Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization

Veli-Matti Leppänena,1, Pipsa Saharinena,b, and Kari Alitaloa,b,1

aWihuri Research Institute, Biomedicum Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland; and bTranslational Cancer Biology Program, Research Programs Unit, University of Helsinki, 00014 Helsinki, Finland

Contributed by Kari Alitalo, March 8, 2017 (sent for review September 28, 2016; reviewed by Joseph Schlessinger and Michel O. Steinmetz)

The endothelial cell (EC)-specific receptor tyrosine kinases mice lacking Tie1 develop severe edema around E13.5 because Tie1 and Tie2 are necessary for the remodeling and maturation of compromised microvessel integrity and defects in lymphatic of blood and lymphatic vessels. -1 (Ang1) growth vasculature and die subsequently (15, 16). Furthermore, Tie1 has factor is a Tie2 agonist, whereas Ang2 functions as a context- critical functions in vascular pathologies, e.g., in tumor angio- dependent agonist/antagonist. The orphan receptor Tie1 modu- genesis and progression (12, 17). lates Tie2 activation, which is induced by association of angio- In EC monolayers, stimulate Tie receptor trans- cis – trans location to cell–cell junctions for Tie2 trans-association, whereas poietins with Tie2 in and across EC EC junctions in . – Except for the binding of the C-terminal angiopoietin domains in the absence of cell cell adhesion the Tie receptors are an- to the Tie2 ligand-binding domain, the mechanisms for Tie2 chored to the (ECM) by Ang1-induced Tie2 cis-association (10, 18). also have been implicated in activation are poorly understood. We report here the structural α β – basis of Ang1-induced Tie2 dimerization in cis and provide Tie2 signaling, and the 5 1 heterodimer enhances Ang1-induced EC adhesion and Tie2 activation (13, 19, 20). The mechanistic insights on Ang2 antagonism, Tie1/Tie2 heterodi- Tie1 and Tie2 heteromeric complexes are promoted by angio- merization, and Tie2 clustering. We find that Ang1-induced poietin stimulation, resulting in Ang1-induced activation of both Tie2 dimerization and activation occurs via the formation of an – β Tie1 and Tie2 (13, 21 23). Several studies have indicated Tie1 as intermolecular -sheet between the membrane-proximal (third) a Tie2 inhibitor (22, 24, 25), whereas recent experiments show Fibronectin type III domains (Fn3) of Tie2. The structures of that Tie1 association with Tie2 is required for Tie2 activation by Tie2 and Tie1 Fn3 domains are similar and compatible with Tie2/ Ang1 and Ang2 in mice (13, 26). Tie1 expression inhibits BIOCHEMISTRY Tie1 heterodimerization by the same mechanism. Mutagenesis Tie2 presentation at the cell surface in sprouting endothelial tip of the key interaction residues of Tie2 and Tie1 Fn3 domains cells, (23), but Tie1 sustains Tie2 signaling in contacting cells decreased Ang1-induced Tie2 phosphorylation and increased (13, 23). Thus, Tie1 exerts its context-dependent effects by the basal phosphorylation of Tie1, respectively. Furthermore, modulating Tie2 activity (13, 22, 23, 26). the Tie2 structures revealed additional interactions between Ligand-induced dimerization is regarded as a common, but the Fn 2 (Fn2) domains that coincide with a mutation of not the only, mechanism for activation of RTK signaling (27). Tie2 in primary congenital glaucoma that leads to defective Angiopoietin monomers form heterogeneous multimeric Tie2 Tie2 clustering and junctional localization. Mutagenesis of the ligands, and it has been suggested that Tie2 activation requires Fn2–Fn2 interface increased the basal phosphorylation of Tie2, receptor clustering (28–30). Also, Tie2 clustering without ligand suggesting that the Fn2 interactions are essential in preformed Tie2 oligomerization. The interactions of the membrane-proximal Significance domains could provide new targets for modulation of Tie recep- tor activity. Tie1 and Tie2 receptor tyrosine kinases are key regulators of blood and lymphatic vessel development and of pathological | angiopoietin | dimerization | homotypic interactions | processes including tumor , atherosclerosis, and crystallography vascular leakage, e.g., in . Tie1 is essential for the Tie2 agonist activity of angiopoietins, and the activated re- eceptor tyrosine kinases (RTKs) expressed in the endothelial ceptors form heteromeric complexes in endothelial cell–cell Rcells (ECs) of blood and lymphatic vessels control the de- junctions. However, little is known about the activation velopment and function of the cardiovascular and lymphatic mechanisms of the Tie receptors. Here we demonstrate that systems. The VEGFs and their endothelial