Isolation and identification of cyclic lipopeptides from Paenibacillus ehimensis, strain IB-X-b.

Antifungal lipopeptides produced by an antagonistic bacterium, Paenibacillus ehimensis strain IB-X-b, were purified and analyzed. The acetone extract of the culture supernatant contained an antifungal amphiphilic fraction stainable with ninhydrin on thin layer chromatography. The fraction was further purified with water-methanol extraction followed by a chromatography on a C18-support. The analysis with LC-MS showed presence of two main series of homologous compounds, family of depsipeptides containing a hydroxy fatty acid, three 2,4-diaminobutyric acid (Dab) residues, five hydrophobic amino acids and one Ser/Thr residue, and cyclic lipopeptides of bacillomycin L and fengycin/plipastatin/agrastatin families. The prevailing compounds in this group are bacillomycin L-C15, fengycin/plipastatin A-C16 together with their homologues responsible for the majority of fungal growth inhibition by P. ehimensis IB-X-b.

Toward the semisynthesis of multidomain transmembrane receptors: modification of Eph tyrosine kinases.

Expressed protein ligation (EPL) is a protein engineering approach that allows the modification or assembly of a target protein from multiple recombinant and synthetic polypeptides. EPL has been previously used to modify intracellular proteins and small integral membrane proteins for structural and functional studies. Here we describe the semisynthetic site-specific modification of the complete, multidomain extracellular regions of both A and B classes of Eph receptor tyrosine kinases. We show that the ectodomains of these receptors can be ligated to different peptides under carefully established experimental conditions, while their biological activity is retained. This work extends the boundaries of the EPL technique for semisynthesis of multidomain, extracellular, disulfide-bonded, and glycosylated proteins and highlights its potential application for reconstituting entire single-pass transmembrane proteins.

Cell-cell signaling via Eph receptors and ephrins.

Eph receptors are the largest subfamily of receptor tyrosine kinases regulating cell shape, movements, and attachment. The interactions of the Ephs with their ephrin ligands are restricted to the sites of cell-cell contact since both molecules are membrane attached. This review summarizes recent advances in our understanding of the molecular mechanisms underlining the diverse functions of the molecules during development and in the adult organism. The unique properties of this signaling system that are of highest interest and have been the focus of intense investigations are as follows: (i) the signal is simultaneously transduced in both ligand-expressing cells and receptor-expressing cells, (ii) signaling via the same molecules can generate opposing cellular reactions depending on the context, and (iii) the Ephs and the ephrins are divided into two subclasses with promiscuous intrasubclass interactions, but rarely observed intersubclass interactions.

The structure of apo tryptophanase from Escherichia coli reveals a wide-open conformation.

The crystal structure of apo tryptophanase from Escherichia coli (space group F222, unit-cell parameters a = 118.4, b = 120.1, c = 171.2 A) was determined at 1.9 A resolution using the molecular-replacement method and refined to an R factor of 20.3% (R(free) = 23.2%). The structure revealed a significant shift in the relative orientation of the domains compared with both the holo form of Proteus vulgaris tryptophanase and with another crystal structure of apo E. coli tryptophanase, reflecting the internal flexibility of the molecule. Domain shifts were previously observed in tryptophanase and in the closely related enzyme tyrosine phenol-lyase, with the holo form found in an open conformation and the apo form in either an open or a closed conformation. Here, a wide-open conformation of the apo form of tryptophanase is reported. A conformational change is also observed in loop 297-303. The structure contains a hydrated Mg(2+) at the cation-binding site and a Cl(-) ion at the subunit interface. The enzyme activity depends on the nature of the bound cation, with smaller ions serving as inhibitors. It is hypothesized that this effect arises from variations of the coordination geometry of the bound cation.

Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: correlation to function in a growth cone collapse assay.

Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide-linked Fc fragments, is best described using a bivalent analyte model as a two-step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30- to 6000-fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A-class ephrin/Eph interactions than in the B-class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions.

Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans.

The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.

Crystal structure of the ephrin-B1 ectodomain: implications for receptor recognition and signaling.

Eph receptors and their ephrin ligands are involved in various aspects of cell-cell communication during development, including axonal pathfinding in the nervous system and cell-cell interactions of the vascular endothelial cells. Recent structural studies revealed unique molecular features, not previously seen in any other receptor-ligand families, and explained many of the biochemical and signaling properties of Ephs and ephrins. However, unresolved questions remain regarding the potential oligomerization and clustering of these important signaling molecules. In this study, the biophysical properties and receptor-binding preferences of the extracellular domain of ephrin-B1 were investigated and its crystal structure was determined at 2.65 A resolution. Ephrin-B1 is a monomer both in solution and in the crystals, while it was previously shown that the closely related ephrin-B2 forms homodimers. The main structural difference between ephrin-B1 and ephrin-B2 is the conformation of the receptor-binding G-H loop and the partially disordered N-terminal tetramerization region of ephrin-B1. The G-H loop is structurally rigid in ephrin-B2 and adopts the same conformation in both the receptor-bound and unbound ligand, where it mediates receptor-independent homodimerization. In the ephrin-B1 structure, on the other hand, the G-H loop is not involved in any homotypic interactions and adopts a new, distinct conformation. The implications of the ephrin-B1 structure, in context of available ephrin-B1 mutagenesis data, for the mechanism of Eph-ephrin recognition and signaling initiation are discussed.

Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3.

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2.ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E alpha-helix, the E-F loop, the underlying H beta-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes.

Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling.

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.

Structure of the semaphorin-3A receptor binding module.

The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. Their distinctive feature is a conserved 500 amino acid semaphorin domain, a ligand-receptor interaction module also present in plexins and scatter-factor receptors. We report the crystal structure of a secreted 65 kDa form of Semaphorin-3A (Sema3A), containing the full semaphorin domain. Unexpectedly, the semaphorin fold is a variation of the beta propeller topology. Analysis of the Sema3A structure and structure-based mutagenesis data identify the neuropilin binding site and suggest a potential plexin interaction site. Based on the structure, we present a model for the initiation of semaphorin signaling and discuss potential similarities with the signaling mechanisms of other beta propeller cell surface receptors, such as integrins and the LDL receptor.

Eph receptors and ephrins.

Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape, and mobility. Eph signaling is crucial for the development of many tissues and organs including the nervous and cardiovascular systems. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell-cell contact initiate unique bi-directional signaling cascades where information is transduced in both the receptor- and the ligand-expressing cells. Recent studies summarized in this review reveal how the signaling process is triggered upon ligand-receptor binding via the formation of a 2:2 circular heterotetramer. This fixes the orientation of the participating molecules and facilitates phosphorylation of their cytoplasmic domains which then interact with downstream signaling factors. The elucidation of the structural details of Eph-ephrin recognition and binding should yield insight into the future development of novel therapeutic agents targeting cardiovascular function, nerve regeneration, and cancer.

Eph signaling: a structural view.

Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape and mobility. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell-cell contact initiate unique bidirectional signaling cascades, with information transduced in both the receptor-expressing and the ligand-expressing cells. Recent structural and biophysical studies summarized in this review reveal unique molecular features not previously observed in any other receptor-ligand families and explain many of the biochemical and signaling properties of Ephs and ephrins. Of particular importance is the insight into how approximation of ligand-expressing and receptor-expressing cells could lead to the formation and activation of highly ordered signaling centers at cell-cell interfaces.