2. MIRAR LA INVESTIGACIÓN DE OTRO MODO
2.2 La construcción de la apuesta metodológica
EphBs/ephrinB recognition and subclass specificity. The recognition specificity of
ephrinB2 and EphB2 is ensured by ligand induced receptor folding (induced fit mechanism). While binding, the dimerization interface centers on the long G-H loop of ephrinB2, which gets buried in a channel on the surface of EphB2. This process is thermodynamically driven by the hydrophobic effect. Loops lining the inside of the channel in EphB2 undergo a secondary-structure rearrangement from disordered to structured, thereby building an extensive interaction surface that is complementary to the ligand G-H loop. The conformational change in EphB2 is strictly localized to the interaction interface and most likely does not involve the intracellular part of the receptor [70,80]. The crystal structure from EphB2/ephrinB2 also gave insights into the molecular basis for subclass specificity [79]. The ligand-receptor high-affinity dimerization interface contains bulky and small polar and hydrophobic side chains on either side. In EphBs these residues are positioned in order to find an energetically favorable conformational state, whereas in EphAs, the side chains are shuffled in position, and therefore would produce an energetically unfavorable conformational state upon ephrinB-binding (e.g. polar residues to face hydrophobic ones). The H-I loop guarantees the formation of tetramers of receptor belonging to the same subclass. However, it is not sure, if a heterogeneous expression of different subclass members would cause heterogeneous clusters which contain more than one type of ligand and/or receptor.
Other structural studies of EphB4/ephrinB2 further suggested a single aa (Leu95) to be responsible for defining ligand selectivity of EphBs. This selectivity could be altered via protein engineering approaches [83]. On the ligand side, further structural studies of unbound ephrinB1 and ephrinA5 underlined the importance of the G-H loop for receptor recognition but also pointed out the uniqueness in the molecular mechanism for receptor-ligand specificity of each cognate pair [84,85].
10 EphB2/ephrin–A5 EphA2/ephrin–A1 EphB2/ephrin–B2 EphB4/ephrin–B2
A
C
D
E
X
FNIIIa EphA/B FNIIIb LBD sushi RBD ephrinA/BX
EGF high-affinity dimerization low-affinity tetramerization 3rd interface - Cys-rich/RBD U = LBD-sushi A = high-affinity dimerization (RBD-LBD) B = LBD-LBD C = RBD-LBD D = sushi-sushi E = FNIIIa-FNIIIain-register LBD-LBD/sushi-sushi array A B C C D E
G
H
I
F
1:1 heterodimer 2:2 heterotetramerstaggered LBD-sushi array U higher-order oligomerization G–H loop J–K loop D–E loop N-terminal C-terminal H-I loop
B
tetramerization interface dimerization interface11
Fig. 1.3 Eph/ephrin complex formation and cluster architecture.
