EGFR Ligands Differentially Stabilize Receptor Dimers to Specify Signaling Kinetics.

Epidermal growth factor receptor (EGFR) regulates many crucial cellular programs, with seven different activating ligands shaping cell signaling in distinct ways. Using crystallography and other approaches, we show how the EGFR ligands epiregulin (EREG) and epigen (EPGN) stabilize different dimeric conformations of the EGFR extracellular region. As a consequence, EREG or EPGN induce less stable EGFR dimers than EGF-making them partial agonists of EGFR dimerization. Unexpectedly, this weakened dimerization elicits more sustained EGFR signaling than seen with EGF, provoking responses in breast cancer cells associated with differentiation rather than proliferation. Our results reveal how responses to different EGFR ligands are defined by receptor dimerization strength and signaling dynamics. These findings have broad implications for understanding receptor tyrosine kinase (RTK) signaling specificity. Our results also suggest parallels between partial and/or biased agonism in RTKs and G-protein-coupled receptors, as well as new therapeutic opportunities for correcting RTK signaling output.

Glioblastoma mutations alter EGFR dimer structure to prevent ligand bias.

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer1,2, and is an important therapeutic target. EGFR inhibitors have been successful in lung cancer, where mutations in the intracellular tyrosine kinase domain activate the receptor1, but not in glioblastoma multiforme (GBM)3, where mutations occur exclusively in the extracellular region. Here we show that common extracellular GBM mutations prevent EGFR from discriminating between its activating ligands4. Different growth factor ligands stabilize distinct EGFR dimer structures5 that signal with different kinetics to specify or bias outcome5,6. EGF itself induces strong symmetric dimers that signal transiently to promote proliferation. Epiregulin (EREG) induces much weaker asymmetric dimers that drive sustained signalling and differentiation5. GBM mutations reduce the ability of EGFR to distinguish EREG from EGF in cellular assays, and allow EGFR to form strong (EGF-like) dimers in response to EREG and other low-affinity ligands. Using X-ray crystallography, we further show that the R84K GBM mutation symmetrizes EREG-driven extracellular dimers so that they resemble dimers normally seen with EGF. By contrast, a second GBM mutation, A265V, remodels key dimerization contacts to strengthen asymmetric EREG-driven dimers. Our results argue for an important role of altered ligand discrimination by EGFR in GBM, with potential implications for therapeutic targeting.

Dimerization of Tie2 mediated by its membrane-proximal FNIII domains.

Tie1 and Tie2, members of the tyrosine kinase family with immunoglobulin and EGF homology domains, are receptor tyrosine kinases found primarily in endothelial cells with key roles in development and maintenance of the vasculature and in angiogenesis. They are attractive targets for therapeutic intervention in tumor angiogenesis, inflammation, and sepsis. Tie2 is regulated directly by the multimeric angiopoietin (Ang) ligands, with Ang1 being its primary activator. Structural studies have shown how Angs bind to the Tie2 ligand-binding region, but do not explain Tie2 activation and suggest a passive role for the Tie2 extracellular region (ECR) in ligand-induced receptor dimerization. Here we show that the Tie2 ECR forms strong dimers even in the absence of bound ligand. Dimerization is mediated by membrane-proximal fibronectin type III (FNIII) domains that were omitted in previous structural studies. We describe a 2.5-Å resolution X-ray crystal structure of the membrane-proximal three Tie2 FNIII domains, Tie2(FNIIIa-c), revealing two possible dimerization modes that primarily involve the third FNIII domain, FNIIIc. Mutating these dimer interfaces implicates one of them (dimer 1) in soluble Tie2 (sTie2) dimerization in solution but suggests that both could play a role in Ang1-induced Tie2 activation, possibly modulated by Tie1. Through small-angle X-ray scattering studies of sTie2 dimers in solution and modeling based on crystal structures, we suggest that Ang1 binding may cross-link Tie2 dimers into higher-order oligomers, potentially explaining how Tie2 is differentially clustered following ligand engagement in different cellular contexts. Our results also firmly implicate FNIII domain-mediated interactions in Tie2 activation, identifying a potential Achilles' heel for therapeutic inhibition.

Intramolecular autoinhibition of checkpoint kinase 1 is mediated by conserved basic motifs of the C-terminal kinase-associated 1 domain.

