Insights Into Eph Receptor Tyrosine Kinase Activation from Crystal Structures of the Epha4 Ectodomain and Its Complex with Ephrin-A5

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Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5

Kai Xua,1, Dorothea Tzvetkova-Robeva,1, Yan Xua, Yehuda Goldgura, Yee-Peng Chanb, Juha P. Himanena, and Dimitar B. Nikolova,2

aStructural Biology Program, Memorial Sloan–Kettering Cancer Center, New York, NY 10065; and bDepartment of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814

Edited* by Dinshaw J. Patel, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved July 24, 2013 (received for review June 10, 2013) Eph receptor tyrosine kinases and their ephrin ligands mediate cell cavity on the surface of the receptor to form high-affinity 1:1 signaling during normal and oncogenic development. Eph signal- ligand–receptor heterodimers (15). The ligand-binding cavity of ing is initiated in a multistep process leading to the assembly of the receptor consists of loops DE, FG, and JK, which were higher-order Eph/ephrin clusters that set off bidirectional signal- shown to exhibit various amounts of conformational flexibility, ing in interacting cells. Eph and ephrins are divided in two sub- depending on the subclass and the individual receptor. Several classes based on their abilities to bind and activate each other and structures of the EphA4 ligand-binding domain (LBD) alone and on sequence conservation. EphA4 is an exception to the general in complex with its ligands have addressed its unusually high li- rule because it can be activated by both A- and B-class ephrin gand promiscuity (8, 16–18). ligands. Here we present high-resolution structures of the com- More recently, the crystal structures of the EphA2 ectodomain plete EphA4 ectodomain and its complexes with ephrin-A5. The (ECD) bound to ephrin-A1 and ephrin-A5 suggested a structural structures reveal how ligand binding promotes conformational basis for the formation of Eph/ephrin signaling clusters at the changes in the EphA4 ligand-binding domain allowing the forma- interfaces of interacting cells (19, 20). Specifically, it was pro- tion of signaling clusters at the sites of cell–cell contact. In addition, posed that these signaling clusters nucleate from high-affinity BIOCHEMISTRY the structural data, combined with structure-based mutagenesis, re- Eph/ephrin heterodimers, which assemble into heterotetramers – veal a previously undescribed receptor receptor interaction that further aggregate into higher-order oligomers. To un- between the EphA4 ligand-binding and membrane-proximal fi- fi derstand the structural basis of EphA4 ligand-induced clustering bronectin domains, which is functionally important for ef cient and explore underlying receptor–receptor and receptor–ligand receptor activation. interactions, we determined and report here the structures of the complete EphA4 ECD, alone and in complexes with its ephrin- crystallography | phosphorylation | protein | transmembrane A5 ligand.

