Author response: Her2 activation mechanism reflects evolutionary preservation of asymmetric ectodomain dimers in the human EGFR family (2013)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The receptor tyrosine kinase Her2, an intensely pursued drug target, differs from other members of the EGFR family in that it does not bind EGF-like ligands, relying instead on heterodimerization with other (ligand-bound) EGFR-family receptors for activation. The structural basis for Her2 heterodimerization, however, remains poorly understood. The unexpected recent finding of asymmetric ectodomain dimer structures of Drosophila EGFR (dEGFR) suggests a possible structural basis for Her2 heterodimerization, but all available structures for dimers of human EGFR family ectodomains are symmetric. Here, we report results from long-timescale molecular dynamics simulations indicating that a single ligand is necessary and sufficient to stabilize the ectodomain interface of Her2 heterodimers, which assume an asymmetric conformation similar to that of dEGFR dimers. This structural parallelism suggests a dimerization mechanism that has been conserved in the evolution of the EGFR family from Drosophila to human. https://doi.org/10.7554/eLife.00708.001 eLife digest ErbB proteins are found in most multi-cellular organisms, and are involved in the regulation of a number of important cellular processes, including proliferation, migration, and differentiation. Humans have four ErbB proteins, which span the plasma membrane of cells. These proteins respond to interactions with molecules outside the cell—such as growth factors and hormones—by sending signals along the appropriate signaling pathway within the cell. ErbB proteins have three portions: an ectodomain that extends outside the cell; a single helix that spans the membrane; and a cytoplasmic domain inside the cell. When a signaling ligand molecule outside the cell binds to the ectodomain of an ErbB protein, this protein must then combine with another ErbB protein to form a dimer before a signal can be sent within the cell. These dimers can include two copies of the same ErbB protein or two different ErbB proteins. However, one of the ErbB proteins—Her2—works in a different way. It cannot bind ligands outside the cell, and it can only send a signal within the cell if it first forms a dimer with an ErbB protein of another type, which itself must be bound to an external ligand. The four ErbB proteins diverged from a common ancestor relatively recently, yet they are now diverse enough to play key roles in a variety of complex signaling networks. In particular, the fact that Her2 cannot bind external ligands, and that it must form a dimer with a different ErbB protein before it can send a signal, has led to suggestions that the role of Her2 is to amplify the signals from other ErbB proteins. Since high levels of Her2 are associated with aggressive forms of breast and ovarian cancer, understanding how it is activated could improve our understanding of these cancers. Arkhipov et al. have now used computer simulations to model how Her2 forms dimers with other ErbB proteins in human cells. They based these simulations on crystal structures of human ErbB proteins and dEGFR, a growth-factor receptor found in fruit flies that closely resembles the ErbB proteins found in humans. They found that the dimers were stable as long as one protein within the dimer was bound to a ligand. Removing this ligand, however, distorted the ectodomain of the host protein, creating a gap that weakened the dimer and prevented Her2 from sending a signal within the cell. Similar results were obtained with the fruit fly dEGFR proteins. These simulations suggest that ErbB proteins form dimers and send signals through a mechanism conserved in evolution. Research in this field might help ongoing efforts to develop new treatments for human tumors characterized by high levels of Her2 expression. https://doi.org/10.7554/eLife.00708.002 Introduction Her2 (also known as Neu or ErbB2), a receptor tyrosine kinase belonging to the human epidermal growth factor receptor (EGFR) family that also includes EGFR/Her1, Her3, and Her4, is an important component of cell-signaling networks, and is implicated in the growth of a variety of cancers (Hynes and Lane, 2005; Riese et al., 2007; Baselga and Swain, 2009; Lemmon and Schlessinger, 2010). The receptors of the EGFR family activate through dimerization (Figure 1A), which is promoted by the binding of ligands from the EGF family (Ushiro and Cohen, 1980; Schreiber et al., 1983; Chung et al., 2010) to