Architecture of Eph Receptor Clusters

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Architecture of Eph receptor clusters

Juha P. Himanena,1, Laila Yermekbayevab,1, Peter W. Janesc,1, John R. Walkerb,1, Kai Xua, Lakmali Atapattuc, Kanagalaghatta R. Rajashankard, Anneloes Mensingac, Martin Lackmannc,2, Dimitar B. Nikolova,2, and Sirano Dhe-Paganonb,e,2

aStructural Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065; bStructural Genomics Consortium, University of Toronto, 101 College Street, Toronto, ON M5G 1L7, Canada; cDepartment of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia; dAdvanced Photon Source, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439; and eDepartment of Physiology, University of Toronto, 101 College Street, Toronto, ON M5G 1L7, Canada

Edited* by Dinshaw J. Patel, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved April 14, 2010 (received for review March 29, 2010)

Eph receptor tyrosine kinases and their ephrin ligands regulate cell the EphB2-ephrinB2 crystal structure suggests a propensity of in- navigation during normal and oncogenic development. Signaling dividual Eph/ephrin complexes to assemble, via direct Eph/Eph of Ephs is initiated in a multistep process leading to the assembly contacts, into heterooligomeric clusters. Furthermore, earlier of higher-order signaling clusters that set off bidirectional signaling findings indicated that Eph-Eph interactions via a C-terminal ec- in interacting cells. However, the structural and mechanistic details todomain region outside the LBD are critical for Eph function of this assembly remained undefined. Here we present high-resolu- during development (3). Presumably such Eph-Eph interfaces tion structures of the complete EphA2 ectodomain and complexes act to recruit non-ligand-bound Eph receptors into Eph clusters with ephrin-A1 and A5 as the base unit of an Eph cluster. The struc- (5). In addition, a random mutagenesis survey of the EphA3 tures reveal an elongated architecture with novel Eph/Eph interac- ectodomain revealed that binding to ephrinA5 requires an inter- tions, both within and outside of the Eph ligand-binding domain, action site located in the CRD. Although having only a modest that suggest the molecular mechanism underlying Eph/ephrin clus- contribution to ligand-binding affinity, mutation of this region tering. Structure-function analysis, by using site-directed muta- severely effected receptor phosphorylation and recruitment of genesis and cell-based signaling assays, confirms the importance signaling molecules (6). Last, a recent report suggested that of the identified oligomerization interfaces for Eph clustering. ephrin-A5 may also interact via the EphA3 fibronectin III repeats (7). Todetermine the structural basis of Eph clustering and explore cell-cell attraction and repulsion ∣ Eph receptor clustering underlying receptor–receptor and receptor–ligand interactions, we determined structures of the complete EphA2 ectodomain, ph receptors and their ephrin ligands control a diverse array alone and in complexes with its cognate ligands ephrin-A5 and Eof cell–cell interactions during patterning of the nervous, ephrin-A1, the latter of which was recently used to elaborate spa- skeletal, and vascular systems (1, 2). Upon ephrin binding, the tiomechanical concepts related to EphA2 clustering (8). These Eph kinase initiates “forward” signaling into receptor-expressing crystal structures, supported by cell-based functional studies, cells, and the ephrin cytoplasmic tail triggers “reverse” signaling show that the CRD mediates Eph/Eph interactions in the assembly into ligand-expressing cells. Ephs and ephrins are divided into of signaling-competent EphA2/ephrin clusters. two subclasses (A and B) on the basis of their affinities for each other. With some exceptions, EphA receptors (EphA1–A10) bind Results and Discussion to A-class ephrins (ephrin-A1–A6), whereas EphB receptors Overall Structures. We crystallized and determined protein struc- (EphB1–B6) interact with the B-subclass ephrins (ephrin-B1– tures comprising the whole—or parts of the—EphA2 extracellular B3). Given that Ephs and ephrins are membrane-bound, their region, either in their apo forms (complete EphA2 ectodomain interaction occurs only at sites of cell–cell contact. In the absence and LBD) or bound to their ephrin ligands ephrin-A1 or -A5 of cell–cell interactions, they exist in loosely associated microdo- (LBD, LBD-CRD, and LBD-CRD-nFN3, Table1). The structure mains, which become more compact and well-ordered when Eph/ of a full-length Ephrin receptor ectodomain (Fig. 1A) reveals an ephrin complexes assemble to generate clearly defined signaling extended structure of tightly packed domains spanning 146 × 52 × centers (2). 55 Å(Fig. S2). The extracellular Eph region contains a conserved N-terminal The fold and atomic details of the Eph CRD, encompassing ligand-binding domain (LBD), an adjacent cysteine-rich domain ∼114 amino acids (residues 201–314) immediately C-terminal (CRD) (3), followed by two fibronectin repeats (FN3) (Fig. S1). to the LBD, can be subdivided into two domains, an N-terminal The cytoplasmic Eph region encompasses a regulatory juxtamem- domain similar to complement control module/short complement brane region connecting the kinase domain, a sterile α motif do- regulator domain, and a TNF receptor like CRD. The N-terminal main, and a postsynaptic density protein (PSD95), Drosophila half (201–260) includes five antiparallel β-strands arranged as a disc large tumor suppressor (DlgA), and zonula occludens-1 pro- β-sandwich, whereby the first residue of the fold (Cys201) is tein (zo-1) binding motif. All ephrins possess a 20 KDa extra- disulfide-anchored to the end of the fourth β-strand (Fig. S3). cellular receptor-binding domain; B-type ephrins also contain A Dali search (9) reveals the top Protein Data Bank (PDB) a short cytoplasmic region. Several crystal structures of complexes between the minimal Author contributions: J.P.H., M.L., D.B.N., and S.D.-P. designed research; J.P.H., L.Y., P.W.J., Eph and ephrin-binding Eph domains have been reported J.R.W., K.X., L.A., K.R.R., and A.M. performed research; M.L. and D.B.N. contributed (reviewed in ref. 4). The crystal structure of the complexed EphB2 new reagents/analytic tools; J.P.H., P.W.J., J.R.W., M.L., D.B.N., and S.D.-P. analyzed data; and ephrin-B2 binding domains revealed two contact surfaces that and J.R.W., M.L., D.B.N., and S.D.-P. wrote the paper. are involved in the assembly of Eph/ephrin tetramers: an expansive The authors declare no conflict of interest. high-affinity ephrin-binding channel, likely responsible for the *This Direct Submission article had a prearranged editor. initial interaction, and a smaller interface that mediates a lower Freely available online through the PNAS open access option. affinity Eph-ephrin contact with an adjacent ephrin molecule (4). 1J.P.H., L.Y., P.W.J., and J.R.W. contributed equally to this work. – In addition to these structurally defined ligand receptor 2To whom correspondence should be addressed. E-mail: [email protected]. interactions, several observations revealed that additional protein edu.au, [email protected], or [email protected]. interfaces are important for the generation and function of This article contains supporting information online at www.pnas.org/lookup/suppl/ Eph/ephrin signaling centers at points of cell-cell contact: First, doi:10.1073/pnas.1004148107/-/DCSupplemental.