receptors (VEGFRs) the membrane-proximal domains of Tie2 mediate homotypic are key regulators of angiogenesis and vascular integrity (1, 2). interactions, which occur via intermolecular β-sheet formation The angiopoietin ligand/Tie receptor pathway is necessary for and are necessary for Tie2 activation. The structural analysis blood and lymphatic vessel remodeling during embryonic and suggests that Tie2/Tie1 heterodimerization occurs by the same postnatal development and for homeostasis of the mature vas- mechanism. The crystal structures provide a model for culature (3, 4). Recently significant interest has focused on tar- angiopoietin-stimulated Tie2 ectodomain dimerization, clus- geting the VEGFR and Tie receptor pathways in antiangiogenic tering, and activation and insights into therapeutic targeting. and antilymphangiogenic therapies (5). Ang1 activation of Tie2 is indispensable for embryonic cardiac Author contributions: V.-M.L. and K.A. designed research; V.-M.L. performed research; development and angiogenesis, and both Ang1 and Ang2 are V.-M.L. analyzed data; and V.-M.L., P.S., and K.A. wrote the paper. necessary for the development of lymphatic and ocular vascula- Reviewers: J.S., Yale University School of Medicine; and M.O.S., Paul Scherrer Institut. ture. In adult tissues, Ang1 is required for vessel stabilization The authors declare no conflict of interest. – after angiogenesis (6 8). Ang2, which is produced by ECs and Freely available online through the PNAS open access option. – stored in their Weibel Palade bodies for rapid release, can Data deposition: The atomic coordinates, and structure factors reported in this paper function as a weak Tie2 agonist or as a context-dependent an- have been deposited in the Data Bank, www.pdb.org (PDB ID codes 5MYA, tagonist that inhibits Ang1-induced Tie2 activation and vascular 5MYB, and 5N06). – 1 stability (9 11). Tie2 is the major signal-transducing receptor of To whom correspondence may be addressed. Email: [email protected] or veli-matti. the angiopoietin/Tie signaling axis, and the homologous Tie1 [email protected]. receptor modulates Tie2 signaling (12, 13). Although Tie1, first This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. identified in human leukemia cells (14), is an orphan receptor, 1073/pnas.1616166114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1616166114 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 binding has been reported (31). The Tie receptors have a unique The dimerization is based on symmetrical interfaces between the extracellular domain (ECD) for ligand binding, a single-pass membrane-proximal Fn3 domains, placing the C termini close to transmembrane domain, a two-partite cytoplasmic protein tyro- each other at a distance of about 25 Å. The C termini point in sine kinase domain, and a C-terminal tail. The ECDs consist of the same direction, whereas the N-terminal Fn-like domains are Ig, epidermal -like, and three fibronectin type III directed away from each other. Dimerization is mediated mainly (Fn) domains (Fn1, Fn2, Fn3) (30, 32). The angiopoietins have by hydrogen bonds between the main-chain atoms of antiparallel an N-terminal region responsible for their multimerization, a β-strands in residues Asp682 to Lys690. Other interactions occur coiled-coil domain for dimerization, and a C-terminal fibrinogen- between symmetry-related Lys700 and Gly701, and the inter- like domain (FLD) that contains the Tie2-binding region (28, 29). acting residues are largely conserved (Fig. 1C). According to the Crystal structures of the angiopoietin/Tie2 ligand-binding domain Pisa server analysis, the interface buries a surface area of about 2 (LBD) complexes demonstrate that Ang1 and Ang2 FLDs bind 580 Å per chain in space group C2; the solvation free energy Tie2 in a similar manner (30, 33). Ang1 is a strong Tie2 agonist, gains of −1.5 and −1.9 kcal/mol for each chain and the related P and Ang2 a weak Tie2 agonist, suggesting that the Tie2 agonism value of 0.54 suggest considerable specificity (Tables S2 and S3). of the native angiopoietins resides in sequences outside the FLDs. In space group P21, the interactions between the antiparallel β-strands are almost identical, but, because of additional inter- Because multimerization of Ang1 and Ang2 is critical for Tie2 2 phosphorylation, it has been suggested that the difference be- actions, the buried surface area is larger, about 700 Å per chain tween the agonistic activities of Ang1 and Ang2 is caused by (Tables S2 and S3). different oligomeric states (34, 35). Indeed, multiple studies in- The Tie2 homodimers in the two crystal