the receptors’ extracellular regions (‘ectodomains’). This activation process relies on a number of allosteric interactions in the extracellular, transmembrane, and intracellular portions of the receptor (Endres et al., 2011, 2013; Arkhipov et al., 2013), which lead to the formation of a specific asymmetric active dimer of the intracellular kinase domains (Zhang et al., 2006). Her2 is unique in the family in that it does not homodimerize under normal conditions, and its ectodomain does not bind ligands. Instead, it activates through heterodimerization with other members of the family (EGFR and Her3, in particular) when they are bound to ligands (Citri and Yarden, 2006; Baselga and Swain, 2009). Figure 1 Download asset Open asset Receptors of the human EGFR family and conformations of their ectodomains. (A) Left: the four members of the human EGFR family, each consisting of an ectodomain, a single-pass transmembrane helix, and an intracellular module that includes a kinase domain. As shown, Her2 bears a closed ligand binding site and does not bind EGF-like ligands. Her3 is kinase-dead, and its intracellular module is thus colored gray. Right: common homo- and heterodimers of the EGFR family. The Her2 homodimer is rendered semitransparent to indicate its instability in normal cell conditions. (B) Schematic of the ligand-free Her2 ectodomain monomer, as observed crystallographically (PDB entries 1N8Z, 2A91, 1S78, 3N85, and 3MZW). Domain II is bent. (C) Schematic of the crystal structure of the 2-ligand EGFR ectodomain dimer (PDB entry 1NJP). The dimer is symmetric, and domain II is straight in both subunits. (D) Schematic of the crystal structure of the dEGFR ectodomain dimer (PDB entry 3LTF). Although Spitz ligands are bound to both subunits, the structure is asymmetric; domain II is straight in one subunit but bent in the other. Domain V and part of domain IV were not resolved in this crystal structure and are not shown in the schematic. The conformations of the bent and straight domain IIs in (B), (C), and (D) are indicated by the black lines. https://doi.org/10.7554/eLife.00708.003 The complexity of signaling underpinned by homo- and heterodimerization of EGFR family members emerged relatively recently in evolution. EGFR families in invertebrates have only one member, but gene duplication gave rise to two-member EGFR families in early vertebrate species, such as fishes. Further gene duplication eventually generated the four members of the mammalian EGFR family (Stein and Staros, 2000). The increase in the number of EGFR family members, accompanied by increasing diversity of the extracellular ligands they interact with, gave rise to a complex signaling network: homo- and heterodimerization of various EGF receptors, induced by ligand binding, began to generate a variety of unique signaling outputs controlled by the identity of the bound ligands. The unique dimerization properties of Her2 are believed to be essential to its critical role as a potent signal amplifier for the other receptors of the EGFR family. The Her3–Her2 heterodimer is particularly prevalent and potent in signaling. The prominence of Her3–Her2 heterodimers is particularly intriguing, since Her2 itself lacks an activating ligand and Her3 is impaired in its kinase activity (Figure 1A). These receptors act primarily through heterodimerization. The unique partnership of Her2 and Her3 has been aptly dubbed ‘the deaf and the dumb’ (Citri et al., 2003). By activating several downstream pathways, including those of MAPK, PI3K, phospholipase C, protein kinase C, and Janus kinase, the Her3–Her2 heterodimer plays a pivotal role in the determination of cell lineage in a variety of tissues in epithelial organs. Experiments have shown that knocking out Her2 and Her3 genes leads to defective development of the heart, mammary gland, and nervous system in mice phenotypes. It is thus not surprising that Her3–Her2 heterodimers are implicated in a number of forms of cancer (Citri et al., 2003) or that Her2 is an important drug target in cancer therapeutics. The structural mechanism underlying Her2’s dimerization properties remains incompletely understood. In particular, the conformations of Her2 homo- and heterodimers remain obscure, and how these conformations relate to the dimerization affinities is uncertain. Moreover, the manner in which ligands binding to Her2’s heterodimerization partners regulate the conformations and stability of the heterodimers is poorly understood. Although various structural and electrostatic factors that may affect Her2 dimerization have been