Precise control of the cell cycle allows for timely repair of genetic material prior to replication. One factor intimately involved in this process is checkpoint kinase 1 (Chk1), a DNA damage repair inducing Ser/Thr protein kinase that contains an N-terminal kinase domain and a C-terminal regulatory region consisting of a ∼100-residue linker followed by a putative kinase-associated 1 (KA1) domain. We report the crystal structure of the human Chk1 KA1 domain, demonstrating striking structural homology with other sequentially diverse KA1 domains. Separately purified Chk1 kinase and KA1 domains are intimately associated in solution, which results in inhibition of Chk1 kinase activity. Using truncation mutants and site-directed mutagenesis, we define the inhibitory face of the KA1 domain as a series of basic residues residing on two conserved regions of the primary structure. These findings point to KA1-mediated intramolecular autoinhibition as a key regulatory mechanism of human Chk1, and provide new therapeutic possibilities with which to attack this validated oncology target with small molecules.

Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase.

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other 'activating' protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found - also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a 'mini' MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

The pellino e3 ubiquitin ligases recognize specific phosphothreonine motifs and have distinct substrate specificities.

The four mammalian Pellinos (Pellinos 1, 2, 3a, and 3b) are E3 ubiquitin ligases that are emerging as critical mediators for a variety of immune signaling pathways, including those activated by Toll-like receptors, the T-cell receptor, and NOD2. It is becoming increasingly clear that each Pellino has a distinct role in facilitating immune receptor signaling. However, the underlying mechanisms by which these highly homologous proteins act selectively in these signaling pathways are not clear. In this study, we investigate whether Pellino substrate recognition contributes to the divergent functions of Pellinos. Substrate recognition of each Pellino is mediated by its noncanonical forkhead-associated (FHA) domain, a well-characterized phosphothreonine-binding module. Pellino FHA domains share very high sequence identity, so a molecular basis for differences in substrate recognition is not immediately apparent. To explore Pellino substrate specificity, we first identify a high-affinity Pellino2 FHA domain-binding motif in the Pellino substrate, interleukin-1 receptor-associated kinase 1 (IRAK1). Analysis of binding of the different Pellinos to a panel of phosphothreonine-containing peptides derived from the IRAK1-binding motif reveals that each Pellino has a distinct phosphothreonine peptide binding preference. We observe a similar binding specificity in the interaction of Pellinos with a number of known Pellino substrates. These results argue that the nonredundant roles that Pellinos play in immune signaling are in part due to their divergent substrate specificities. This new insight into Pellino substrate recognition could be exploited for pharmacological advantage in treating inflammatory diseases that have been linked to the aberrant regulation of Pellinos.

Structural insights into the role and targeting of EGFRvIII.

The epidermal growth factor receptor (EGFR) is a well-known oncogenic driver in lung and other cancers. In glioblastoma multiforme (GBM), the EGFR deletion variant III (EGFRvIII) is frequently found alongside EGFR amplification. Agents targeting the EGFR axis have shown limited clinical benefits in GBM and the role of EGFRvIII in GBM is poorly understood. To shed light on the role of EGFRvIII and its potential as a therapeutic target, we determined X-ray crystal structures of a monomeric EGFRvIII extracellular region (ECR). The EGFRvIII ECR resembles the unliganded conformation of EGFR, including the orientation of the C-terminal region of domain II. Domain II is mostly disordered, but the ECR structure is compact. We selected a nanobody with preferential binding to EGFRvIII relative to EGFR and structurally defined an epitope on domain IV that is occluded in the unliganded intact EGFR. These findings suggest new avenues for EGFRvIII targeting in GBM.

Structural basis for inhibition of the epidermal growth factor receptor by cetuximab.

Recent structural studies of epidermal growth factor receptor (EGFR) family extracellular regions have identified an unexpected mechanism for ligand-induced receptor dimerization that has important implications for activation and inhibition of these receptors. Here we describe the 2.8 angstroms resolution X-ray crystal structure of the antigen binding (Fab) fragment from cetuximab (Erbitux), an inhibitory anti-EGFR antibody, in complex with the soluble extracellular region of EGFR (sEGFR). The sEGFR is in the characteristic "autoinhibited" or "tethered" inactive configuration. Cetuximab interacts exclusively with domain III of sEGFR, partially occluding the ligand binding region on this domain and sterically preventing the receptor from adopting the extended conformation required for dimerization. We suggest that both these effects contribute to potent inhibition of EGFR activation.

A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth.

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development.

Interaction of antibodies with ErbB receptor extracellular regions.