structures share the dicate that the strongly activating Ang1 has higher tendency to same overall structure, except that only one of the two Tie2 in the C2 dimer has the Fn1 domain (Fig. S1). The 376 form large oligomers than the weak agonist Ang2, although the α – significance of differential oligomerization is poorly understood C atoms in the two Tie2 Fn2 3 dimers superimpose with an (28, 29, 35, 36). The need for oligomerization of angiopoietin rmsd of 1.445 Å. The crystal packing indicates that the Fn1 domains ligands suggests that Tie2 clustering is required for its activation, were proteolytically removed before crystallization in space group but the mechanism(s) mediating Tie2 dimerization or higher- P21, whereas in space group C2 the other Fn1 domain is either order clustering are not known. disordered or present at low occupancy, because crystal packing in We report here the crystal structures of Tie2 and Tie1 mem- C2 should accommodate it, but only weak residual density is seen brane proximal domains and their structural and functional anal- (Fig. S2). Superposition of the Fn1 domain-bearing chain in space group C2 with the chain lacking the Fn1 domain creates a model of ysis, which provide mechanistic insight into ligand-induced – D Tie2 activation and to the mode of Tie1/Tie2 heterodimerization. aTie2Fn13 homodimer (Fig. 1 ). A recent EM analysis of full- length Tie2 ECDs did not indicate Tie2 dimerization, but the shape – Results and length of the extended structure of Tie2 Fn-like domains 1 3in – space group C2 are consistent with those in the EM analysis Structure of Tie2 Fn-Like Domains 1 3. To better understand the (11.8 nm vs. 12 nm) (37). Although data from small-angle X-ray mechanism of Tie2 dimerization and activation, we expressed – ∼ μ – – A scattering (SAXS) of an Fn2 3protein(at 80 M) indicated a Tie2 Fn-like domains 1 3 (Fn1 3) (Fig. 1 ) in insect cells and structure that fits best with our crystal structure of an Fn3 domain- crystallized the purified protein in space groups C2 and P21. The mediated Tie2 dimer, the Tie2 ECD and Tie2 Fn domains were crystal structure in space group C2 was determined at 2.9-Å monomericinsolutionwhenanalyzedbysize-exclusionchroma- resolution using multiple isomorphous replacement with anom- tography multiangle laser light scattering (SEC-MALLS) (Fig. S3 alous scattering (MIRAS) phases, and the structure in space A–E). This finding suggests that the unliganded receptor has a group P21 was determined at 2.6-Å resolution with molecular relatively weak tendency for the Fn-domain–mediated interactions replacement using the structure in the C2 space group as a and that the formation of the putative homotypic interactions by search model (Table S1). Both structures revealed a homodimer the full-length receptor requires spatial (and dimensional) con- of the Tie2 Fn-like domains in the asymmetric unit (Fig. 1B). straint in the plasma membrane and/or is ligand dependent.

Homotypic Fn3 Interactions Are Required for Ligand-Induced Tie2 Activation. We tested the model of Fn3-mediated homotypic in- teractions of Tie2 by targeting the dimerization interface with a V685Y/V687Y double mutation and by expressing the WT and mutant receptors in HeLa cells for ligand-stimulation experi- ments. Two conserved valine residues were changed to bulkier tyrosine residues to introduce steric hindrance across the Fn3– Fn3 interface (Fig. 2 A and B). A model of the Tie2 Fn3 mutant indicates that the neighboring Tyr674 and Tyr697 residues force the tyrosine mutant conformations toward the Fn3–Fn3 interface (Fig. 2B). Although Comp-Ang1 stimulation of the WT Tie2 in- duced strong Tie2 phosphorylation and colocalization with Tie1 in cell–cell junctions of receptor-transfected HeLa cells, phosphory- lation of the mutant Tie2 was much reduced, strongly suggesting that the mutation interfered with Tie2 dimerization (Fig. 2 C and D) (10, 18). Neither these Fn3 mutations nor the other Tie2 or Tie1 mutations described below had any overt effect on receptor folding or expression in HeLa cells (Fig. S3 F and G). The Fn1– Fig. 1. Homodimerization of Tie2 Fn-like domains. (A) A schematic model Fn2 junction in Tie2, unlike the Fn2–Fn3 junction, has no specific of Tie2 with the LBD and the three membrane-proximal Fn-like domains labeled. (B) Comparison of the homodimer structures of Tie2 Fn-like domains interactions between the two domains, consistent with its apparent flexibility and susceptibility to proteolysis (Fig. S4 A–C). In com- in space groups C2 and P21. The structures are shown as cartoon diagrams – with the Fn-like domains labeled and the N