discussed in the literature (Cho et al., 2003; Franklin et al., 2004; Garrett et al., 2003; Alvarado et al., 2009, 2010; Liu et al., 2012), further structural elucidation is needed for a better molecular understanding of Her2 activation. Crystal structures of the ectodomains of human EGFR-family receptors (Lemmon, 2009) provide a starting point for elucidation of the structural mechanisms of Her2 heterodimerization. In these structures (Figure 1B,C), the ectodomains (each consisting of four domains numbered I, II, III, and IV) are found either as monomers or as symmetric homodimers with ligands bound to both dimer subunits (Garrett et al., 2002; Lu et al., 2010; Ogiso et al., 2002; Liu et al., 2012). In the homodimer structures, domain II constitutes most of the ectodomain’s dimer interface (Figure 1C), and its conformation is critical to ectodomain dimerization. A bent domain II is found in all ligand-free, inactive ectodomains of the EGFR-family receptors (Figure 1B), while a straight domain II is associated with the (ligand-bound) active dimer (Figure 1C). The domain II of Her2 appears to be constitutively bent (Cho et al., 2003; Garrett et al., 2003; Franklin et al., 2004; Alvarado et al., 2009), which is consistent with its poor homodimerization. Unfortunately, no crystal structure is available for human EGFR family heterodimers with only a single ligand bound, as is the case for Her2 heterodimers. Intriguingly, recent crystal structures of Drosophila EGFR (dEGFR) ectodomains reveal asymmetric dimers that bear only one fully formed ligand binding site, with the other partially closed, regardless of whether one or both subunits are ligand-bound (Alvarado et al., 2009, 2010). These dEGFR structures hint at a possible structural mechanism for Her2 heterodimerization, but translating the dEGFR results to human Her2 is difficult due to differences between these receptors. First, dEGFR dimers assume an asymmetric conformation even when ligands are bound to both dimer subunits, whereas all existing human EGFR-family dimer structures exhibit a symmetric conformation. Second, the ectodomains of Her2 and other members of the human EGFR family each comprise four domains, whereas the dEGFR ectodomain includes a fifth domain, consisting of 166 residues immediately N-terminal to the transmembrane helix. Here we investigate the dimerization of Her2 using molecular modeling and long-timescale molecular dynamics (MD) simulations. We modeled and simulated the EGFR–Her2 and Her3–Her2 heterodimers as well as the Her2 homodimer. The heterodimers were found to be stable when a ligand was bound to the EGFR or Her3 subunit. Our simulations further showed that, when the single bound ligand was removed from a Her2 heterodimer, a substantial gap developed in the dimer interface. A similar gap was also observed in our simulation of the Her2 homodimer, which explains the weak homodimerization of Her2. Structural analysis shows that such a gap arises from the bending of the domain IIs in both subunits due to the absence of bound ligands. These observations are strikingly similar to the findings from the crystal structures of dEGFR homodimers (Alvarado et al., 2009, 2010): the ligand-free, but not the ligand-bound, dEGFR dimer exhibits a gap in the dimer interface, which explains the reduced dEGFR dimerization in the absence of ligands. In agreement with the recent experimental data showing that a single bound ligand is sufficient to activate an EGFR dimer (Liu et al., 2012), our simulations demonstrate that the activation mechanism of the receptors of the human EGFR family and of Her2 in particular conserves Drosophila EGFR’s capacity to form stable asymmetric ectodomain dimers upon the binding of a single ligand. Results Simulations reproduce the gap in the interface of the dEGFR dimer The general approach of this study was to use long-timescale MD simulations to infer from crystal structures of EGFR ectodomain dimers the structures of dimers with different constituent receptors or with different bound ligands. To examine the validity of this approach, we first applied it to dEGFR, for which the structures of both the ligand-bound and ligand-free ectodomain dimers (Alvarado et al., 2009, 2010) have been resolved. Our simulations correctly demonstrated that, once the ligands are removed from the ligand-bound structure, a substantial gap between the two subunits develops, leading to a reduced dimerization interface. Unlike the symmetric human ‘2-ligand’ EGFR dimer (in which both subunits bear fully formed and occupied ligand binding sites; Figure 