ph receptors, the largest family of receptor tyrosine kinases, Results and Discussion Eand their ephrin ligands regulate a variety of cell–cell inter- Structure of the EphA4 ECD. The structure of the complete EphA4 actions during development and in the adult organism (1–3). receptor ECD (residues 22–543) (Fig. 1A) was determined by Because both receptors and ligands are membrane-bound, their molecular replacement at 2.08-Å resolution and refined to an interactions at sites of cell–cell contact initiate unique bidirectional signaling cascades (4). The signaling downstream of Significance the Eph is referred to as “forward,” and the signaling down- “ ” stream of the ephrins is referred to as reverse. Our paper describes the high-resolution structures of the The 16 Eph receptors and 9 ephrins are divided into two fi complete EphA4 receptor tyrosine kinase ectodomain and its subclasses based on sequence homology and binding af nities. fi – complex with ephrin-A5. These structures reveal for the rst Receptor ligand binding within each subclass is fairly pro- time how ephrin binding promotes Eph conformational miscuous, whereas cross-subclass signaling happens rarely (5). changes allowing for the formation of ordered signaling clus- The best-known exception to this general rule is EphA4, which ters at the sites of cell–cell contact. In addition, the structural has long been known to bind and be activated by both A- and data, combined with structure-based mutagenesis and cell- B-class ligands (6, 7). Because of its unique properties, EphA4 is based assays, reveal a previously undescribed receptor–re- an attractive target for studying the structural details of subclass ceptor interaction between the EphA4 ligand-binding and specificity (8). fi fi membrane-proximal bronectin domains, which is functionally Eph and ephrins were originally identi ed as axon guidance important for efficient Eph activation. Based on the data we molecules; they have now been implicated in a vast array of cell suggest a mechanism for Eph preclustering and recruitment of communication events. Those include bone morphogenesis and unliganded Eph into existing Eph/ephrin signaling clusters. homeostasis, immunological and inflammatory host responses, ’ stem cell plasticity, learning and memory, and Alzheimer s dis- Author contributions: K.X., J.P.H., and D.B.N. designed research; K.X., D.T.-R., Y.X., Y.G., ease (9). However, currently, the most intensely studied function Y.-P.C., and J.P.H. performed research; K.X., D.T.-R., Y.X., J.P.H., and D.B.N. analyzed data; of the Eph/ephrin system is that during development and pro- and K.X., J.P.H., and D.B.N. wrote the paper. gression of different cancers. Many A- and B-class receptors The authors declare no conflict of interest. were shown to be overexpressed in various tumor types (10–13) *This Direct Submission article had a prearranged editor. and to regulate critical steps of blood vessel formation (vasculo- The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org genesis) and remodeling (angiogenesis) and hence tumor growth. (PDB ID codes 4M4P and 4M4R). Structural studies on the minimal binding domains of Eph 1K.X. and D.T.-R. contributed equally to this work. receptors and ephrins revealed important details of receptor– 2To whom correspondence should be addressed. E-mail: [email protected]. ligand recognition (2, 14). The initial binding force comes from a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. penetration of a long, hydrophobic ephrin loop into a hydrophobic 1073/pnas.1311000110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1311000110 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 Fig. 1. Structures of the EphA4-ECD and the EphA4-ECD/ephrin-A5 complexes and comparison with EphA2. (A)(Left) Structure of the unbound EphA4-ECD (blue). The glycosylation moiety at N408 is drawn as spheres. (Center) Structure of the EphA4-ECD (green and magenta) in complex with ephrin-A5 (cyan and yellow). Two EphA4 and two ephrin-A5 molecules are shown, connected via the clustering interface. (Right) Two EphA4 and two ephrin-A5 molecules from the same crystal connected via the heterotetramerization interface. Magnified views of the clustering and heterotetramerization interfaces are shown in the rectangular areas defined by the dashed lines, with the contacting residues drawn in stick and labeled. (B) (Left) Structure of unbound EphA2-ECD (green) with the individual domains indicated and the glycosylation moiety drawn (19). (Center) Structure of the EphA2-ECD (green and magenta) bound to ephrin-A5 (cyan and yellow). Two EphA2 and two ephrin molecules are shown, connected via the clustering interface. (Right) Two EphA2 and two ephrin molecules from the same crystal connected via the heterotetramerization interface. CRD, cysteine-rich domain; FN1, fibronectin 1; FN2, fibronectin 2; LBD, ligand-binding domain.