and C termini labeled where parison with the analysis of the Fn1 domain of the Ang1 Tie2 applicable. (C) A surface-and-stick representation of the Tie2 Fn3 interactions. LBD complex at 4.5-Å resolution (33), we observed a sequence The Tie2 residues in the Fn3 surface model are colored according to the rate register difference of one to three residues between the Fn1 of evolutionary conservation from cyan (variable) to magenta (conserved). structures (Fig. S4D). However, by superimposing the Fn1 do- (D) A model of homodimer of the Tie2 Fn-like domains 1–3. Asn-linked mains in the Tie2 Fn1–3 homodimer and Ang1–Tie2 LBD com- N-acetylglucosamine moieties are shown as cyan spheres. plex structures (33), we were able to construct a structural model

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1616166114 Leppänen et al. Downloaded by guest on September 23, 2021 Tie1 tyrosine mutant (3Y; V681Y, L691Y, and I693Y) in an analogous manner to the Tie2 Fn3 double mutant. Val681 was mutated to tyrosine to mimic the Tie2 Fn3 domain and to direct the side-chain conformers of I693Y and L695Y toward the interface. Another mutant (5M) with additional D689R and D696R substitutions was designed to disrupt the interactions further. The Tie1 mutants increased the basal phosphorylation of Tie1 and made Tie1 insensitive to Tie2-mediated Comp-Ang1 stimulation (Fig. 4 C and D). Interestingly, the 5M mutant did not affect Tie1/Tie2 heterodimerization according to the cell-surface crosslinking assay, and Tie1 baseline phosphorylation was not af- fected in Tie1 singly transfected cells, suggesting that the Tie1 Fn3 mutations affected only Tie1/Tie2 kinase crosstalk (Fig. S6 A–C).

A Model for Tie2 Clustering. In addition to the very similar inter- actions between the Fn3 domains in the C2 and P21 structures, we observed structurally analogous interactions between the Fn2 domains of neighboring homodimers in the crystal, sug- gesting that both types of interactions exhibit natural protein contacts (Fig. 5A). Combining Fn3-guided homodimerization of Tie2 with Fn2-guided interactions between homodimers creates an array of Tie2 molecules that could represent the structural Fig. 2. Tie2 Fn3-mediated homotypic interaction. (A) A bottom view of the di- A merization interface in Tie2 Fn3. The dimerization is mediated by hydrogen bonds basis of Tie2 clustering (Fig. S7 ). The symmetrical interactions between the main-chain atoms of antiparallel β-strands between two neighbor- between the Fn2 domains involve multiple hydrogen bonds be- ing Tie2 molecules. (B)AsinA with Val685 and Val687 mutated to tyrosines. The tween highly conserved Arg-Trp-Arg motifs and the main-chain tyrosine side-chain conformations are the most common ones in the backbone atoms of residues 582–585 in a hairpin loop of the neighboring conformation-dependent rotamer library in PyMOL. (C) GFP (green) and phospho- chain (Fig. S7 B and C). Interestingly, in a primary congenital Tie2 (Tyr992; red) immunofluorescence staining of HeLa cells transfected with glaucoma (PCG) family, a TEK mutation (Y611C) is located Tie1-GFP and Tie2-WT or the Tie2-Fn3 mutant. Note the junctional colocalization close to this Fn2′–Fn2′′ interface of neighboring homodimers, BIOCHEMISTRY of Tie1 and phospho-Tie2 in Comp-Ang1 (cA1) stimulated WT Tie2 transfected resulting in haploinsufficiency because of the loss of Tie2 func- cells and that the phospho-Tie2 (Tyr992) antibody is known to bind also to the tion (Fig. 5B) (38). Tyr611 is a buried residue that packs against homologous region in Tie1. (D) Western blot analysis of Tie2 phosphorylation in the hairpin loop, making interactions with the Arg-Trp-Arg the transfected HeLa cells. Cells were stimulated with COMP-Ang1 (cA1) for 1 h or motif. Mutation of Tyr611 to cysteine would remove a poten- were left unstimulated. Error bars indicate SEM. **P ≤ 0.01. Student’s t test; n = 3. tially important hydrogen bond to His606 in the neighboring Fn2 loop. We next created a Q588R/V612R double mutant to ′ ′′ of the Ang1-bound Tie2 ECD homodimer that provided mecha- target the Fn2 -Fn2 interface and analyzed the effect of the nistic insight for Tie2 activation in cis (Fig. 3 A and B). Interestingly, Fn2 mutations on Tie2 activation in Tie2 singly transfected and Tie1/Tie2 doubly transfected HeLa cells (Fig. 5 C and D and Fig. the Tie2 LBDs are too far apart for a dimeric angiopoietin ligand to D E bridge the receptors for successful dimerization and activation. S7 and ). The mutant showed a significant increase in basal Instead, a dimeric ligand would compete with multimeric angio- poietin for Tie2 binding (Fig. 3B).