10860–10865 ∣ PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1004148107 Downloaded by guest on September 24, 2021 Table 1. Data collection and refinement statistics LBD + LBD-CRD + LBD-CRD-nFN3 + Dataset LBD EphrinA1 EphrinA1 EphrinA5 LBD-CRD-nFN3-cFN3 EphA2 residues 23-202 23-202 23-326 27-435* 23-531 Expression host Sf9 Sf9 Sf9 HEK293 Sf9 Crystallization 25% PEG 3350, 0.1 M 14.9% PEG 4000, 10% PEG 3350, 20% PEG 3350, 200 mM 3% PEG 4 K, 0.1 M buffer Ammonium Sulfate, 0.1 M Na Citrate 0.16 M Ammonium Citrate, NaAc, 0.1 M CaCo pH 5.5 0.1 M Bistris pH 5.5 pH 5.6, 20% Ammonium Phosphate 0.1 M Na Citrate pH 5.8 with 0.5 M NDSB 256 Isopropanol PDB ID code 3C8X 3CZU 3MBW 3MX0 3FL7 Space group P 31 21 P 65 22 C 2221 P21 P 21 21 2 Unit cell 92.518, 92.518, 99.431, 99.431, 58.265, 215.787, 57.859, 89.049, 59.358, 89.992, (a, b, c, α,ß,γ) 41.291, 90, 90, 120 204.879, 90, 90, 120 107.263, 90, 90, 90 198.150, 90, 96.22, 90 136.476, 90, 90, 90 Beamline RIGAKU FR-E RIGAKU FR-E APS SBC-CAT 19 ID APS NE-CAT 24-ID-C APS GMCA-CAT 23-ID-B Wavelength 1.54178 1.54178 0.98792 0.98 0.97948 Resolution 35.0-1.95 25.0-2.65 39.0-2.80 45.0-3.50 41.0-2.50 Unique reflections 14,579 18,122 16,953 23,129 25,966 Data redundancy† 6.8 (5.0) 20.8 (21.2) 5.3 (5.3) 5.5 (5.2) 4.1 (4.0) Completeness 99.9 (98.6) 100 (100) 99.7 (100) 91.4 (71.3) 99.7% (98.1%) I∕σI 15.44 (1.828) 35.74 (5.05) 23.65 (2.17) 21.6 (6.2) 22.43 (2.94) R sym 0.151 (0.849) 0.109 (0.714) 0.066 (0.668) 0.11 (0.22) 0.070 (0.375) R R ∕R p:i:m: and meas r:i:m: 0.061 (0.415) 0.025 (0.158) 0.033 (0.333) Refinement Resolution 35.0-1.95 24.86-2.65 34.10-2.81 45.0-3.50 40.59-2.50 Reflections used 14,205 17,120 16,891 22,383 42,331 All atoms 1,394 (0,114) 26.03 (39, 73) 3,305 (90,28) 8,380 3,741 (18, 46) (hetero, solvent) R ∕R ‡ 16 3∕22 6193∕22 5225∕27 0246∕29 4 30 2∕39 7 24 6∕29 9 work free ...... ( . . ) . . rmsd bond length 0.016 0.009 0.009 0.006 0.009 rmsd bond angle 1.52 1.23 1.15 1.10 1.24 Mean B factor 24.22 42.45 79.92 138.34 35.47 Ramachandran plot Favored 96.8 95.7 96.5 79.4 93.6 Allowed 100 100 100 94.6 100 Disallowed 0 0 0 58 0 *Ordered and modeled. †Highest resolution shell shown in parenthesis. ‡R free calculated with 5% of the data. BIOCHEMISTRY