1C), the 2-ligand dEGFR dimer is asymmetric: although both ectodomain subunits are ligand-bound, only one bears a fully formed ligand binding site and a straight domain II (Figure 2A). The ‘1-ligand’ dEGFR dimer (in which only one subunit is ligand-bound) is essentially identical to the 2-ligand dimer, except for the missing ligand. The gap between the two subunits is closed in both dEGFR dimers. Conversely, the ligand-free dEGFR dimer is symmetric, featuring a bent domain II in both subunits, and a gap in the dimer interface. We removed both ligands from the crystal structure of the 2-ligand dEGFR dimer (PDB entry 3LTF) and simulated the resulting system. In two independent simulations, a substantial gap emerged in the dimer interface, and the resulting conformation resembled that of ligand-free dEGFR crystal structures (Figure 2A,B). Figure 2C shows a significant reduction in the surface area buried within the dimer interface, consistent with the buried areas in the crystal structures of the ligand-free dEGFR dimers. We note that the dimer conformation drifted away from that of the ligand-free crystal structure after ∼2 µs. This reflected the high flexibility of the simulated dimer, likely a result of the truncation of the domain IVs in the resolved crystal structure from which the simulations were initiated and the lack of inter-subunit contact between the two domains. The fact that our dEGFR simulations were able to reproduce the gap in the dimer interface found in the crystal structures of ligand-free dEGFR dimers lends support to the approach we adopted for the analysis of Her2 dimerization. Figure 2 Download asset Open asset Simulations reproduce the gap in the interface of the dEGFR ectodomain dimer. (A) The schematic shows the 2-ligand dEGFR ectodomain dimer on the left. In simulations initiated from this structure with both ligands removed, gap opened in the dimer interface, as indicated by a V-shaped outline in the right diagram. In the ligand-free dimer, domain II is bent in both subunits. The simulation snapshots are shown below the schematic diagrams. (B) The simulated dEGFR dimer from (A), at t = 0.5 µs, is compared with the crystal structure of the ligand-free dEGFR dimer (tan). Molecular renderings (A, B) omit domains IV and V for clarity (although the crystallographically resolved portion of domain IV was present in simulations). (C) The surface area buried within the dimer interface (counting the contributions from domains I, II, and III). The results of three independent simulations are shown, two starting from the 2-ligand crystal structures (after removal of the ligands), and one from the ligand-free crystal structure. https://doi.org/10.7554/eLife.00708.004 Models of the EGFR–Her2 heterodimer and Her2 homodimer The 2-ligand EGFR homodimer has been resolved crystallographically (Garrett et al., 2002; Ogiso et al., 2002; Lu et al., 2010) and studied previously using MD simulations (e.g., Tynan et al., 2011; Zhang and Wriggers, 2011). Given the close homology between the EGFR-family receptors (‘Materials and methods’), this crystal structure may serve as a starting point for the modeling of Her2 homo- and heterodimers. The first model we constructed was that of the ligand-bound EGFR–Her2 heterodimer, in which the EGFR subunit is ligand-bound and the Her2 subunit is ligand-free. We assumed that a 1-ligand EGFR homodimer is structurally similar to this EGFR–Her2 heterodimer and thus may serve as a template. To obtain a structure of the 1-ligand EGFR homodimer, we removed one ligand from the crystal structure of the 2-ligand EGFR homodimer (PDB entry 3NJP [Lu et al., 2010]) and simulated the remaining complex (Figure 3A). In all three independent simulations performed, the dimer assumed an asymmetric conformation, in which the ligand-free subunit differs from the ligand-bound one. In the former, the space previously occupied by the bound ligand between domains I and III is closed and domain II is bent (Figure 3B), in agreement with a recent MD study (Tynan et al., 2011). It is notable that the removal of one of the two ligands from the ectodomain dimer did not lead to substantial decrease in the area of the dimer interfaces (Figure 3E), suggesting that, in agreement with recent experimental findings (Liu et al., 2012), a single bound ligand is sufficient to maintain a stable ectodomain dimer of EGFR. By comparison, no significant conformational changes were observed in the control simulations of the 2-ligand EGFR dimer. Figure 3 Download asset Open asset Bending of domain II. (A) A schematic showing