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ABSTRACT: The EphA2 receptor tyrosine kinase is over-expressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand carbohydrates and two residues of EphA2: Asp78 and Lys136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.
Journal of Biological Chemistry 05/2013; · 4.77 Impact Factor
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ABSTRACT: The Eph receptors and their ephrin ligands play crucial roles in a large number of cell-cell interaction events, including those associated with axon pathfinding, neuronal cell migration and vasculogenesis. They are also involved in the patterning of most tissues and overall cell positioning in the development of the vertebrate body plan. The Eph/ephrin signaling system manifests several unique features that differentiate it from other receptor tyrosine kinases, including initiation of bi-directional signaling cascades and the existence of ligand and receptor subclasses displaying promiscuous intra-subclass interactions, but very rare inter-subclass interactions. In this review we briefly discuss these features and focus on recent studies of the unique and expansive high-affinity Eph/ephrin assemblies that form at the sites of cell-cell contact and are required for Eph signaling initiation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.
Biochimica et Biophysica Acta 04/2013; · 4.66 Impact Factor
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ABSTRACT: Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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ABSTRACT: The ADAM10 transmembrane metalloprotease cleaves a variety of cell surface proteins with importance in disease, including ligands for receptor tyrosine kinases of the erbB and Eph families. ADAM10-mediated cleavage of ephrins, the ligands for Eph receptors, is suggested to control Eph/ephrin-mediated cell-cell adhesion and segregation, important during normal developmental processes, and implicated in tumour neo-angiogenesis and metastasis. We previously identified a substrate binding pocket in the ADAM10 C domain that binds the EphA/ephrin-A complex thereby regulating ephrin cleavage. We have now generated monoclonal antibodies specifically recognising this region of ADAM10, which inhibit ephrin cleavage and Eph/ephrin-mediated cell function, including ephrin-induced Eph receptor internalisation, phosphorylation and Eph-mediated cell segregation. Our studies confirm the important role of ADAM10 in cell-cell interactions mediated by both A- and B-type Eph receptors, and suggest antibodies against the ADAM10 substrate-recognition pocket as promising therapeutics, acting by inhibiting cleavage of ephrins and potentially other ADAM10 substrates.
Journal of Cell Science 10/2012; · 6.11 Impact Factor
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Yee-Peng Chan,
Min Lu,
Somnath Dutta,
Lianying Yan,
Jennifer Barr,
Michael Flora,
Yan-Ru Feng,
Kai Xu, Dimitar B Nikolov,
Lin-Fa Wang,
Georgios Skiniotis,
Christopher C Broder
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ABSTRACT: The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF(GCNt)), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F(0) precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF(GCNt)) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF(GCNt) constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF(GCNt) and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses.
Journal of Virology 08/2012; 86(21):11457-71. · 5.40 Impact Factor
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ABSTRACT: Hendra virus and Nipah virus are recently discovered and closely related emerging viruses that now comprise the genus henipavirus within the sub-family Paramyxoviridae and are distinguished by their broad species tropism and in addition to bats can infect and cause fatal disease in a wide variety of mammalian hosts including humans. The high mortality associated with human and animal henipavirus infections has highlighted the importance and necessity of developing effective immunization strategies. The development of suitable animal models of henipavirus infection and pathogenesis has been critical for testing the efficacy of potential therapeutic approaches. Several henipavirus challenge models have been used and recent successes in both active and passive immunization strategies against henipaviruses have been reported which have all targeted the viral envelope glycoproteins.
