Additionally it is possible that there surely is a temporal legislation which different EphA2 signaling pathways are activated through the primary infections (canonical, which would stimulate pathogen internalization via endocytosis) and reactivation (noncanonical, which would boost cellular oncogenic potential). In this survey, we present the structure from the HHV-8 gH/gL destined to EphA2 and display the fact that gH/gL induces dimerization of EphA2 portrayed by cells, aswell as morphological changes at cellular level, resembling the action mode of ephrin ligands. C terminus: the LBD shaded in crimson, the CRD in green, and 2 FN-like domains in yellowish and orange, respectively. The EphA2 DIN in the LBD and the CIN in the CRD are indicated. In the absence of ligand, EphA2 exists in an equilibrium between monomers (1) and dimers (2), with the unliganded EphA2 dimers stabilized via the CIN . (3) At low ligand concentration or in the presence of soluble, m-ephrin ligands or agonist peptides , each Eph receptor interacts with 2 ephrin moleculesits cognate ligand with high Rabbit polyclonal to ZMYM5 affinity and via low affinity interactions with ephrin from the other complex, forming the so-called tetrameric assembly made of 2 receptor (EphA2 dimer stabilized via DIN) and 2 ligand molecules. (4) At higher ligand concentrations, emulated by addition of dimeric or preclustered soluble ephrin ligands, the EphA2 molecules from the tetrameric assembly interact with EphA2 from other tetramers via the CIN, giving rise to larger oligomeric structures, i.e., clusters. (5) HHV-8 gH/gL is drawn as gray/blue rectangles. Data presented in this manuscript demonstrate that soluble gH/gL PSI induces formation of EphA2 dimers stabilized via DIN, similar to the effect of m-ephrinA2 or agonist peptides (3). The figure was created in BioRender.com. CIN, clustering surface; CRD, cysteine-rich domain; DIN, dimerization interface; Eph, PSI erythropoietin-producing human hepatocellular carcinoma cell line; ephrin, Eph family receptor interacting protein; FN, fibronectin; gH/gL, glycoproteins H and L; HHV-8, human herpesvirus 8; LBD, ligand-binding domain.(PDF) pbio.3001392.s002.pdf (211K) GUID:?078EC7BC-AA0E-46D0-B2AE-F27507EE781B S3 Fig: Secondary structure topology diagram of EphA2 LBD, gL, and ephrin-A1. Secondary structure elements are represented by arrows (-strands), rectangles (-helices), and rounded rectangles PSI ( helices (B, I, J)). The dashed lines indicate regions not resolved in the structures. The vertical dotted lines designate the two 5-stranded -sheets adopting a jelly roll fold in EphA2 LBD and a 3- and 5-stranded sheets forming a -sandwich in ephrin-A1. The conserved residues R103EphA2 and E119ephrin-A1, which are important for high-affinity interaction, are represented as red and blue circles, respectively. Cysteine residues establishing disulfide bonds (yellow lines) are represented with yellow circles. The secondary structure diagrams for EphA2 LBD and HHV-8 gL are drawn based on the structure presented in this paper (PDB 7B7N), while ephrin-A1 ligand was represented as in the structure (PDB 3HEI) . EphA2 LBDgray and pink shaded areas indicate the structural elements involved in interactions with gH/gL and ephrin-A1, respectively. The ephrin uses an 18-residue long and mostly hydrophobic loop that connects strands G and Hthe GHephrin loopfor insertion into a complementary hydrophobic cavity presented at the surface of the receptor EphA2 LBD . The GHephrin loop carries a conserved E119ephrin-A1 (red circle) that establishes polar interactions, critical for PSI high-affinity binding, with a strictly conserved R103EphA2 (blue circle) on the loop connecting strands G and H in EphA receptors, designated also as a GH loop (GHEphA2) . In gL and ephrin-A1, gray shaded areas highlight the structural elements involved in interactions with EphA2 LBD. DIN, dimerization interface; ephrin, Eph family receptor interacting protein; gH/gL, glycoproteins H and L; HHV-8, PSI human herpesvirus 8; LBD, ligand-binding domain.(PDF) pbio.3001392.s003.pdf (263K) GUID:?D396E259-A818-4C8E-AF16-C1334EE7E48A S4 Fig: EphA2 LBD binds to HHV-8 gH/gL in 1:1 stoichiometry. SEC-MALS traces are shown for HHV-8 gH/gL alone (blue curve), EphA2 ectodomain or LBD alone (red curves), and gH/gL mixed with EphA2 (purple, dashed curve). Molecular weights are indicated on the chromatograms, demonstrating that the tertiary complexes are composed of 1 1 molecule of HHV-8 gH/gL bound to 1 1 molecule of EphA2 ectodomain or EphA2 LBD. The underlying data can be found in S2 Data. gH/gL, glycoproteins H and L; HHV-8, human herpesvirus 8; LBD, ligand-binding domain; SEC-MALS, size exclusion chromatography coupled with multi-angle light scattering.(PDF).