Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2–Tie2 complex

Abstract

The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition-unique for a receptor kinase-where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.

Structural basis for angiopoietin-1–mediated signaling initiation

“…It is a compact protein with overall dimensions of ∼50 × 40 × 35 Å and has three domains A, B, and P (according to fibrinogen nomenclature) colored in red, cyan, and yellow, respectively. The P domain was identified previously as the site of receptor binding (12,13). This region displays little secondary structure and contains mostly long, extended coil regions (Fig.…”

“…Large-scale protein expression was performed from stably expressing HEK293 cells in a BioFlo310 bioreactor (New Brunswick Scientific) or roller-bottle culture with typical yields averaging 10 mg/L for Ang2-TAG and Tie2. Fusion proteins were purified as described (12,13). Full-length angiopoietins were purified via anti-myc (9E10) affinity chromatography and eluted with low-pH buffer [100 mM glycine (pH 3.0), 150 mM NaCl] before buffer exchange with HBS [20 mM Hepes (pH 7.0), 150 mM NaCl].…”

“…Alternatively, the receptor antagonist Ang2 has no effect on the inhibitory Tie1/Tie2 complex and is, therefore, unable to promote Tie2 activation. It remains unclear, however, exactly how the Ang ligands differentially effect the Tie1/ Tie2 interactions and Tie2 activation despite the available structural models of Ang2 and the Ang2/Tie2 complex (12,13).…”

“…All angiopoietin family members, Ang1 to -4, contain an amino-terminal "superclustering" and coiled-coil domain, followed by a carboxyl-terminal fibrinogen domain. Several studies demonstrate that the fibrinogen domain mediates the interaction with Tie2, whereas the coiled-coil and the superclustering motifs, on the other hand, are required for ligand oligomerization, a prerequisite for receptor clustering and activation (9)(10)(11)(12)(13)(14). Indeed, all angiopoietins exist primarily as tetramers, hexamers, and higherorder oligomers in solution.…”

“…Indeed, all angiopoietins exist primarily as tetramers, hexamers, and higherorder oligomers in solution. Nonetheless, chimeric Ang ligands illustrate that only the highly conserved receptor-binding fibrinogen domain is necessary and sufficient for their unique functional signaling characteristics (13)(14)(15).…”