A Model of Tie1/Tie2 Heterodimerization. To study the mechanism of Tie1 and Tie2 heterodimerization, we expressed the membrane- proximal Tie1 Fn3 in insect cells and crystallized the purified protein for structure determination. The structure was solved at 2.6-Å resolution by molecular replacement using the Tie2 Fn3 domain as a search model (Table S1). Interestingly, the structure is a strand-swapped homodimer in which the two Fn-like domains exchange their C-terminal β-strands (Fig. S5 A and B). Strand swapping occurs by extension of the sixth β-strand toward its counterpart to form an antiparallel β-sheet that extends over Ile722–Gly724 in the bridge region where the two strands meet. This swapping may be an important feature of Tie1 function, but a model derived from SAXS data indicates that Tie1 Fn3 is a monomer rather than a dimer in solution (Fig. S5C). Thus, the strand-swapped dimerization may instead be a crystallization artifact of an isolated domain. Comparison of the Tie1 and Tie2 Fn3 domain structures re- veals very similar folds in both, despite their low sequence identity (Fig. 4A), suggesting that structural homology between Fig. 3. Structural basis of Tie2 dimerization and activation in cis.(A)A model of the ligand-bound homodimer of Tie2 ECDs based on the structure the Tie receptors extends to the Fn3-mediated dimerization and – activation. Superposition of Tie1 Fn3 domain with an Fn3 domain of the Ang1-bound Tie2 LBD Fn1 complex (PDB ID code 4K0V) and the homodimer of the Tie2 Fn-like domains. The Ang1 receptor-binding domain in the Tie2 homodimer indicates that Tie1/Tie2 heterodimerization (RBD), colored in red, and the two chains of Tie2 ECDs, colored in blue and also could be mediated by interactions between the antiparallel β B orange, are shown as cartoon and semitransparent surface models. Note the -strands in the Fn3 domains (Fig. 4 ). The model also suggests distance between the Tie2 LBDs. (B) A schematic model of Ang2 antagonism. additional favorable interactions, including a salt bridge between The model of the Ang1 tetramer was adopted from angiopoietin tetramers Tie2 Asp682 and Tie1 Arg697 (Fig. 4B). We tested the model of analyzed by EM (29). The Ang2-activating antibody ABTAA binds to the Fn3-mediated Tie2/Tie1 heterodimerization by analyzing Tie1 Ang2 fibrinogen-like domains and creates a tetrameric Ang2 suitable for phosphorylation in Tie1/Tie2-transfected HeLa cells. We created a Tie2 dimerization and activation (43).

Leppänen et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 domains in KIT and VEGFR-2 (39, 40). In VEGFR-2, the C termini also are about 25 Å apart, and in these three RTKs the membrane-proximal domains contribute symmetrical interactions around an axis with twofold symmetry (39, 40). Interestingly, the interactions between the Tie2 Fn3 domains occur via in- termolecular hydrogen bonding between antiparallel β-strands. Although this binding creates an intermolecular β-sheet with only a few side-chain–mediated interactions, the interacting residues are largely conserved. Mutation of two conserved valines in the interacting β-strands in Fn3 to bulkier tyrosines was made to disorient the transmembrane and kinase domains for optimal transphosphorylation upon ligand-stimulated Tie2 activation. These mutations reduced Comp-Ang1–stimulated Tie2 phosphor- ylation, thus confirming the importance of the intermolecular β-sheet formation for Tie2 activation. In the accompanying study in this issue of PNAS, Moore et al. describe the same intermolecular interactions in an independent crystal structure of Tie2 Fn-like domains and show reduced Ang1-induced Tie2 phosphorylation with the Y697A mutation located in the interface (41). Receptor activation via intermolecular β-sheet formation, as seen in this di- mer, is unique to RTKs, although intermolecular β-sheet formation represents a common mode of protein–protein interactions (42). We constructed a model of the ligand-bound Tie2 ECD homodimer based on our Tie2 Fn1–3 homodimer and the pub- lished Ang1–FLD/Tie2–LBD complex (33) structures. The model indicates that the ligand-binding sites are too far apart for a dimeric angiopoietin to promote the formation of a Tie2 dimer in the cis orientation. Since Ang2 is mainly a covalent dimer, whereas Ang1 is multimeric in nonreducing conditions, the model provides an