how the model of the 1-ligand EGFR dimer (right) is generated by simulation after the removal of one EGF ligand from the crystal structure of the 2-ligand dimer (left). The resulting structure is asymmetric: domain II of the ligand-free subunit is bent and the binding site is closed, whereas domain II of the ligand-bound subunit is straight and the binding site is open (as is the case in both subunits of the 2-ligand dimer). The angle θ, which characterizes the bending of domain II, is measured between the Cα atoms of EGFR residues 194, 239, and 296. (B) Bending of domain II. A simulation snapshot of the ligand-free subunit (red) from the 1-ligand EGFR dimer is overlaid with the crystal structures of a subunit from the 2-ligand EGFR dimer (green). EGFR residues 240–309 are used for reference. (C) An overlay of the ligand-free subunit of the 1-ligand EGFR dimer, generated by simulation (red), with the crystal structure of Her2 monomer (cyan). In the interest of clarity, domain IV is not shown. (D) The angle θ (illustrated in [A]) is shown as function of time in the three independent simulations of the 1-ligand EGFR homodimer, for the ligand-free subunit (middle) and for the ligand-bound subunit (right). The value of θ in the crystal structure of Her2 monomer is indicated by a straight line. (E) The buried surface area at the interfaces of 1-ligand and 2-ligand EGFR dimers in simulation. https://doi.org/10.7554/eLife.00708.005 The conformation of the ligand-free subunit in the simulated 1-ligand EGFR homodimer resembles the crystal structure of Her2 monomer (Figure 3C), especially in the bending of domain II. (The bent domain II is also observed in the crystal structures of ligand-free EGFR monomers [PDB entries 1NQL and 1YY9; Ferguson et al., 2003; Li et al., 2005].) The bending in domain II can be characterized by the angle θ formed by the top, middle, and bottom regions of the domain II dimerization interface (Figure 3A,D). In our simulations, this angle was ∼150° to 160° in the ligand-free subunits and ∼170° in the ligand-bound subunits, with the former values being very close to that of the Her2 crystal structure (156°). The EGFR–Her2 heterodimer was thus modeled by superimposing a crystal structure of the monomeric Her2 ectodomain (PDB entry 3BE1 [Bostrom et al., 2009]) onto the ligand-free EGFR subunit in the 1-ligand EGFR dimer using the portion of domain II directly involved in dimerization (EGFR residues 240–309; see ‘Materials and methods’). We subsequently modeled the ligand-free EGFR–Her2 heterodimer and the Her2 homodimer using a similar approach. In the former case, the ligand was removed from our model of the EGFR–Her2 heterodimer and the resulting ligand-free heterodimer was then simulated. In the simulations, domains I, II, and III of the EGFR subunit again assumed a ‘Her2-like’conformation. This model of the ligand-free EGFR–Her2 heterodimer was then used as a template for the Her2 homodimer, in the same way that the model of the 1-ligand EGFR homodimer was used as a template for the EGFR–Her2 heterodimer (‘Materials and methods’). Dynamics of the dimer interfaces in the EGFR–Her2 heterodimer and Her2 homodimer We subsequently simulated the EGFR–Her2 heterodimer and Her2 homodimer starting from these models. In these simulations, the ligand-bound EGFR–Her2 heterodimer remained stable (Figure 4A), the dimer interface was largely intact, and the ligand wedged between domains I and III of the EGFR subunit, preventing any bending in this subunit’s domain II. In a gap opened in the dimer interface of the ligand-free EGFR–Her2 heterodimer was observed in two independent between the N-terminal portions of the domain IIs (Figure This as domain II of the EGFR subunit bent away from the dimer interface upon the removal of its bound ligand. a gap at the dimer interface was also observed in two independent simulations of the Her2 homodimer (Figure of the buried surface area that the of the gap in the simulations (Figure but on was in the ligand-free EGFR–Her2 heterodimer and Her2 homodimer in the ligand-bound Figure Download asset Open asset Simulations of the EGFR–Her2 heterodimer and the Her2 homodimer. (A) The EGFR–Her2 heterodimer with the ligand bound to EGFR (B) The ligand-free EGFR–Her2 heterodimer or homodimer (C) The Her2 homodimer. snapshots from the simulations. of the surface area buried within the dimer interface (counting the contributions from domains I, II, and as a function of simulation time EGFR–Her2 and results of two independent simulations are the conformation of domain II and the dimer interface. of the bent and straight domain II are by the black and the gap in the dimer interface is indicated by a V-shaped outline when clarity, domain IV is not shown. 