Current topics in microbiology and immunology 04/2012; 359:197-223. · 4.93 Impact Factor
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ABSTRACT: Members of the ephrin cell-surface protein family interact with the Eph receptors, the largest family of receptor tyrosine kinases, mediating bi-directional signaling during tumorogenesis and various developmental events. Surprisingly, ephrin-B2 and -B3 were recently identified as entry receptors for henipaviruses, emerging zoonotic paramyxoviruses responsible for repeated outbreaks in humans and animals in Australia, Southeast Asia, India and Bangladesh. Nipah virus (NiV) and Hendra virus (HeV) are the only two identified members in the henipavirus genus. While the initial human infection cases came from contact with infected pigs (NiV) or horses (HeV), in the more recent outbreaks of NiV both food-borne and human-to-human transmission were reported. These characteristics, together with high mortality and morbidity rates and lack of effective anti-viral therapies, make the henipaviruses a potential biological-agent threat. Viral entry is an important target for the development of anti-viral drugs. The entry of henipavirus is initiated by the attachment of the viral G envelope glycoprotein to the host cell receptors ephrin-B2 and/or -B3, followed by activation of the F fusion protein, which triggers fusion between the viral envelop and the host membrane. We review recent progress in the study of henipavirus entry, particularly the identification of ephrins as their entry receptors, and the structural characterization of the ephrin/Henipa-G interactions.
Seminars in Cell and Developmental Biology 02/2012; 23(1):116-23. · 6.65 Impact Factor
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ABSTRACT: The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G) and class I fusion (F) envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins.
Viruses 02/2012; 4(2):280-308. · 1.50 Impact Factor
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ABSTRACT: Hendra virus and Nipah virus, comprising the genus Henipavirus, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. Two surface glycoproteins, the attachment (G) and fusion (F), mediate host cell entry. The crystal structures of the Hendra G glycoprotein alone and in complex with the ephrin-B2 receptor reveal that henipavirus uses Tryptophan 122 on ephrin-B2/B3 as a "latch" to facilitate the G-receptor association. Structural-based mutagenesis of residues in the Hendra G glycoprotein at the receptor binding interface document their importance for viral attachments and entry, and suggest that the stability of the Hendra-G-ephrin attachment complex does not strongly correlate with the efficiency of viral entry. In addition, our data indicates that conformational rearrangements of the G glycoprotein head domain upon receptor binding may be the trigger leading to the activation of the viral F fusion glycoprotein during virus infection.
PLoS ONE 01/2012; 7(11):e48742. · 4.09 Impact Factor
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ABSTRACT: Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.
Glycobiology 12/2011; 22(4):572-84. · 3.58 Impact Factor
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ABSTRACT: We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization.
Chemistry & biology 03/2011; 18(3):361-71. · 6.52 Impact Factor
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ABSTRACT: Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A- and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligand-binding domain at 2.3A resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.
Biochemical and Biophysical Research Communications 09/2010; 399(4):555-9. · 2.48 Impact Factor
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Juha P Himanen,
Laila Yermekbayeva,
Peter W Janes,
John R Walker,
Kai Xu,
Lakmali Atapattu,
Kanagalaghatta R Rajashankar,
Anneloes Mensinga,
Martin Lackmann, Dimitar B Nikolov,
Sirano Dhe-Paganon
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ABSTRACT: Eph receptor tyrosine kinases and their ephrin ligands regulate cell navigation during normal and oncogenic development. Signaling of Ephs is initiated in a multistep process leading to the assembly of higher-order signaling clusters that set off bidirectional signaling in interacting cells. However, the structural and mechanistic details of this assembly remained undefined. Here we present high-resolution structures of the complete EphA2 ectodomain and complexes with ephrin-A1 and A5 as the base unit of an Eph cluster. The structures reveal an elongated architecture with novel Eph/Eph interactions, both within and outside of the Eph ligand-binding domain, that suggest the molecular mechanism underlying Eph/ephrin clustering. Structure-function analysis, by using site-directed mutagenesis and cell-based signaling assays, confirms the importance of the identified oligomerization interfaces for Eph clustering.
