Synthesis of Novel Diaziridinyl Quinone Isoxazole Hybrids and Evaluation of Their Anti-Cancer Activity as Potential Tubulin-Targeting Agents.

Two series of diaziridinyl quinone isoxazole derivatives were prepared and evaluated for their cytotoxic activity against MCF7, HeLa, BT549, A549 and HEK293 cell lines and interaction with tubulin. Compounds (6A-M: ) showed promising activity against all the 5 human cancer cell lines. Compounds 6A: , 6E: and 6 M: were potent [IC50 ranging between 2.21 µg to 2.87 µg] on ER-positive MCF7 cell line similar to the commercially available drug molecule Doxorubicin. The results from docking models are in consistent with the experimental values which demonstrated the favourable binding modes of compounds 6A-M: to the interface of α- and ß-tubulin dimer.

Characterizing Plexin GTPase Interactions Using Gel Filtration, Surface Plasmon Resonance Spectrometry, and Isothermal Titration Calorimetry.

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.

Plexin structures are coming: opportunities for multilevel investigations of semaphorin guidance receptors, their cell signaling mechanisms, and functions.

Plexin transmembrane receptors and their semaphorin ligands, as well as their co-receptors (Neuropilin, Integrin, VEGFR2, ErbB2, and Met kinase) are emerging as key regulatory proteins in a wide variety of developmental, regenerative, but also pathological processes. The diverse arenas of plexin function are surveyed, including roles in the nervous, cardiovascular, bone and skeletal, and immune systems. Such different settings require considerable specificity among the plexin and semaphorin family members which in turn are accompanied by a variety of cell signaling networks. Underlying the latter are the mechanistic details of the interactions and catalytic events at the molecular level. Very recently, dramatic progress has been made in solving the structures of plexins and of their complexes with associated proteins. This molecular level information is now suggesting detailed mechanisms for the function of both the extracellular as well as the intracellular plexin regions. Specifically, several groups have solved structures for extracellular domains for plexin-A2, -B1, and -C1, many in complex with semaphorin ligands. On the intracellular side, the role of small Rho GTPases has been of particular interest. These directly associate with plexin and stimulate a GTPase activating (GAP) function in the plexin catalytic domain to downregulate Ras GTPases. Structures for the Rho GTPase binding domains have been presented for several plexins, some with Rnd1 bound. The entire intracellular domain structure of plexin-A1, -A3, and -B1 have also been solved alone and in complex with Rac1. However, key aspects of the interplay between GTPases and plexins remain far from clear. The structural information is helping the plexin field to focus on key questions at the protein structural, cellular, as well as organism level that collaboratoria of investigations are likely to answer.

NMR structure of a heterodimeric SAM:SAM complex: characterization and manipulation of EphA2 binding reveal new cellular functions of SHIP2.

The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural basis for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex toward alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM, whereas EphB members may not; correspondingly, we demonstrate binding of EphA1, but not of EphB2, to SHIP2. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting receptor coordinated chemotactic cell migration.

Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs).

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 ß3-ß4 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.

Structure and function of the intracellular region of the plexin-b1 transmembrane receptor.

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 A crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120(GAP).H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.

Thermodynamic characterization of two homologous protein complexes: associations of the semaphorin receptor plexin-B1 RhoGTPase binding domain with Rnd1 and active Rac1.

Plexin receptors function in response to semaphorin guidance cues in a variety of developmental processes involving cell motility. Interactions with Rho, as well as Ras family small GTPases are critical events in the cell signaling mechanism. We have recently determined the structure of a cytoplasmic domain (RBD) of plexin-B1 and mapped its binding interface with several Rho-GTPases, Rac1, Rnd1, and RhoD. All three GTPases associate with a similar region of this plexin domain, but show different functional behavior in cells. To understand whether thermodynamic properties of the GTPase-RBD interaction contribute to such different behavior, we have examined the interaction at different temperatures, buffer, and pH conditions. Although the binding affinity of both Rnd1 and Rac1 with the plexin-B1 RBD is similar, the detailed thermodynamic properties of the interactions are considerably different. These data suggest that on Rac1 binding to the plexin-B1 RBD, the proteins become more rigid in the complex. By contrast, Rnd1 binding is consistent with unchanged or slightly increased flexibility in one or both proteins. Both GTPases show an appreciable reduction in affinity for the dimeric plexin-B1 RBD indicating that GTPase binding is not cooperative with dimer formation, but that a partial steric hindrance destabilizes the dimer. However, a reduced affinity binding mode to a disulphide stabilized model for the dimeric RBD is also possible. Consistent with cellular studies, the interaction thermodynamics imply that further levels of regulation involving additional binding partners and/or regions outside of the RhoGTPase binding domain are required for receptor activation.

Insights into oncogenic mutations of plexin-B1 based on the solution structure of the Rho GTPase binding domain.

The plexin family of transmembrane receptors are important for axon guidance, angiogenesis, but also in cancer. Recently, plexin-B1 somatic missense mutations were found in both primary tumors and metastases of breast and prostate cancers, with several mutations mapping to the Rho GTPase binding domain (RBD) in the cytoplasmic region of the receptor. Here we present the NMR solution structure of this domain, confirming that the protein has both a ubiquitin-like fold and surface features. Oncogenic mutations T1795A and T1802A are located in a loop region, perturb the average structure locally, and have no effect on Rho GTPase binding affinity. Mutations L1815F and L1815P are located at the Rho GTPase binding site and are associated with a complete loss of binding for Rac1 and Rnd1. Both are found to disturb the conformation of the beta3-beta4 sheet and the orientation of surrounding side chains. Our study suggests that the oncogenic behavior of the mutants can be rationalized with reference to the structure of the RBD of plexin-B1.

Binding of Rac1, Rnd1, and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 effector domain.

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, beta-strands 3 and 4 and a short alpha-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 beta-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.

Development of bacteriorhodopsin analogues and studies of charge separated excited states in the photoprocesses of linear polyenes.

Development of bacteriorhodopsin (bR) analogues employing chromophore substitution technique for the purpose of characterizing the binding site of bR and generating bR analogues with novel opto-electronic properties for applications as photoactive element in nanotechnical devices are described. Additionally, the photophysical and photochemical properties of variously substituted diarylpolyenes as models of photobiologically relevant linear polyenes are discussed. The role of charge separated dipolar excited states in the photoprocesses of linear polyenes is highlighted.