Rfree of 22.5% (Table S1). It reveals an extended arrangement of respectively) (19, 20). As in all characterized Eph/ephrin com- tightly packed domains spanning 150 × 50 × 55 Å. The overall plexes, the high-affinity ligand/receptor interface (total buried structure of unbound EphA4 is similar to that of EphA2 (19) area of ∼2,360 Å2) centers around the GH loop of ephrin-A5, as illustrated in Fig. 1 A and B,andthetwoECDscanbe which is inserted in a channel on the surface of EphA4. This superimposed with an rmsd between equivalent Ca positions of binding is dominated by van der Waals contacts between two ∼2.7 Å. The Eph ECD contains an N-terminal LBD (rmsd be- largely hydrophobic surfaces. Adjacent to the channel/GH loop tween the EphA2 and EphA4 structures for this region is 0.7 Å), interactions, a second, structurally separate, polar contact area followed by a cysteine-rich domain (CRD) (rmsd between the encompasses the ephrin-docking site along the upper surface of EphA2 and EphA4 CRD is 0.8 Å), two fibronectin type III the receptor. The specific interaction residues in the high-affinity repeats (FN1 and FN2) (rmsd between the EphA2 and EphA4 is “heterodimerization interface” (24 EphA4-LBD residues and 22 1.0 Å for FN1 and 0.8 Å for FN2). The Eph CRD, encompassing ephrin-A5 residues) are listed in Fig. S2A. ∼114 amino acids (residues 201–314) immediately C-terminal to EphA4 clustering. Upon ephrin binding, Eph/ephin clusters form the LBD, can be subdivided into two domains: an N-terminal between interacting cells via extensive Eph/Eph, Eph/ephin, and domain similar to complement control module/short comple- ephrin/ephrin interactions. The crystal structure of the EphA4/ ment regulator domain and a TNF receptor-like cysteine-rich ephrin-A5 complex visualizes these interactions. Specifically, module (19, 20). The LBD, CRD, and FN1 fold into a rigid, rod- there are two distinct EphA4/ephrin-A5 heterotetrameric for- like structure with little interdomain flexibility. The connection mations generated via two distinct EphA4/EphA4 interfaces between FN1 and FN2, on the other hand, is flexible, and these that, when combined, generate a continuous EphA4/ephrin-A5 domains are positioned differently in the ligand-bound and un- assembly (Fig. S1). bound structures (Fig. 1A). EphA4 is glycosylated on positions The first of these heterotetramers (Fig. 1B, Right) is generated N235, N340, and N408 as predicted, and the carbohydrate was by Eph–Eph interactions that encompass only residues within the built in the model. LBD. This interface is simply referred to as “heterotetramerization interface” (15, 19). It is relatively small, burying a total of Structure of EphA4/Ephrin-A5 Complex. The structure of the EphA4 ∼620 Å2, and contains van der Waals contacts, hydrogen bonds, ECD (residues 22–543) bond to ephrin-A5 (resides 24–196) (Fig. andsaltbridgesbetweensixEphresiduesineachinteracting 1A) was determined by molecular replacement at 3.15-Å reso- partner as illustrated in Fig. 1A, Right, Inset,andFig. S2B. lution and refined to an Rfree of 28.5% (Table S1). The second EphA4/ephrin-A5 heterotetrameric assembly (Fig. High-affinity Eph/ephrin heterodimer. The structure of the EphA4- 1B, Center) is generated via Eph–Eph interactions involving the ECD/ephrin-A5 complex confirms the model that functional LBD and the CRD. It is referred to as “clustering interface” Eph/ephrin signaling clusters assemble from high-affinity Eph/ (19). The LBD region of the clustering interface, which involves ephrin heterodimers, which aggregate into heterotetramers and the GH loop, beta strand J, and the JK loop, is mostly polar and higher-order oligomers (2) (Fig. S1). Indeed, the EphA4/ephrin- includes hydrogen bonds and three salt bridges as illustrated in A5 heterodimer is overall quite similar to the EphA2/ephrin-A1 Fig. 1A, Center, Inset,andFig. S2C. The CRD region of the and EphA2/ephrin-A5 structures (rmsd values of 2.5Å and 4.3Å, clustering interface involves a leucine zipper-like assembly with