Fig. 1. EphA2/ephrin-A1 (5) structures, structural alignment, and ligand binding. (A) Left, backbone representation of superimposed structures. Right,all structures are shown from the same perspective in ribbon format. Each structure is labeled according to its PDB code and differentially colored. (B) The EphA2-EphrinA1 high-affinity heterodimer interface. Stereoscopic view of the interface with domains labeled and shown in ribbon format. Ligand residues that are within 4 Å of the LBD are shown as sticks; LBD residues as lines. Water molecules in the vicinity are shown as red spheres, hydrogen bonds as black dashed lines.

Himanen et al. PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 ∣ 10861 Downloaded by guest on September 24, 2021 hit with this domain is that of 2Z3R with a Z score of 4.5, an rmsd only the N-terminal globular LBD (Fig. 1B). Overall, the of 2.7 Å over 56 Cα atoms, with 13% sequence identity, including EphA2/ephrin-A1/5 heterodimers are very similar to known the cysteines involved in the disulphide bridges matching the Eph Eph/ephrin structures involving only the Eph LBD (4, 9). The sequences 201–247 and 230–260. The C-terminal half of the Eph high-affinity ligand/receptor interface centers around the G-H CRD comprises two β-strands and six tightly packed random loop of ephrin-A1 or -A5, which is inserted in a channel on the sur- coils, including four disulfide bridges, the first of which anchors face of EphA2 (Fig. 1B and Fig. S6). Four antiparallel β-strands the first residue of this half to the sixth CRD β-strand. It resem- define the two sides of the channel and two strands line its back. bles the TNF receptor CRD, closely matching the Death Recep- The ligand binds by attaching the side of its β-sandwich to the tor 5 (PDB ID 2H9G) with a Z score of 4.9, an rmsd of 2.4 over 46 α outside surface of the channel and inserting its long G-H loop into C atoms, and 17% sequence identity, including conservation of the channel, which then becomes buttressed by a receptor loop – – – three disulphide bridges (262 273, 276 290, and 293 307). The closing in from the top. The binding is dominated by van der Waals two CRD halves are tightly packed against each other, with the contacts between two predominantly hydrophobic surfaces, be- N- and C-terminal residues of the CRD occurring on opposite cause the ligand buries Gln109, Phe111, Thr112, Pro113, sides of the long axis of the domain. Apart from some negatively Phe114, Thr115, Leu116, and Gly117 (Fig. S6). Gln109 interacts charged patches, the CRD surface is predominantly neutral. not only with the sides of the channel but also with Phe100 and The N-terminal fibronectin-type-3 domain (nFN3) adopts a Pro101 from the long EphA2 loop at the top of the interface. typical immunoglobulin-like fold (Fig. 1A and Fig. S4A), most Pro113 is in direct contact with the Cys70-Cys188 disulfide bridge closely homologous to Integrin Beta-4 (1QG3) and Plectin-1 in EphA2. Adjacent to the channel/G-H-loop interactions, a sec- (3F7P) FN3 (rmsd ∼2.0 Å over 91 Cα atoms, DALI search). ond, structurally separate, contact area encompasses the ephrin- Of note, lack of significant binding clefts at either ends of the A1/5 docking site along the upper surface of the receptor. Here the domain suggests that nFN3 does not bind small-molecule ligands β (Fig. S4B). Although cFN3 has the same topology as nFN3, it is ephrin -sandwich interacts via a network of hydrogen bonds and structurally distinct with an rmsd of 7.7 Å extending over 68 Cα salt bridges (Eph-ephrin: Arg103-Glu119; Arg159-Asp86; Asp53- atoms (Fig. S4A): Its β3-β4 and β5-β6 loops are split open to Lys107) (Fig. S6). reveal an aromatic-lined cleft that might represent a membrane- surface binding pocket (Fig. S4B). Structural homologs to the Comparisons of Bound and Unbound EphA2-Ectodomain (ECD) and cFN3 include Neural Cell Adhesion Molecule 2, Fibronectin, EphrinA5. Interestingly, the overall structure of the ephrin-bound α EphA2-ECD is very similar to that of the unbound protein, with and Tenascin, with rmsd values from 1.6 to 2.0 over 82 C atoms. α A The elongated architecture of EphA2 is stabilized by extensive an rmsd between equivalent C positions of 0.9 Å (Fig. 1 ). In- interdomain interactions. The first and last LBD domain residues deed, the most significant conformational changes involve loops (25–27 and 199–200) together with the β5-β6 loop form an inter- within the ephrin-binding interface. The fact that there is little 2 action surface with the CRD, which buries 1;211 Å surface area conformational differences in the various crystal lattices implies and is stabilized by six hydrogen or salt bonds (Fig. S5A). Like- a very rigid rod-like architecture of the Eph ectodomain, at least 2 wise, a buried 701 Å interface and a salt bridge stabilizes the in the region encompassing the LBD, CRD, and nFN3, which is CRD–nFN3 interaction (Fig. S5B). The association between not modulated by ephrin binding. Likewise, ephrin-A5/1 does not nFN3 and cFN3 seems more flexible and, apart from a single hy- undergo significant structural rearrangements upon EphA2 bind- drogen bond, not stabilized by buried protein surfaces. ing and can be superimposed onto the structure of the unbound molecule with rmsd between equivalent Cα positions of ∼0.4 Å. High-Affinity Eph/Ephrin Heterodimer. Functional Eph/ephrin signal- The only significant conformational changes upon complex for- ing clusters assemble from high-affinity Eph/ephrin heterodimers, mation involve the rearrangement of the Eph-binding (G-H) which aggregate into heterotetramers and higher-order oligomers loop, which becomes structurally complementary to the ephrin- (1). The two EphA2/ephrin-A1 and one EphA2/ephrin-A5 com- binding channel on the Eph-LBD surface. plexes elucidated in our study show strikingly similar structural arrangements (Fig. 1A, alignment): The two EphA2/ephrin-A5 Eph/Ephrin Heterotetramers. The full EphA2/ephrin-A5 ectodo- heterodimers in the asymmetric unit of the corresponding crystals main complex forms a heterotetramer in solution (Fig. S7), and differ only by an rmsd of 0.3 Å between equivalent Cα positions. As crystal packing reveals two potential Eph/ephrin heterotetrameric expected, high-affinity EphA2/ephrin-A1/5 interactions involve assemblies (Fig. 2). These two heterotetramers are generated