Antibodies to the extracellular region of the ErbB receptors have played key roles in the development of a mechanistic understanding of this family of receptor tyrosine kinases. An extensively studied class of such antibodies inhibits activation of ErbB receptors, and these antibodies have been the focus of intense development as anti-cancer agents. In this review we consider the properties of ErbB receptors antibodies in light of the current structure-based model for ErbB receptor homo- and hetero-dimerization and activation. Crystal structures of the Fab fragments from five different inhibitory antibodies in complex with the extracellular regions of EGFR and ErbB2 have been determined. These structures highlight several different modes of binding and mechanisms of receptor inhibition. Information about antibody interactions with the structurally well-characterized soluble extracellular regions of ErbB receptors can be combined with the rich knowledge of the effects of these antibodies in cultured cells, and in vivo, to provide insights into the conformation and activation of ErbB receptors at the cell surface.

Structural basis for EGF receptor inhibition by the therapeutic antibody IMC-11F8.

Therapeutic anticancer strategies that target and inactivate the epidermal growth factor receptor (EGFR) are under intense study in the clinic. Here we describe the mechanism of EGFR inhibition by an antibody drug IMC-11F8. IMC-11F8 is a fully human antibody that has similar antitumor potency as the chimeric cetuximab/Erbitux and might represent a safer therapeutic alternative. We report the X-ray crystal structure of the Fab fragment of IMC-11F8 (Fab11F8) in complex with the entire extracellular region and with isolated domain III of EGFR. We compare this to our previous study of the cetuximab/EGFR interaction. Fab11F8 interacts with a remarkably similar epitope, but through a completely different set of interactions. Both the similarities and differences in binding of these two antibodies have important implications for the development of inhibitors that could exploit this same mechanism of EGFR inhibition.

Pellino proteins contain a cryptic FHA domain that mediates interaction with phosphorylated IRAK1.

Pellino proteins are RING E3 ubiquitin ligases involved in signaling events downstream of the Toll and interleukin-1 (IL-1) receptors, key initiators of innate immune and inflammatory responses. Pellino proteins associate with and ubiquitinate proteins in these pathways, including the interleukin-1 receptor associated kinase-1 (IRAK1). We determined the X-ray crystal structure of a Pellino2 fragment lacking only the RING domain. This structure reveals that the IRAK1-binding region of Pellino proteins consists largely of a previously unidentified forkhead-associated (FHA) domain. FHA domains are well-characterized phosphothreonine-binding modules, and this cryptic example in Pellino2 can drive interaction of this protein with phosphorylated IRAK1. The Pellino FHA domain is decorated with an unusual appendage or "wing" composed of two long inserts that lie within the FHA homology region. Delineating how this E3 ligase associates with substrates, and how these interactions are regulated by phosphorylation, is crucial for a complete understanding of Toll/IL-1 receptor signaling.

Insulin and epidermal growth factor receptor family members share parallel activation mechanisms.

Insulin receptor (IR) and the epidermal growth factor receptor (EGFR) were the first receptor tyrosine kinases (RTKs) to be studied in detail. Both are important clinical targets-in diabetes and cancer, respectively. They have unique extracellular domain compositions among RTKs, but share a common module with two ligand-binding leucine-rich-repeat (LRR)-like domains connected by a flexible cysteine-rich (CR) domain (L1-CR-L2 in IR/domain, I-II-III in EGFR). This module is linked to the transmembrane region by three fibronectin type III domains in IR, and by a second CR in EGFR. Despite sharing this conserved ligand-binding module, IR and EGFR family members are considered mechanistically distinct-in part because IR is a disulfide-linked (αß)2 dimer regardless of ligand binding, whereas EGFR is a monomer that undergoes ligand-induced dimerization. Recent cryo-electron microscopy (cryo-EM) structures suggest a way of unifying IR and EGFR activation mechanisms and origins of negative cooperativity. In EGFR, ligand engages both LRRs in the ligand-binding module, "closing" this module to break intramolecular autoinhibitory interactions and expose new dimerization sites for receptor activation. How insulin binds the activated IR was less clear until now. Insulin was known to associate with one LRR (L1), but recent cryo-EM structures suggest that it also engages the second LRR (albeit indirectly) to "close" the L1-CR-L2 module, paralleling EGFR. This transition simultaneously breaks autoinhibitory interactions and creates new receptor-receptor contacts-remodeling the IR dimer (rather than inducing dimerization per se) to activate it. Here, we develop this view in detail, drawing mechanistic links between IR and EGFR.

Epidermal growth factor receptor dimerization and activation require ligand-induced conformational changes in the dimer interface.