interesting possible explanation why Ang2 functions as a weak agonist (28, 35, 36). In EM, both Ang1 and Ang2 exist from dimers up to hexamers, but although the ma- jority of Ang2 showed low-order “mushroom-like” states, the majority of Ang1 existed as high-order oligomers (29, 35). Unlike the native Ang2, tetrameric Bow–Ang2 (Ang-F2-Fc-F2) is a po- Fig. 4. Model of Tie1/Tie2 heterodimerization. (A) Structural comparison of tent Tie2 agonist (26, 29), suggesting that the extended “reach” the Fn3 domains in Tie1 and Tie2. (B) A Tie2 homodimer-guided model of of receptor-binding sites in these multimers is crucial for pro- Tie1/Tie2 heterodimer of the Fn3 domains. (C) A model of the Tie1 moting Tie2 dimerization in cis. This dimerization may be fa- Fn3 mutant 5M in the Tie1/Tie2 heterodimer. Three aliphatic residues were cilitated by the flexibility of the Fn1–Fn2 junction revealed by changed to tyrosine residues in the Tie1 Fn3 3Y mutant (magenta). The 5M mutant also has two aspartates changed to arginines (red). (D) Western blot comparing our structures with that in the accompanying paper by analysis of Tie1 phosphorylation in HeLa cells doubly transfected with Moore et al in this issue of PNAS (41). Furthermore, the recently Tie2 and Tie1-WT or the Tie1-Fn3 mutant 3Y or 5M. Cells were stimulated published Ang2-binding and Tie2-activating antibody ABTAA with COMP-Ang1 (cA1) for 1 h or were left unstimulated. Error bars indicate presumably binds two Ang2 low-order oligomers, possibly homo- SEM. *P ≤ 0.05; **P ≤ 0.01; ns, not significant; Student’s t test, n = 3. dimers, in an optimal angle for Tie2 activation (Fig. 3B)(43). Tie1 is an orphan receptor, but Ang1 activates Tie1 in Tie2/ Tie1 heteromeric receptor complexes (21–23). Prompted by our Tie2 phosphorylation in both singly and doubly transfected cells. observation of Fn3-mediated homotypic Tie2 interactions, we Interestingly, the Fn2 mutant could be stimulated with Comp- solved the Tie1 Fn3 crystal structure for comparison. Structural Ang1 in the presence of Tie1 but not in the absence of Tie1. comparison of the Tie2 and Tie1 Fn3 domains revealed very Alignment of Ang1-bound homodimers of the Tie2 ECDs cre- similar folds despite their low sequence identity, suggesting that β ates a model for Tie2 clustering (Fig. 5E). Notably, multiple Tie1 also interacts with Tie2 through intermolecular -sheet Tie2 homodimers can interact without steric clashes, and the formation. To analyze the role of Tie1 Fn3 in Tie2/Tie1 hetero- model brings the neighboring LBDs close to each other. dimerization, we created the Tie1 Fn3 tyrosyl mutants 3Y and 5M in which the mutations correspond to those in Tie2. Both alter- Discussion ations increased the basal phosphorylation level of Tie1, making Although the structural features of the - Tie1 insensitive to Comp-Ang1 stimulation in cells coexpressing Tie2. Interestingly, the Tie1 Fn3 mutations did not affect Tie1/Tie2 binding domain interactions with the Tie2 LBD have been de- heterodimerization in the crosslinking assay, and the increased scribed, the structural basis for Ang1-induced Tie2 activation, baseline activation was Tie2-dependent. These results indicate that Tie1/Tie2 heterodimerization, and the context-dependent an- the Tie1/Tie2 heterodimerization is likely to involve interactions tagonism by Ang2 has been largely lacking. Our crystal structures between the membrane-proximal domains and that these interac- and functional analysis of mutant receptors show that the tions restrict aberrant Tie2–Tie1 cross-activation. We cannot membrane-proximal Fn3 domains in Tie2 and Tie1 contribute exclude alternative interactions for Tie1 and Tie2 Fn3 domains, to Tie2 homodimerization and Tie2/Tie1 heterodimerization, and additional interactions, such as putative electrostatic inter- whereas the Tie2 Fn2 domain seems to mediate Tie2 clustering. actions between the Tie2 LBD and the corresponding domain in In addition, our results provide a structural explanation for the Tie1 (25), may stabilize the heterodimerization despite the Tie1 need of Ang1 oligomers in Tie2 activation. Fn3 mutations. Crystal structures of the Tie2 Fn-like domains revealed a In addition to Fn3-mediated Tie2 homodimerization, the two mechanism of Fn3-mediated Tie2 dimerization in which the crystal structures of the Tie2 Fn-like domains revealed sym- Fn3 domains bring their C termini close to each other in a way metrical interactions between the Fn2 domains that could me- similar to that described for the membrane-proximal Ig-like diate Tie2 clustering. The interactions are almost identical in