Fig. 2. Eph/ephrin assemblies. Left, ribbon diagram of four molecules each of receptor and ligand are shown with receptors colored differently for clarity. Right, two assemblies are shown; the first (“heterotetramerization assembly”) is mediated only by the LBD, the second (“clustering assembly”) is mediated by both LBD and CRD. This figure was generated from the EphA2/ephrin A5 complex but also applies to the other, ephrin-A1, complex structures.

10862 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1004148107 Himanen et al. Downloaded by guest on September 24, 2021 LBD/ephrin-B2 structure (10), although the precise interfaces and interactions are quite distinct. Indeed, whereas Eph-Eph contacts are not observed in the EphB2-LBD/ephrin-B2 tetra- mer, both Eph/ephrin and Eph/Eph interactions facilitate EphA2/ephrinA1/5 tetramers (Fig. 3). Moreover, ephrin glycosy- lation units may also contribute to this interaction (Fig. 3C and PDB ID code 3MBW). The total surface area buried in the 2 EphA2/ephrinA1/5 heterotetramers (2;088 Å ) is somewhat smaller than the surface area buried in the EphB2/ephrin-B2 het- 2 erotetramers (2;532 Å ). Because the presence of Eph/ephrin contacts suggests that the formation of this heterotetrameric complex is dependent on ephrin binding, and because the architecturally similar B-class assembly has been referred to as “low affinity Eph/ephrin heterotetramers” (1), we will also refer to these EphA2/ephrinA1/5 and EphA2/EphA2 interfaces simply as the “heterodimerization” interfaces. This assembly is seen in all three of our EphA2/ephrin structures, even in the absence of the CRD domain. The second heterotetrameric assembly (Fig. 2B) is generated only via Eph-Eph interactions, suggesting that its formation is not dependent on ephrin binding. In fact, these interactions are conserved in all CRD-containing structures. We will therefore refer to these Eph/Eph interactions and interfaces as “clustering.” The EphA2/ephrin clustering interactions involve two distinct Eph/Eph interfaces—one in the LBD (Fig. 4 A and B) and one in the CRD (Fig. 4C). The LBD-mediated clustering interface is almost entirely po- lar, involving several salt bridges and hydrogen bonds (Fig. 4B). At the center of this Eph-Eph interface Lys116 from one of the Eph molecules makes a salt bridge with Glu117 and Asp104 from the other, whereas the side chain of Thr144 hydrogen-bonds with the main chain carbonyl of Pro147. Because the interface Fig. 3. Eph/ephrin heterotetramerization assembly. (A) Ribbon and (B)sur- is twofold symmetric, the reverse salt bridge and hydrogen bond face representations of the LBD-ephrin-A5 heterotetramer with each domain are also present (Fig. 4). The CRD-mediated clustering interface colored differently. (C) Detailed stereoscopic view of the 2∶2 assembly. involves a leucine-zipper-like assembly with a large number of van der Waals interactions. The hydrophobic zipper is formed C via two distinct EphA2/EphA2 interfaces and, when combined, by residues Pro221, Leu223, Leu254, Val255,and Ile257 (Fig. 4 ). Upon formation of the clustering interface, approximately would generate a continuous Eph/ephrin assembly (Fig. 2A). 2 850 Å of surface area is buried in each Eph molecule—large The first of these heterotetramers is generated by Eph-Eph enough for this interface to be considered biologically relevant. and Eph-ephrin interactions that encompass only Eph residues Moreover, the interaction surfaces are composed of conserved within the LBD. Indeed this Eph-LBD/ephrin heterotetramer residues across human Eph receptors, further underlying its is also observed in all our complex structures (Figs. 2B and 3). potential functional significance (Fig. S8). Architecturally, these EphA2-LBD/ephrinA1/5 complexes are Interestingly, both the Eph-Eph heterodimerization and BIOCHEMISTRY similar to the circular heterotetramers observed in the EphB2- clustering interfaces are present in the free Eph-ECD crystals