1 Download asset This cannot be in does support may the for Download as Download as Download as The simulation of the ligand-free EGFR–Her2 ectodomain heterodimer with Her2 colored and EGFR A gap between the N-terminal portion of each subunit’s domain II the simulation. In of their and on dimer the observed in our simulations are of the one in the crystal structure of ligand-free dEGFR dimer (Alvarado et al., 2009, 2010). The dEGFR gap results in a reduced dimer interface area of compared to in the and 2-ligand dEGFR the gap is closed (Figure This reduction of the dimer interface area is believed to for the relatively weak dimerization of the ligand-free dEGFR, which is that of ligand-bound Figure shows that the reduction of the dimer interface is observed in our simulations of the ligand-free EGFR–Her2 heterodimer and Her2 when the gap is the surface area buried within the interface is compared to in the ligand-bound We thus suggest that the same structural mechanism that ligand-free dEGFR homodimers (Alvarado et al., 2009, 2010) also ligand-free EGFR–Her2 dimerization and Her2 homodimerization. the other ligand binding in the EGFR subunit to domain II which the of the gap and the dimer interface of EGFR–Her2 heterodimer (Figure This is of the recent finding (Liu et al., that one bound ligand is sufficient to activate an EGFR dimer. Her3–Her2 heterodimer the dimerization partners of Her2, Her3 is particularly notable in that the Her3–Her2 heterodimers are potent signaling and in that the normal and signaling through Her2 and Her3 relies on Her3–Her2 dimerization and Swain, 2009; 2009). We thus studied the Her3–Her2 heterodimer in similar to our study of EGFR–Her2 heterodimers and the Her2 homodimer. We first modeled the Her3–Her2 heterodimer using the resolved crystal structure of the Her3 monomer (Cho and and our model of the EGFR–Her2 heterodimer as (‘Materials and methods’). The EGF-like domain of the Her3 ligand was then the Her3 binding site between domains I and III, in with the of The resulting model of the Her3–Her2 heterodimer was then first with bound, in which case the dimer interface remained stable (Figure of simulation the ligand was removed, and the resulting ligand-free heterodimer was then simulated the ligand, the Her3 subunit conformational changes similar to those for the ligand-free EGFR subunit, including a bending of domain II. As a a gap again emerged in the dimer interface. In the first of simulations the gap in the Her3–Her2 heterodimer was partially closed, but and in a significant decrease in the buried surface area in the dimer interface. In the the gap was open once the ligand was removed (Figure These observations suggest that Her3–Her2 heterodimerization, similar to that of is promoted by the of the dimer interface ligand Figure Download asset Open asset Her3–Her2 (A) from the simulations of the Her3–Her2 heterodimer with and (right) bound to the of the simulation with bound to Her3, was removed, and the resulting system was and further simulated the ligand. A gap opened in the dimer interface, as by the snapshot on the clarity, these omit domain (B) The surface area buried within the dimer interface, the contributions only from domains I, II, and III, as a function of independent of two simulations each and are shown. of the dimer interfaces in the asymmetric dimers Our simulations suggest that ligand binding the formation of asymmetric dEGFR or EGFR-family dimers by specific interactions between the domain II N-terminal regions of the two subunits. We found that the asymmetric dimers we EGFR–Her2 and Her3–Her2 heterodimers, the 1-ligand EGFR homodimer, and the and 2-ligand dEGFR key interactions between subunits. In particular, between (in EGFR or Her3 of the ligand-bound subunit and and (in Her2 Figure of the ligand-free subunit (Figure were particularly The of these is consistent with the crystal structures of the dEGFR asymmetric dimers after a at the be consistent with the same In these are not present in the crystal structure or in simulations of the symmetric 2-ligand EGFR dimer, and they are by the gap in the dimer interface of all ligand-free dimers we the ligand-free dEGFR crystal structure and our simulations of ligand-free EGFR and Her2 homodimers and EGFR–Her2 Our observations suggest an important role for of the ligand-bound subunit in 1-ligand dimers. the is conserved in Her3, Her4, and dEGFR, which bind ligands,