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1616166114 Leppänen et al. Downloaded by guest on September 23, 2021 Fig. 5. Model for Tie2 clustering. (A) A cartoon presentation of the Fn2′–Fn2′′ interaction between the neighboring Tie2 chains. (B) A close-up view of the symmetrical interactions between the Fn2 do- mains centered on Arg581. Tyr611 is highlighted in red. Y611C corresponds to the TEK variant in a PCG family, which results in haploinsufficiency because of protein loss of function (38). (C) Western blot analysis of Tie2 phosphorylation in the WT or Fn2- mutant (Gln588Arg, Val612Arg)-transfected HeLa cells. Tie2 single-transfected and Tie1/Tie2 double- transfected cells were stimulated with COMP-Ang1 (cA1) for 1 h or were left unstimulated. (D) Quanti- tative analysis of C. Error bars indicate SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001: ns, not significant; Student’s t test. n = 3. (E) A model for clustering of Ang1 RBD-bound homodimers of Tie2 ECDs based on the array of the Fn-like homodimers in A and in Fig. S7A.

both structures, and residues around the Arg-Trp-Arg motif, Tie2 association may represent distinct ligand-binding modes in BIOCHEMISTRY which interacts with a hairpin loop of the neighboring chain, are trans vs. in cis. Consistent with our model of Tie2 cis association, conserved. Site-directed mutagenesis of the Fn2–Fn2 interactions several studies indicate that artificial angiopoietin dimers, such as increased the basal phosphorylation of Tie2, suggesting that the Fc-tagged dimers, are inactive as Tie2 ligands (29, 30, 34). How- Fn2–Fn2 interactions may be involved in Tie2 oligomerization in ever, Oh et al. have reported a specific Tie2-activating Ang1 dimer the absence of ligands rather than in ligand-induced Tie2 activa- (CA1-3), which is comprised of the linker and the FLD domains of tion. There is growing evidence that various transmembrane re- Ang1 fused to a dimeric Comp domain (46). CA1-3 stimulation ceptors have a preformed, but inactive, oligomeric structures on shows prominent Tie2 activation in EC–EC junctions, indicating the cell surface, and Tie2 also has been shown to exist as pre- that a dimer of Ang1 FLD and the linker domain is capable of FLD formed oligomers in the absence of ligands (27, 31, 44). Ligand domain presentation in the correct angle for receptor in trans in- binding then will induce a conformational change for receptor teraction (Fig. S7F). Comp-Ang1 stimulates strong Tie2 activation activation. The difference between the Fn2–Fn2 interface in our in EC–EC junctions in trans and in the rear of migrating cells in cis, analysis and dimer 1 in the paper by Moore et al. (41) could rep- although the pentameric bundle of Comp-Ang1 is only about 10 nm resent such a change, because mutational analysis also implicates in diameter (10, 34, 35). Therefore, it is likely to represent a ligand- dimer 1 interactions in Ang1-stimulated Tie2 activation. Although binding mode whereby the multimericity may bridge neighboring the Fn3-mediated dimers in both papers are identical, a lateral shift Tie2 homodimers in cis in addition to Tie2 interactions in trans. of Fn3-mediated homodimers by only a few nanometers is required Decreased junctional Tie2 phosphorylation upon site-directed for the adjustment of the Fn2–Fn2 and dimer 1 interfaces [Protein mutagenesis of the Fn3 interface suggests that Tie2 clustering in Data Bank (PDB) ID codes 5MYB and 5UTK]. EC–EC junctions may involve arrays of Tie2 homodimers. On Interestingly, a mutation in the human TEK gene in the se- the other hand, Tie1 associates with Tie2 in EC–EC junctions quence encoding Tie2 Fn2 domain was discovered in a cohort of and thereby can regulate the context-dependent differences PCG patients who did not carry mutations in other known in Tie2 signaling during angiogenesis (13, 23, 26). We show that disease-causing genes (Fig. 5B) (38). This mutation, Y611C, Tie1/Tie2 heterodimerization may involve interactions between occurs in the symmetrical interface between the Fn2 domains of the Fn3 domains, but it is not clear how Tie1 interacts with the the