of transmembrane receptors have important functions in guiding axon growth in the developing nervous system (1-3). Plexins also function in several developmental processes such as cardiovascular development and angiogenesis (4 -8), in the invasive growth of epithelial cells (9), and in the immune system (10). The extracellular part of plexin shares significant homology with that of the hepatocyte growth factor receptor as well as with semaphorins, the principal family of ligands for plexins. The cytoplasmic region of plexins is also well conserved (11). Two segments with homology to Ras GTPase-activating proteins (GAPs) 5 are linked by a region that has been identified as the location for the binding of several Rho family GTPases.Plexins are unique in that they are the first documented example of a transmembrane receptor that interacts directly with small GTPases. Most A-and B-family plexins bind activated Rac1 and Rnd1 (12-15), Rho family GTPases that are known regulators of cytoskeletal dynamics and cell adhesion (16). Plexin-A1 has also been shown to bind active RhoD (17), another Rho family GTPase involved in actin remodeling and endosomal dynamics and possibly in receptor down-regulation (18). The role of these plexin-Rho GTPase interactions has remained unclear, however, as have the characteristics of the binding region. Does the Rho GTPase binding segment provide a GTPase regulatory property, functioning akin to a guanine nucleotide dissociation inhibitor by sequestering certain Rho family GTPases, or does it act as an effector pro...

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

“…A dimer of the plexin B1 RBD core has been reported previously (18). The interface is mediated by the extra ␤-hairpin outside of the ubiquitin-like fold, which form a short four-stranded ␤-sheet between the two monomers (Fig.…”

“…1 A and B) (18). The ubiquitin-like RBD core (residues 1,478-1,578) is connected to C1 and C2 by two linkers, the N-terminal linker (residues 1,451-1,477) and Cterminal linker (residues 1,579-1,650), respectively ( Fig.…”

“…A recent study shows that plexin D1 RBD binds another Rho GTPase Rnd2, whereas the Rho GTPase specific for the class C plexin has yet to be identified (17). The structures of the core region of the plexin B1 RBD and its complex with Rnd1 have been solved [PDB ID: 2REX, (18,19)]. The RBD core adopts an ubiquitin-like fold and binds Rnd1 through one edge of its ␤-sheet.…”

“…The RBD core adopts an ubiquitin-like fold and binds Rnd1 through one edge of its ␤-sheet. Mutational analyses suggest that Rac1 and RhoD bind to the RBD in the same mode (18).…”

Binding the Atypical RA Domain of Ste50p to the Unfolded Opy2p Cytoplasmic Tail Is Essential for the High-Osmolarity Glycerol Pathway

“…Increasing evidence shows that RA domains have a much wider spectrum of binding partners than just Ras-like small GTPases. Examples include the RA domain from the tumor suppressor Nore 1, which binds intramolecularly its C1 domain (Harjes et al, 2006); the Ras-binding domain (RBD) domain of plexin B1 receptor, which associates with Rho GTPases in an atypical way (Tong et al, 2007); and the RBD of Raf, which binds the SH2 domain of the Grb10 adaptor protein (Nantel et al, 1998). The Ste50p-RA domain has been previously shown to interact with the small GTPase Cdc42p, a member of Rho type of the Ras superfamily.…”