(A) Comparison of the structures from the unligated EphA2 (cyan) and EphB2 (magenta) ligand-
binding domain. Secondary structure elements are labeled on the ribbon diagram (arrows). Both the EphA2 and EphB2 structure are close to congruence. The main structural differences between EphA2 and the EphB receptors are the conformations of the D-E, J-K and H-I loops. The H-I loop length is the only clear feature that distinguishes the sequences of the A- and B-class molecules (with EphA receptors H-I loop four residues shorter in length). In the EphB2/ephrinB2 crystal structure, this H-I loop is involved in forming the low-affinity tetramerization interface [79]. The J-K and D-E loops are responsible for forming the ligand-binding cleft. Although in the unligated B-class receptors they adopt more open conformations [86], in EphA2 they form a compact ligand-binding channel - even in the absence of the ligand [87]. The tetramerization and dimerization interface is indicated by stippled lines [79]. (B) Conformational changes in A- and B-class Eph receptors upon ephrin binding. Left: comparison of the structures of unligated EphB2 (yellow), ephrin-bound EphB2 (green), and a 12-mer antagonistic peptide-bound EphB2 (magenta) [87,88]. Right: comparison of the structures of unligated (green) and ephrin-A1 bound (blue) EphA2. In contrast to EphA2, EphB2 undergoes significant structural rearrangements upon ligand/peptide binding (see arrows). (C) Comparison of the structures of the known A- and B-class Eph receptor/ephrin complexes except for EphA4/ephrinB2. EphB2: Val27-Arg207; ephrin-B2: Ile31–Gly168; ephrin-A5: Val28–Met165; EphB4: Glu17–Lys196. The
EphA2/ephrin-A1 heterodimer is architecturally similar to the B-class complexes. (D) Schematic
presentation of the 1:1 receptor - ligand contact of opposing cells. The circle indicates the cross- sectional view taken to depict receptor/ligand complex formation in panels (E) - (I). For simplification reasons, receptor and ligand are presented in a truncated form (indicated by X) only displaying the
ectodomains. (E) Initial Eph/ephrin contact produces a high-affinity interaction of the receptor and
ligand-binding domain. The G-H loop of ephrinB2 gets buried in a channel on the surface of EphB2- LBD leading to secondary structure rearrangements as shown in (B). (F) Formation of the tetrameric EphB2/ephrinB2 complex involving the class-specificity H-I loop and the surrounding surface as indicated in (A). This complex is thought to be the minimal functional unit to activate receptors [79]. (G) Hypothetical higher-order cluster formation model of Ephs/ephrins based on various structural and functional studies. Note, that a third interaction site is proposed between the ligand and receptor which belongs to neighboring tetrameric Eph/ephrin complexes [89] and interactions between the Cys-rich domains [90]. (H,I) Latest model to propose an extracellular steric seeding mechanism for the formation of array-like networks of EphA2 [72]. A switch from a parallel staggered array, produced by interactions of the LBD and sushi domain of adjacent unligated EphA2 receptors, to ephrin-bound in- register arrays, characterized by LBD-LBD/sushi-sushi and FNIIIa-FNIIIa interactions, is proposed. The Cys-rich and FNIII domain are highlighted to be required for higher-order cluster formation. Similar other results support this model [91]. Ribbon diagrams in (A,B,C) are adapted from [87].
EphAs/ephrinA recognition and subclass specificity. The first study to investigate the structural basis of EphAs/ephrinAs was done by Himanen and colleagues presenting the crystal structure of an EphA2/ephrinA1 complex [87]. Although these structures are overall similar to their B-class counterparts (Fig. 1.3B,C), they reveal important differences that define subclass specificity. EphAs/ephrinAs interactions involve smaller rearrangements in the interacting partners, better described by a “lock-and-key”- type binding mechanism, in contrast to the “induced fit” mechanism defining the B-class molecules (Fig. 1.3B). In retrospective, the fact that no small-molecule antagonists have been found for any EphBs so far highlights the biological relevance of their different binding modes.
Eph binding promiscuity. Cross-subclass interactions were revealed by the physiologically relevant receptor/ligand pair EphB2/ephrinA5 (Fig 1.3C) [70]. EphB2/ephrinA5 complex is a
12
heterodimer architecturally distinct from the tetrameric EphB2/ephrinB2 structure. The authors concluded that bi-directional signaling is the result of a combination of Eph/ephrin interactions - both intrasubclass and intersubclass. Thus, even interactions that are of lower affinity could significantly cause signaling responses depending on the number or density of interacting molecules. In this context, the authors also put up for debate that receptor activation might not require the precise positioning of nearby Eph receptors.
EphA4, one of the most studied Eph receptors, shows cross-subclass binding promiscuity. Published structures differ considerably from each other and strikingly different explanations for the exceptional cross-subclass specificity and affinity were proposed [71,77,92]. One report addressing these contradictory findings showed that the receptor has an unprecedented ability to adopt two distinct, well-ordered structures even in the unbound state. These results suggest that the ligand promiscuity of EphA4 is directly correlated with the structural flexibility of the ligand-binding surface of the receptor [93].