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Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD∙Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases. [ABSTRACT FROM AUTHOR]

The synthesis, electronic absorption, fluorescence (λf, λex, Φf, τf) and photoisomerization (Φt→c, photostationary state composition) properties of 3-(4-nitrophenylethenyl-E)-NH-indole (1), 3-(4-nitrophenylethenyl-E)-N-ethylindole (2) and 3-(4-nitrophenyl ethenyl-E)-N-benzenesulfonylindole (3) in organic solvents of varying polarity are reported. The absorption maximum of these compounds undergoes a moderate red shift with increasing solvent polarity. However, the fluorescence maximum becomes highly red shifted with increasing solvent polarity. Whereas 1 and 2 show broad fluorescence bands, 3 exhibits dual fluorescence. Further, 1 and 2 fluoresce much more efficiently than 3. Correlation of the Stokes shift with solvent polarity parameters such as Δf and ET(30) and excited-state dipole moment indicate a highly polar excited state for 1–3. Time-resolved fluorescence studies show that the fluorescence decays are single- and multi-exponential type, depending on the solvent polarity. Further, 1 and 2 do not show photoisomerization on irradiation. However, 3 is photoactive and shows efficient photoisomerization in non-polar heptane. The sensitivity (ρ) of the photoreaction is determined in various solvent in terms of the Hammett plot, which showed that the excited states involved are electron deficient in nature and consequently stabilized more by an electron sufficient polar solvent and electron donating substituent. These results led us to suggest the existence of three types of excited states, namely the locally excited state, the intramolecular charge-transfer excited state and the conformationally relaxed intramolecular charge-transfer excited state in the photoprocesses of these compounds. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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 spectros- copy 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-Bi is not involved in this interaction. Instead, all three GTPases interact with the same region, β-strands 3 and 4 and a short α-helical segment of the plexin domain. The 2.0 A 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 β-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 [ABSTRACT FROM AUTHOR]

Plexins are unique, as they are the first example of a transmembrane receptor that interacts directly with small GTPases, a family of proteins that are essential for cell motility and proliferation/survival. We and other laboratories have determined the structure of the Rho GTPase-binding domain (RBD) of several plexins and also of the entire intracellular region of plexin-B1. Structures of plexin complexes with Rho GTPases, Rac1 and Rnd1, and a structure with a Ras GTPase, Rap1b, have also been solved. The relationship between plexin-Rho and plexin-Ras interactions is still unclear and in vitro biophysical experiments that characterize the protein interactions of purified components play an important role in advancing our understanding of the molecular mechanisms that underlie the function of plexin. This chapter describes the use of gel filtration (also known as size-exclusion chromatography or SEC), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) in studies of plexin-small GTPase interactions with plexin-B1:Rac1 as an example. Together with other assays and manipulations (e.g., by mutagenesis or protein domain truncation/deletion), these in vitro measurements provide an important reference for the role and extent of the interactions.