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311000110 Xu et al. Downloaded by guest on September 23, 2021 a large number of van der Waals interactions. The hydrophobic now reveals that ephrin-A5 binding also directly facilitates the zipper is formed by residues L226, Y246, L254, and V255. formation of Eph/ephrin clusters by inducing conformational Overall, the clustering interface buries 1,780 Å2 (Fig. S2C). changes in the LBD of EphA4. Specifically, as illustrated in Fig. 2, The EphA4/ephrin-A5 heterotetramerization and clustering upon ephrin-A5 binding, the EphA4 JK and DE loops undergo interfaces are analogous to the interfaces observed in the EphA2/ major conformational rearrangements: both the Eph DE beta ephrin-A5 structures (19), which are shown, for comparison, in hairpin and the JK loop bend toward the incoming ephrin GH Fig. 1B, Right and Center, respectively). There are differences loop to form an intimate interaction interface which leads to between the heterotetramerization and clustering interfaces ob- large structural changes in this Eph region, including the for- served in the EphA4/ephrin-A5 and the EphA2/ephrin-A5 struc- mation of a new alpha helix in the JK loop. These rearrange- tures, most notably that the heterotetramerization interface is much ments result in the formation of complementary interaction smaller in the EphA4/ephrin-A5 structure (∼620 Å2 vs. ∼2,000 Å2 surfaces in adjacent Eph receptors, facilitating the Eph/Eph in the EphA2/ephrin-A5 structure) and that it only involves Eph– interactions at the clustering interface (Fig. 2, Right Inset). Un- Eph interaction. Nevertheless, as can be seen in Fig. 1, the overall bound EphA2 would not be able to form a similar clustering heterotetramerization and clustering arrangements are similar, and interface because of steric overlap of the JK loop (residues D151 they lead to analogous architectures of the continuous EphA4/ and E152) in one receptor with the beta strand J (residues K144, ephrin-A5 and EphA2/ephrin-A5 assemblies (Fig. S1). D146, and T147) of its neighbor. As mentioned above, both of Eph activation as an ordered multistep process. The structures repor- these regions are integral parts of the clustering interface in the ted here, combined with the previously reported EphA2/ephrin- ligand/receptor complex. The same ligand-induced conforma- A5 and EphA2/ephrin-A1 structures (19, 20), support a “seed- tional change in the EphA4 LBD was also observed in the studies ing” mechanism for the assembly of Eph/ephrin clusters: First, reporting the structures of the EphA4-LBD and the EphA4- upon cell–cell contact, high-affinity 1:1 Eph/ephrin heterodimers LBD/ephrin-A2 and ephrin-B2 complexes (8, 16, 18), but be- are formed. Then, the ligand-bound Eph receptors use two sep- cause the isolated Eph LBD does not cluster, its significance was arate interfaces for the assembly of signaling-competent clusters not appreciated. (see Fig. 3). The heterotetramerization interface is responsible for receptor dimerization and formation of 2:2 ligand–receptor het- LBD-FNIII Interactions in the Unbound EphA4. The herein reported erotetramers (15). Once Eph receptors are in ligand-induced close structure of the unbound EphA4 ECD reveals a previously contact, they can independently use the clustering interface to undescribed interaction between the EphA4 LBD and the FNIII bind other receptor dimers (or 2:2 Eph/ephrin complexes), thus region of a neighboring EphA4 molecule (Fig. 3). The primary BIOCHEMISTRY forming “trimers of receptor dimers.” This process would then go binding is between two surface loops of the N-terminal EphA4 on until large-sized assemblies, consisting of hundreds of recep- FNIII repeat (FN2) and the CD, DE, EF, GH, and JK loops and tors, are formed. Fig. S1 shows the packing of the EphA4/ephrin- D, E, G, and M strands of the EphA4 LBD. It is noteworthy that A5 complexes in the crystals, illustrating a continuous 2D EphA4/ this LBD surface partially overlaps with the surface that binds ephrin-A5 assembly. The precise size or composition of these the ephrin ligands, and Fig. 3B illustrates the similarities between assemblies in vivo, however, is still unknown. the EphA4-LBD/ephrin-A5 and EphA4-LBD/EphA4-FN2 binding. A second adjacent Eph–Eph contact area involves CRD res- Ephrin Binding Induces Conformational Changes in the EphA4 LBD, idues forming a salt bridge (Fig. 3A, Lower Inset). The total binding Allowing for Formation of Eph/Ephrin Signaling Clusters. Based on interface is large (2,460 Å2) and includes multiple van der Waals previous analysis of EphA2-ECD/ephrin structures (19), it has contacts, hydrogen bonds, and salt bridges (all interacting residues been suggested that the role of the ligand is to increase the local are listed in Fig. S2D), indicating that it is biologically relevant. concentration of the receptors on cell surface above the critical Moreover, an Eph LBD-FN2 interaction is also present in the value for efficient clustering. The EphA4/ephrin-A5 structure structure of the unbound EphA2 ECD (19), but the interface, as

