Plexin-Bs enhance their GAP activity with a novel activation switch loop generating a cooperative enzyme.

Plexins receive guidance cues from semaphorin ligands and transmit their signal through the plasma membrane. This family of proteins is unique amongst single-pass transmembrane receptors as their intracellular regions interact directly with several small GTPases, which regulate cytoskeletal dynamics and cell adhesion. Here, we characterize the GTPase Activating Protein (GAP) function of Plexin-B1 and find that a cooperative GAP activity towards the substrate GTPase, Rap1b, is associated with the N-terminal Juxtamembrane region of Plexin-B1. Importantly, we unveil an activation mechanism of Plexin-B1 by identifying a novel functional loop which partially blocks Rap1b entry into the plexin GAP domain. Consistent with the concept of allokairy developed for other systems, Plexin-B activity is increased by an apparent substrate-mediated cooperative effect. Simulations and mutagenesis suggest the repositioned JM conformation is stabilized by the new activation switch loop when the active site is occupied, giving rise to faster enzymatic turnover and cooperative behavior. The biological implications, essentially those of a threshold behavior for cell migration, are discussed.

K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function.

Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the ß2 and ß3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

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.

Eliciting neutralizing antibodies with gp120 outer domain constructs based on M-group consensus sequence.

One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-OD×3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-OD×3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-OD×3 was superior to gp120-OD, suggesting that gp120-OD×3 is a promising prototype for further gp120 OD-based immunogen development.

Binding and function of phosphotyrosines of the Ephrin A2 (EphA2) receptor using synthetic sterile α motif (SAM) domains.

The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr(921), Tyr(930), and Tyr(960), has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr(921) and Tyr(930) enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling.

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.