Proceedings of the National Academy of Sciences 06/2010; 107(24):10860-5. · 9.68 Impact Factor
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ABSTRACT: The Tie family of endothelial-specific receptor tyrosine kinases is essential for cell proliferation, migration, and survival during angiogenesis. Despite considerable similarity, experiments with Tie1- or Tie2-deficient mice highlight distinct functions for these receptors in vivo. The Tie2 receptor is further unique with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists; angiopoietin-1 and -4 are constitutive agonists. To address the role of Tie1 in angiopoietin-mediated Tie2 signaling and determine the basis for the behavior of the individual angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and -2 localization and association. We provide evidence for Tie1-Tie2 complex formation on the cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by angiopoietin-1 and -2. Based on the available data, we propose a unified model for angiopoietin-induced Tie2 signaling.
Molecular cell 03/2010; 37(5):643-55. · 14.61 Impact Factor
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ABSTRACT: Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B-class receptors. Here, we present the crystal structures of an A-class complex between EphA2 and ephrin-A1 and of unbound EphA2. Although these structures are similar overall to their B-class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A-class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a 'lock-and-key'-type binding mechanism, in contrast to the 'induced fit' mechanism defining the B-class molecules. This model is supported by structure-based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell-based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2-specific homotypic cell adhesion interactions.
EMBO Reports 07/2009; 10(7):722-8. · 7.36 Impact Factor
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ABSTRACT: 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.
Protein Science 10/2008; 17(10):1740-7. · 2.80 Impact Factor
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ABSTRACT: Nipah virus (NiV) and Hendra virus are the type species of the highly pathogenic paramyxovirus genus Henipavirus, which can cause severe respiratory disease and fatal encephalitis infections in humans, with case fatality rates approaching 75%. NiV contains two envelope glycoproteins, the receptor-binding G glycoprotein (NiV-G) that facilitates attachment to host cells and the fusion (F) glycoprotein that mediates membrane merger. The henipavirus G glycoproteins lack both hemagglutinating and neuraminidase activities and, instead, engage the highly conserved ephrin-B2 and ephrin-B3 cell surface proteins as their entry receptors. Here, we report the crystal structures of the NiV-G both in its receptor-unbound state and in complex with ephrin-B3, providing, to our knowledge, the first view of a paramyxovirus attachment complex in which a cellular protein is used as the virus receptor. Complex formation generates an extensive protein-protein interface around a protruding ephrin loop, which is inserted in the central cavity of the NiV-G beta-propeller. Analysis of the structural data reveals the molecular basis for the highly specific interactions of the henipavirus G glycoproteins with only two members (ephrin-B2 and ephrin-B3) of the very large ephrin family and suggests how they mediate in a unique fashion both cell attachment and the initiation of membrane fusion during the virus infection processes. The structures further suggest that the NiV-G/ephrin interactions can be effectively targeted to disrupt viral entry and provide the foundation for structure-based antiviral drug design.
Proceedings of the National Academy of Sciences 07/2008; 105(29):9953-8. · 9.68 Impact Factor
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ABSTRACT: 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.
Current Opinion in Cell Biology 11/2007; 19(5):534-42. · 12.90 Impact Factor
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ABSTRACT: 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.
Protein Science 04/2007; 16(3):355-61. · 2.80 Impact Factor
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ABSTRACT: The advent of the multiwavelength anomalous diffraction phasing method has significantly accelerated crystal structure determination and has become the norm in protein crystallography. This method allows researchers to take advantage of the anomalous signal from diverse atoms, but the dominant method for derivative preparation is selenomethionine substitution. Several generally applicable, high-efficiency labeling protocols have been developed for use in the bacterial, yeast, and baculovirus/insect cell expression systems but not for mammalian tissue culture. As a large number of proteins of biomedical importance can only be produced in yields sufficient for X-ray diffraction experiments in mammalian expression systems, it becomes all the more important to develop such protocols. We therefore evaluated several variables that play roles in determining incorporation levels and report here a simple protocol for selenomethionine modification of proteins in mammalian cells routinely yielding >90% labeling efficiency.
Protein Science 09/2006; 15(8):2008-13. · 2.80 Impact Factor