Fig. 2. Ligand-induced conformational changes in the EphA4 LBD facilitate receptor clustering. The LBDs of two Ephs (green and magenta) bound to two ephrins (yellow and cyan) in the EphA4-ECD/ ephrin-A5 complex are shown in a view centered on the LBD region of the heterotetramerization interface (as in Fig. 1A, Center, top of the rectangular Inset). Superimposed on the magenta ephrin-bound EphA4 LBDs is the structure of the LBD of the unbound EphA4 (blue). The superimposed area is magniﬁed and shown in the rectangular area de- ﬁned by the dashed line. Upon ligand engagement, the JK loop of EphA4 changes conformation (under- goes coil-to-helix transition), and both the JK and DE loops move toward the ephrin-A5 GH loop, frap- ping the entrance of the pocket for the GH loop. These rearrangements create the complementary interacting surfaces forming the Eph–Eph heterotetramerization interface (Left Inset). Ligand-free EphA4 (Right Inset) cannot participate in similar Eph–Eph heterotetramerization interactions because of a steric clash mediated by the unbound conformation of the EphA4 JK loop. The clashing residues are shown in stick (Right Inset).

Xu et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 Fig. 3. LBD–FNIII interactions in the unbound EphA4. (A) Two EphA4 molecules (orange and blue) interacting in the crystals of ligand-free EphA4-ECD. The main Eph–Eph interface, which presumably mediates EphA4 preclustering, involves the LBD of one EphA4 molecule interacting with the membrane-proximal FNIII domain (FN2) of a neighboring EphA4 molecule. A second, smaller Eph–Eph interface involves residues in the Eph CRD. The two interface regions are magniﬁed and shown in the rectangular area deﬁned by the dashed line, with the contacting residues shown in stick and labeled. The glycosylation moiety is shown as spheres. (B)(Left) EphA4-LBD (magenta) bound to ephrin-A5 (yellow) in the crystals of the EphA4-ECD/ephrin-A5 complex. (Center) EphA4-LBD (blue) bound to the EphA4-FN2 of a neighboring molecule (orange) in the crystals of unliganded EphA4-ECD. (Right) EphA2-LBD (green) bound to the EphA2-FN2 of a neighboring molecule (red) in the crystals of unliganded EphA2-ECD (19).