Fig. 4. Clustering assembly. (A) Ribbon display of the clustering assembly with each EphA2 molecule colored differently; (B) detailed stereoscopic view of the LBD- mediatedclustering interaction. Middle, ribbon diagramof two molecules of the unbound EphA2—dimer in light orange and pale green. (C) Detailed view of CRD- mediated clustering. Residues that mediate clustering are shown in stick format and labeled. Dashes represent van der Waals or hydrogen bond interactions.

Himanen et al. PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 ∣ 10863 Downloaded by guest on September 24, 2021 and in all complex structures (Eph-LBD-CRD-nFN3/ephrin-A5 and Eph-LBD-CRD/ephrin-A1). This observation suggests high affinity and that the continuous Eph-ECD/Eph-ECD assemblies are formed independent of ephrin binding at high enough Eph concentrations.

Role of the Eph/Eph Interfaces for Stability and Function of Eph/Ephrin Signaling Clusters. To compare the relative contributions of receptor–receptor interactions within the LBD and CRD of EphA2, we designed GFP-tagged EphA2 deletion mutants lacking either domain: ΔLBD is truncated between residues 28–198, and in ΔCRD residues 201–325 are replaced by a Gly-Ser-Gly-Ser lin- ker. We tested these mutants functionally, by transfecting mutant or WT EphA2-GFP cDNAs into HEK293 cells and analyzed their capacity to support ligand-independent Eph kinase activation that is induced upon transient overexpression. Immunoblot analysis of anti-GFP immunoprecipitated receptors demonstrated markedly reduced relative phosphorylation levels of both deletion mutants compared to WT EphA2, confirming the invol- vement of both domains in Eph-Eph clustering (Fig. 5A). Impor- tantly, ligand-independent activation of the ΔCRD mutant was most strongly affected, confirming its critical role in Eph signaling initiation as was suggested previously for EphA3 (3, 5). To evaluate the contribution of ligand-independent clustering to EphA2 activation, we interrogated EphA2 point mutants in a cell-based EphA2 phosphorylation assay. The mutants were designed to substitute hydrophobic residues in the leucine-zipper- like clustering interface predicted from the crystal structure with positively charged ones. They included Arg substitutions at positions Leu223 (single mutation), Leu223 and Leu254 (double mutation), or Leu223, Leu254, and Val255 (triple mutation). Analysis of HEK293Tcell clones stably expressing WTor mutant EphA2 by flow cytometry confirmed that all of these exogenous receptors were expressed on the cell surface at similar levels and were capable of ephrin-A5 binding (Fig. S9). Anti-phosphotyrosine Western blot analysis of ephrin-A5-Fc stimulated cells re- vealedsignificantly reducedactivation ofEphA2mutantcompared to WT-EphA2: As expected, double and triple substitutions affected activation stronger than single substitutions (Fig. 5B). In contrast, substitutions of charged residues within the LBD region of the clustering interface did not affect ephrin-induced EphA2 phosphorylation, suggesting that increased hydrophobicity Fig. 5. Cellular studies. (A) Immunoprecipitates from HEK293 cells trans- of the interface may compensate for loss of the salt bridge caused by Δ B fected with increasing amounts of GFP-tagged WT EphA2, LBD-EphA2, these mutations (Fig. 