Structural studies have shown that ligand-induced epidermal growth factor receptor (EGFR) dimerization involves major domain rearrangements that expose a critical dimerization arm. However, simply exposing this arm is not sufficient for receptor dimerization, suggesting that additional ligand-induced dimer contacts are required. To map these contributions to the dimer interface, we individually mutated each contact suggested by crystallographic studies and analyzed the effects on receptor dimerization, activation, and ligand binding. We find that domain II contributes >90% of the driving energy for dimerization of the extracellular region, with domain IV adding little. Within domain II, the dimerization arm forms much of the dimer interface, as expected. However, a loop from the sixth disulfide-bonded module (immediately C-terminal to the dimerization arm) also makes a critical contribution. Specific ligand-induced conformational changes in domain II are required for this loop to contribute to receptor dimerization, and we identify a set of ligand-induced intramolecular interactions that appear to be important in driving these changes, effectively "buttressing" the dimer interface. Our data also suggest that similar conformational changes may determine the specificity of ErbB receptor homo- versus heterodimerization.

Structural Basis for MARK1 Kinase Autoinhibition by Its KA1 Domain.

The kinase associated-1 (KA1) domain is found at the C-terminus of multiple Ser/Thr protein kinases from yeast to humans, and has been assigned autoinhibitory, membrane-binding, and substrate-targeting roles. Here, we report the crystal structure of the MARK1 kinase/UBA domain bound to its autoinhibitory KA1 domain, revealing an unexpected interface at the αD helix and contacts with both the N- and C-lobes of the kinase domain. We confirm the binding interface location in kinetic studies of variants mutated on the kinase domain surface. Together with other MARK kinase structures, the data implicate that the KA1 domain blocks peptide substrate binding. The structure highlights the kinase-specific autoinhibitory binding modes of different KA1 domains, and provides potential new avenues by which to intervene therapeutically in Alzheimer's disease and cancers in which MARK1 or related kinases are implicated.

Molecular Basis for Necitumumab Inhibition of EGFR Variants Associated with Acquired Cetuximab Resistance.

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521-31. ©2017 AACR.

Comparison of Saccharomyces cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site.

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.

Pleckstrin homology domains and the cytoskeleton.

Pleckstrin homology (PH) domains are 100-120 amino acid protein modules best known for their ability to bind phosphoinositides. All possess an identical core beta-sandwich fold and display marked electrostatic sidedness. The binding site for phosphoinositides lies in the center of the positively charged face. In some cases this binding site is well defined, allowing highly specific and strong ligand binding. In several of these cases the PH domains specifically recognize 3-phosphorylated phosphoinositides, allowing them to drive membrane recruitment in response to phosphatidylinositol 3-kinase activation. Examples of these PH domain-containing proteins include certain Dbl family guanine nucleotide exchange factors, protein kinase B, PhdA, and pleckstrin-2. PH domain-mediated membrane recruitment of these proteins contributes to regulated actin assembly and cell polarization. Many other PH domain-containing cytoskeletal proteins, such as spectrin, have PH domains that bind weakly, and to all phosphoinositides. In these cases, the individual phosphoinositide interactions may not be sufficient for membrane association, but appear to require self-assembly of their host protein and/or cooperation with other anchoring motifs within the same molecule to drive membrane attachment.

The EGFR family: not so prototypical receptor tyrosine kinases.

The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed "prototypical" RTKs. Many years of mechanistic studies, however, have revealed that--far from being prototypical--the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive "receptor-mediated" dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface.

Complex relationship between ligand binding and dimerization in the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) plays pivotal roles in development and is mutated or overexpressed in several cancers. Despite recent advances, the complex allosteric regulation of EGFR remains incompletely understood. Through efforts to understand why the negative cooperativity observed for intact EGFR is lost in studies of its isolated extracellular region (ECR), we uncovered unexpected relationships between ligand binding and receptor dimerization. The two processes appear to compete. Surprisingly, dimerization does not enhance ligand binding (although ligand binding promotes dimerization). We further show that simply forcing EGFR ECRs into preformed dimers without ligand yields ill-defined, heterogeneous structures. Finally, we demonstrate that extracellular EGFR-activating mutations in glioblastoma enhance ligand-binding affinity without directly promoting EGFR dimerization, suggesting that these oncogenic mutations alter the allosteric linkage between dimerization and ligand binding. Our findings have important implications for understanding how EGFR and its relatives are activated by specific ligands and pathological mutations.