illustrated in Fig. 3B,ismuchsmaller(980Å2 total buried area), (using anti-Fc mAb; Materials and Methods) was added to HEK293 and it was not discussed in the corresponding publication. cells, transfected with either WT EphA4 or the EphA4 mutants, We used structure-based mutagenesis, in combination with in 30 min before lysis. Anti-phosphotyrosine immunoprecipitates vitro binding assays and a cell-based EphA4 activation assay, to from lysates were analyzed by Western blotting with an anti- evaluate the significance of this Eph LBD-FNIII interface. EphA4 mAb. The overall EphA4 expression levels were evalu- Specifically, we generated mutations in FN2 (R454A/Y455A, or ated by probing the total cell lysates with anti-EphA4. The M2) and the CRD (E238A, or M3) designed to weaken the Eph/ phosphorylation levels (corrected for EphA4 expression level) Eph interface by abolishing hydrogen bonds and salt bridges as for WT and mutant EphA4 are shown in Fig. 4 as a function of well as a mutation (N504D/T507D, or M1) designed to strength- the concentration of ephrin-A5. The results document that the en the Eph/Eph interface by introducing two additional salt FN2–LBD interaction is functionally relevant, regulating the bridges. Mutations of these surface-exposed EphA4 residues level of EphA4 activation following ephrin stimulation. Muta- would not affect ligand binding because these regions are neither tions that weaken the unliganded Eph–Eph interactions decrease part of the Eph/ephrin heterodimerization interface nor part of EphA4 activation, whereas mutations that strengthen the FN2– the Eph/Eph heterotetramerization and clustering interfaces LBD interface increase EphA4 activation. Indeed, the M1 Eph (see above). mutant exhibits greatly elevated basal phosphorylation levels Pull-down protein-binding assays shown in Fig. 4B docu- even in the absence of ligand stimulation. This suggests that mented that the M2 mutation decreases LBD binding to the the FN2–LBD interaction mediates ligand-independent Eph mutated FN2, whereas the M1 mutation increases it (M3 was not association or “preclustering,” thus presumably facilitating tested in this assay because the mutation is outside the LBD). fast and efficient receptor activation upon contact with ligand- The mutations were then introduced into full-length EphA4, expressing cells. and the engineered proteins were transfected into HEK293 cells. Similar LBD–FN2 interactions have not been reported for EphA4 activation was measured by quantifying the phosphory- B-class ephrins, and the potential LBD–FN2 interaction as ob- lated fraction of EphA4 following stimulation with ephrin-A5. served in the EphA2 structure is significantly weaker than the one Specifically, varying amounts of preclustered ephrin-A5-Fc in EphA4. Thus, this strong, unliganded Eph–Eph interaction

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311000110 Xu et al. Downloaded by guest on September 23, 2021 (25, 26) to regulate, for example, Eph receptor phosphorylation (27) and pathway selection by spinal motor axons (28). Conclusion The herein reported structures of the complete EphA4 ECD and its complex with ephrin-A5 reveal several important character- istics of Eph signaling: (i) They confirm the general mechanism of assembly of Eph/ephrin signaling clusters, suggested from the EphA2 ECD/ephrin structures, which uses three types of Eph/ ephrin interfaces: a high-affinity Eph/ephrin heterodimerization interface and lower-affinity heterotetramerization and clustering interfaces. (ii) They provide direct visualization of how ephrin binding induces conformational changes in the Eph LBD facilitating the clustering interactions required for formation of the expansive Eph/ephrin signaling centers at the sites of cell–cell contact. (iii) They reveal a previously undescribed homotypic interaction between unliganded neighboring Eph molecules which could facilitate both Eph preclustering before ephrin binding and an increase in the size of the signaling clusters via recruitment of unliganded Ephs, thus promoting efficient and potent signaling initiation. Materials and Methods Fig. 4. Mutations in the EphA4 preclustering interface functionally affect Cloning and Mutagenesis. Human EphA4 ECD (residues 22–543) and human forward signaling. The effects of various mutations in the EphA4 pre- ephrin-A5 (residues 24–169) were cloned into a modified pAcGP67 baculo- clustering interface were evaluated in pull-down and kinase activation virus expression vector (BD Bioscience), with GP67 secretion signal and hu- assays. Mutants M3 and M2 (E238A and R454A/Y455A, respectively) weaken man Fc fragment as C-terminal tag. Recombinant baculovirus was generated the interface, whereas mutant M1 (N504D/T507D) strengthens the interface. by cotransfecting the expression plasmid along with linearized BaculoGold (A) Pull-down assay between LBD and mutant FN2 of EphA4: The Fc-tagged DNA (Pharmingen) into SF9 cells. The human EphA4 full-length protein and BIOCHEMISTRY EphA4 FN2 domains with mutations that were designed to either enhance the mutants were cloned into a pcDNA3.1 + hygromycin vector (Invitrogen) (M1) or weaken (M2) the preclustering interface were used to pull down the for stable expression in a HEK293 cell line. Mutations were introduced by EphA4 LBD. WT EphA4 FN2 was used as positive control. The M1 FN2 binds site-directed mutagenesis (Stratagene) and were sequenced. to the LBD better than WT, whereas the M2 FN2 binding is weaker than WT. (B) EphA4 LBD binding to FN2 domains of different Ephs and to ephrins: The Protein Expression and Crystallization. Hi5 cells were infected with passage 3 EphA4-LBD was tested in a pull-down assay with Fc-tagged FN2 of different baculoviruses (for EphA4 or ephrin-A5 expression) at a multiplicity of in- Eph family members as labeled above lanes 1–4. EphA4 LBD binds only the fection of 10. The medium containing the secreted fusion proteins was EphA4 FN2. Fc-tagged ephrin-A1 and ephrin-B2 were used as positive control collected 72 h postinfection and loaded onto a Protein A-Sepharose affinity (lanes 7–8), confirming that ephrin-A1 binds EphA4 better than ephrin-B2. column. Recombinant proteins were eluted with low-pH buffer containing (C) Cell-based EphA4 kinase activation assay: HEK293 cells stably expressing 150 mM NaCl and 100 mM glycine (pH 3.0). The Fc tag was cleaved by WT or mutant full-length EphA4 were treated with increasing concen- thrombin proteolysis and removed by Protein A-Sepharose. Both proteins trations of ephrin-A5-Fc for 10 min and harvested. The activated (phos- fi fi phorylated) EphA4 in the cell lysate was immunoprecipitated with an were further puri ed by gel ltration chromatography. EphA4 was con- anti-phosphotyrosine antibody and was then detected with an anti-EphA4 centrated to 10 mg/mL in Hepes-buffered saline (HBS) and crystallized by antibody in Western blots. The Western blot films were scanned, and the hanging-drop vapor diffusion at room temperature against a well solution signal intensity was quantified and plotted. The amount of phosphorylated of 22% PEG 400, 0.1 M 2-(N-morpholino)ethanesulfonic acid (Mes) buffer pH EphA4 was normalized for the total EphA4 amount in the total cell lysates. All intensity readings were calculated as the ratio to the basal (ligand- independent, resting) EphA4 phosphorylation level. The experiments were performed in triplicate, and the error bars are shown in the plot.