neighboring Tie2 homodimers in our model of Tie2 clus- arrays of Tie2 homodimers in trans or how angiopoietins bridge tering and in the dimer 1 interface in the Tie2 structure by Tie2 association across EC–EC junctions. The context-dependent Moore et al. (41). Although the mutant Tie2 was properly lo- differences in angiopoietin-activated Tie2 signaling pathways may calized at the plasma membrane in resting ECs, it did not undergo depend on differences in other subcellular protein constituents, normal Ang1-stimulated Tie2 clustering or junctional localization such as integrins in the ECM contacts and VE-PTP in EC–EC (38). Although the nature of the impaired interactions remains contacts (10, 13, 19, 47). Further structural and functional in- elusive, the disease-causing mutation supports the evidence for the vestigation is required to understand how Tie1 acts to modulate Fn2-mediated Tie2 clustering. Similarly, in the Eph family tyrosine the effects of Ang1 and Ang2 on Tie2 and the mechanism of kinase receptors, the membrane-proximal Fn-like domains are Ang2 antagonism in the Tie2 trans association. known to stimulate intermolecular interactions (45). Ligand-mediated dimerization of RTKs involves weak homo- Angiopoietins provide a rare example of soluble ligands that typic interactions between the membrane-proximal domains engage receptor oligomers on the same cell in cis or bridge their which allow precise positioning of the C-terminal regions and the receptors across cell–cell contacts in trans (10, 18). Ang1 stimula- transmembrane domains in the correct orientation that enables tion of Tie2 in trans vs. in cis seems to induce partly different sig- activation of the cytoplasmic tyrosine kinase domains (27, 39, 40, naling pathways. Ang1 binding to Tie2 in EC–ECM contacts 48, 49). We have shown previously that targeting such interac- represents the cis association mode, which preferentially acti- tions in VEGFR-3 with an antibody that does not block ligand vates the Erk and DokR pathways (10, 18). In contacting ECs, binding can still block the formation of VEGFR-3 homodimers both angiopoietin ligands induce Tie2 translocation to cell–cell and VEGFR-3/VEGFR-2 heterodimers, and signaling (50). Sim- junctions, but Ang2 induces only weak Tie2 activation (10, 18). ilarly, antibodies targeted to the membrane-proximal domain of

Leppänen et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 KIT inhibit its activation (51). Our results here indicate that and the purified proteins were crystallized for structure determination. Full ligand-induced Tie2 activation also involves homotypic inter- details of protein purification and X-ray and SAXS data collection and actions of the membrane-proximal domains. Importantly, site- analysis are described in SI Methods. directed mutagenesis of the Tie2 Fn3–Fn3 interactions inhibited ligand-stimulated Tie2 phosphorylation, whereas mutagenesis Cell Culture and Phosphorylation of Tie1 and Tie2. WT and mutant Tie1 and of the Fn2–Fn2 interactions increased the basal phosphor- Tie2 were cloned into pMXs retroviral expression vector that was used to ylation of Tie2. Also, site-directed mutagenesis of the Tie1 transduce HeLa cells as described by Saharinen et al. (10). Full details of cell Fn3 interactions in Tie1/Tie2 heterodimerization experiments culture and analysis are described in SI Methods. increased the basal Tie1 phosphorylation. The targeting of Tie receptor membrane-proximal domains thus may provide unique ACKNOWLEDGMENTS. We thank Tapio Tainola, Seppo Kaijalainen, Tanja therapeutic approaches for the modulation of Tie receptor Laakkonen, Kirsi Mänttäri, and Jarmo Koponen for excellent technical assistance activation. and Drs. Kathryn Ferguson and Mark Lemmon for valuable comments. This work was funded by the Jenny and Antti Wihuri Foundation, the Academy of Finland (Grants 307366, 292816, and 273817), the Sigrid Juselius Foundation, the Cancer Methods Society of Finland, the Leducq Foundation (Grant 11CVD03), the Helsinki Uni- Crystallization and Structure Determination. Human Tie2 Fn1–3 (residues 443– versity Hospital’s Government Special State Subsidy for Health Sciences, the 735) and human Tie1 Fn3 (residues 641–738) were expressed in insect cells, Novo Nordisk Foundation, and the Jane and Aatos Erkko Foundation.

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