5 ). Together,these findings in live cells con- or ΔCRD-EphA2 were immunoblotted with α-EphA2 and α-phosphotyrosine firm the relevance of the Eph–Eph interactions observed in the antibodies. Densitometry quantified EphA2 phosphorylation relative to crystal structures for the formation of functional signaling clusters. EphA2 expression, by using samples with most similar EphA2 levels (lanes To directly assess the effect of EphA2 point mutations on 2, 5, and 8). (B) Activation of WT and mutated EphA2 in HEK293 cells by pre- clustering via the CRD but in the absence of LBD-mediated in- clustered ephrin-A5. Single ¼ L223R, double ¼ L223R;L254R, and triple ¼ teractions, we transfected HEK293Tcell clones, expressing WTor L223R;L254R;V255R. (C) HEK293 cells stably expressing WT or point mutated Δ Δ L-R-mutated EphA2 with GFP-tagged EphA2 lacking the ligand- EphA2, or control cells, were transfected with LBD-EphA2-GFP ( LBD) and binding domain (ΔLBD-EphA2-GFP). This “reporter” allowed stimulated with clustered ephrinA5-Fc. Protein A sepharose pull-downs of ephrinA5-Fc associated receptors were Western blotted with α-EphA2 and us to monitor coclustering via its CRD with ephrin-A5-bound α-GFP antibodies. The graph shows the amount of ΔLBD pulled down WT EphA2 or with the L-R-substitution mutants. Anti-GFP (anti-GFP blot) via association with full-length EphA2, relative to full-length Western blot analysis of ephrin-bound EphA2 demonstrated that EphA2, quantified by densitometry. (D) Parental HEK293 cells or derived the relative level of coprecipitated GFP-tagged reporter was clones stably expressing WT EphA2 or EphA2 point mutants as indicated were notably reduced in cells expressing L-R-substituted EphA2 transfected with ΔLBD-EphA2-GFP. Alexa594-ephrinA5 conjugated Dyna- receptors, indicating that CRD interface point mutations, most beads were added to the cells for 5 min before cultures were rinsed and prominently the triple Arg substitution, disrupt the ability for fixed for microscopy. Panels show representative images of EphA2-GFP CRD-mediated clustering (Fig. 5C). and Alexa594-ephrinA5 fluorescence, white arrows indicating beads with recruited EphA2-GFP, yellow arrows indicating beads in contact with cells We validated the functional importance of these findings by lacking recruitment. Insets show higher magnification images of boxed re- analyzing with confocal fluorescence microscopy in HEK293T gions. The average proportion of beads in contact with cells that recruited cell clones the recruitment of ephrin-binding-compromised ΔLBD-EphA2-GFP was determined for each cell line (WT EphA2, triple or ΔLBD-EphA2-GFP to full-length WT or mutant receptors. double mutant or control cells) and is shown in the graph (SEM). Localized recruitment and clustering of ΔLBD-EphA2-GFP to Alexa594ephrinA5-coated beads added to the cells was discern- Eph/Eph interaction via the intact CRD (Fig. 5D). By comparison, ible from GFP fluorescence marking the outline of the beads: cells expressing triple L-R EphA2 point mutants and, to a lesser Thus, in cells expressing full-length WT EphA2, but not in control extent, double Arg substitutions revealed significantly reduced (293) cells, bead-associated GFP fluorescence confirmed a robust GFP fluorescence around the ephrin-coated beads (Fig. 5D),