might be unique to EphA4 and might be responsible, at least in part, for the ligand promiscuity of the receptor—EphA4 is more preclustered than other Ephs and easier to activate. Eph signaling clusters often span cell surface areas larger than the areas of immediate Eph/ephrin contact (21, 22). In addition, it has been shown that Ephs that are not ligand-bound can sometimes be recruited to the Eph/ephrin clusters (21, 23, 24). The observed LBD–FNIII interactions provide a structural basis for the observed functional participation of unliganded Ephs in the Eph/ephrin signaling assemblies, and Fig. 5 presents a sche- – matic of two interacting cells where homotypic Eph LBD FNIII Fig. 5. The homotypic, head-to-tail Eph–Eph (LBD–FN2) interactions ob- interactions increase the size of the signaling site on the Eph- served in the crystals of unbound EphA4-ECD presumably represent Eph expressing cell by recruiting unliganded Ephs to the Eph/ preclustering interactions and could also explain the observation that Ephs ephrin complexes. form clusters well beyond the area of immediate ephrin contact (22). Pro- The observed head-to-tail homotypic interactions between vided is a schematic representation of two interacting cells where the Eph EphA4 molecules represent yet another example of the subtle clusters on the Eph-expressing cells are larger than the Eph/ephrin contact area, mediated by interactions between the LBDs of ephrin-free Ephs and ways Eph/ephrin signaling is controlled. Such interactions might the FNIII regions of their neighbors. The Eph molecules are colored in green, work in concert with in cis Eph/ephrin interactions that have and the ephrins are in red. Phosphorylated intracellular tyrosines are shown been reported in some cell types coexpressing Eph and ephrins as small circles.

Xu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 6.0, 3% DMSO. For production of the EphA4/ephrin-A5 complex, purified (pH 7.4), 150 mM NaCl, 1% (wt/vol) Nonidet P-40, and 1 mM EDTA. Activated EphA4 and ephrin-A5 were mixed in a 1:2 molar ratio and incubated on ice receptor was immunoprecipitated with anti-phosphotyrosine antibody for 1 h. The complex was purified on a SD200 gel filtration column (GE (Upstate Biotechnology) and Protein A-Sepharose beads, resolved on SDS/ Healthcare) and concentrated to 10 mg/mL in HBS. The complex was crys- PAGE, and blotted onto PVDF. Membranes were then blotted with anti- tallized by sitting-drop vapor diffusion at room temperature against a well EphA4 antibody (R&D Biotechnology). The Western blot films were scanned solution of 2.0 M sodium acetate, 0.1 M sodium acetate buffer pH 4.5. For and quantified by spot densitometry using ImageQuantTL software (GE data collection, the crystals were frozen in a cryobuffer containing an ad- Healthcare Biosciences). All intensity readings were normalized for the ditional 25% (vol/vol) glycerol. X-ray diffraction data were collected at EphA4 amount in total cell lysate and calculated as the ratio to the WT basal beamline 24ID-C (Northeastern Collaborative Access Team, Advanced Pho- activity (ligand-independent phosphorylation level). The experiments were ton Source) and processed with HKL2000 (29). Both structures were determined by molecular replacement with EphA2 [Protein Data Bank (PDB) ID done in triplicate, and the error bars are shown in Fig. 4. is 3FL7] (19) and EphA4/ephrin-B2 complex (PDB ID is 2WO3) (16) as search templates, using the Phaser program (30) in the Phenix suite (31). Sub- Pull-Down Experiments. Pull-down experiments were performed essentially as sequent refinement proceeded with iterative rounds of model adjustments, described earlier (5). Briefly, 5 μg of recombinant EphA4 LBD were incubated using the programs Coot (32) and PhenixRefine (31). Crystallographic details with the Fc-tagged recombinant ephrins or with isolated Eph FN2 domains and statistics are listed in Table S1. at room temperature for 30 min in 500 μL binding buffer containing 20 mM

Hepes (pH 8.2), 150 mM KCl, and 2 mM MgCl2. Protein A-Sepharose Fast Cell Manipulations and Transfections. HEK293 was grown in DMEM (Invi- Flow beads (GE Healthcare) were washed with the binding buffer, added to trogen) supplemented with 10% (vol/vol) FBS, 100 units/mL penicillin, and the reaction mixture, and mixed at room temperature for 30 min. The beads 100 μg/mL streptomycin. Cells were consistently transfected at 80–90% were then harvested by centrifugation and washed twice with 500 μLofthe conﬂuence in six-well plates using Lipofectamine 2000 (Invitrogen). binding buffer, and the bound proteins were separated on a 10–20% polyacrylamide gel. Cell-Based EphA4 Kinase Activation Assay. For the EphA4 activation assays, HEK293 cells were stably transfected with full-length EphA4 or Eph mutants Illustrations. Figures were prepared using Adobe Illustrator (Adobe). All with alterations in the preclustering interface (M1, N504D/T507D; M2, R454A/ molecular representations were produced with PyMOL (33). Y455A; and M3, E238A). Clones with similar expression levels, tested by anti-EphA4 antibody in Western blots, were chosen for the assay. EphA4- ACKNOWLEDGMENTS. We thank Dr. Rüdiger Klein for protein expression expressing cells were then challenged with ephrin-A5-Fc, which was clustered constructs and advice, Momchil Kolev for technical support, Marina Himanen using anti-human IgG antibody at 25 nM concentration. After 10 min of for hand-drawn illustrations, and K. R. Rajashankar for help with data col- incubation with ligand, cells were washed with PBS and harvested. Total cell lection. This research was supported by National Institutes of Health Grant lysate was prepared by lysing cell pellets in a buffer containing 20 mM Hepes R01NS038486 (to D.B.N.).

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