10864 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1004148107 Himanen et al. Downloaded by guest on September 24, 2021 confirming that residues 223, 254, and 255 of EphA2 mediate Summary receptor–receptor interactions and facilitate ephrin-independent Ephrin-dependent Eph receptor clustering and subsequent receptor clustering in intact cells. downstream signaling cause cytoskeleton reorganization that leads to the contact-dependent cell–cell attraction or repulsion Implications for Eph Signaling Initiation that is involved in tissue patterning (13). Our study reveals the Ligand-induced activation of receptor tyrosine kinases (RTKs) is structure of the functional Eph/ephrin assemblies at the cell a tightly regulated process that is not yet well understood at the surface and suggests a mechanism for Eph receptor clustering molecular level. It seems likely that the different RTK families and activation that involves bivalent homotypic interactions utilize different molecular mechanisms for coupling ligand between the LBD and CRD domains in neighboring receptors. binding with activation of the catalytic kinase domain. The recep- Previously, we demonstrated that the CRD plays a critical role tor structures reported here reveal that the extracellular region of in the formation of Eph/ephrin “signalosomes” (3, 5) and now Eph RTKs are composed of four individual structural domains reveal the specific molecular regions involved, as well as the un- that together fold into a unique rod-like structure. The Eph derlying structural mechanisms. Focusing on EphA2, we deter- ectodomain is rigid, and ligand binding does not cause significant mined a series of EphA2 ectodomain structures containing the conformational changes in any of the individual domains or CRD, including that of the full EphA2 extracellular region, both significant structural rearrangements between them. Thus, ligand- alone and in complex with A-class ephrin ligands. The same induced conformational changes in the receptor extracellular CRD-mediated EphA2 assemblies are observed under all differ- domain do not seem to be the molecular mechanism driving ent crystallization conditions and space groups, in both the pre- Eph signal transduction. sence and absence of bound ligand. Importantly, we document Our structures reveal that the Eph CRD is a unique protein- the physiological relevance of the proposed activation mechanism interaction/dimerization module, which cooperates with ligand- by using structure-based mutagenesis in a variety of cell-based dependent clustering to mediate the assembly of continuous signaling systems, including EphA2 phosphorylation and cell- oligomers, a process that could also happen independently of surface localization and clustering. ligand binding at high receptor concentrations. We confirmed Plasmid construction, host-cell growth, protein purification, this concept by structure-based mutagenesis in combination crystallization, structure determination, and cell-culture studies with cell-based signaling and receptor-visualization assays, also are described in SI Methods. explaining the previously observed recruitment of non-ligand- bound receptors into signaling clusters (5). The presence at the Note Added in Proof. cell surface of highly ordered receptor assemblies is a unique While this manuscript was under consideration, Y. Jones and feature of the Eph receptors and has not been observed in any colleagues published similar structure findings (23). other receptor kinase family. Another unique characteristic of Eph/ephrin signaling is the ACKNOWLEDGMENTS. We thank Christine Butler for cloning plasmids, Alma dependence of Eph activation and downstream signalling on Seitova for generating recombinant baculovirus, Linda Hii and Dorothea membrane-attached and preculstered ephrin ligands. Continuous Robev for generating EphA2 mutants, and Yehuda Goldgur for help with Eph/Eph and Eph/ephrin assemblies in our crystals therefore sug- data collection and analysis. This work was supported by National Institutes of Health Grants NS38486 (to D.B.N.) and GM75886 (to J.P.H.) and National gest that the function of ephrin ligands might be to increase local Health and Medical Research Council Grant 487922 (to M.L.). The NE-CAT receptor concentration so that ordered Eph/ephrin assemblies beamlines are supported by Award RR-15301 from the National Center for can be formed on the cell surface. Indeed, it has been shown that Research Resources at the National Institutes of Health. Argonne Advanced treatment of cells with antibodies recognizing the Eph ectodo- Photon Source use is supported by the United States Department of Energy under Contract DE-AC02-06CH11357. The Structural Genomics Consortium is main can also induce Eph receptor activation and initiation of a registered charity (#1097737) that receives funds from the Canadian downstream signaling (11). Institutes for Health Research, the Canadian Foundation for Innovation, Finally, EphA2 clustering has been associated with tissue Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, invasion by cancer cells. Indeed, nearly half of human breast Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & cancers overexpressed the receptor (12). Our studies confirm Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innova- BIOCHEMISTRY that, at high concentrations, Eph receptors could cluster indepen- tion Systems, the Swedish Foundation for Strategic Research, and the dent of ligand, potentially leading to transforming phenotypes. Wellcome Trust.

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Himanen et al. PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 ∣ 10865 Downloaded by guest on September 24, 2021