The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer
, and is an important therapeutic target. EGFR inhibitors have been successful in lung cancer, where mutations in ...the intracellular tyrosine kinase domain activate the receptor
, but not in glioblastoma multiforme (GBM)
, where mutations occur exclusively in the extracellular region. Here we show that common extracellular GBM mutations prevent EGFR from discriminating between its activating ligands
. Different growth factor ligands stabilize distinct EGFR dimer structures
that signal with different kinetics to specify or bias outcome
. EGF itself induces strong symmetric dimers that signal transiently to promote proliferation. Epiregulin (EREG) induces much weaker asymmetric dimers that drive sustained signalling and differentiation
. 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.
Abstract
Natural photosynthetic protein complexes capture sunlight to power the energetic catalysis that supports life on Earth. Yet these natural protein structures carry an evolutionary legacy of ...complexity and fragility that encumbers protein reengineering efforts and obfuscates the underlying design rules for light-driven charge separation. De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build new enzymes for efficient solar-to-fuel energy conversion. Here, we report the rational design, X-ray crystal structure, and electron transfer activity of a multi-cofactor protein that incorporates essential elements of photosynthetic reaction centers. This highly stable, modular artificial protein framework can be reconstituted in vitro with interchangeable redox centers for nanometer-scale photochemical charge separation. Transient absorption spectroscopy demonstrates Photosystem II-like tyrosine and metal cluster oxidation, and we measure charge separation lifetimes exceeding 100 ms, ideal for light-activated catalysis. This de novo-designed reaction center builds upon engineering guidelines established for charge separation in earlier synthetic photochemical triads and modified natural proteins, and it shows how synthetic biology may lead to a new generation of genetically encoded, light-powered catalysts for solar fuel production.
Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the ...Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of “conformational disruptors” to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.
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•Pseudokinases share autoinhibitory interactions of their closest kinase relatives•Structural dynamics of pseudokinases closely resemble those of canonical kinases•ROR1 ATP-binding site can be targeted with small molecules despite being occluded•Inhibitor binding disrupts pseudokinase conformation and may inhibit signaling
Sheetz et al. ask how Wnt-binding receptor tyrosine kinases can signal through intracellular “pseudokinase” domains. They find that these domains resemble autoinhibited kinases but are dynamic enough to regulate signaling through conformational switching. They find small molecules that can alter pseudokinase conformation, suggesting routes for modulating this receptor class therapeutically.
Tyrosine kinase inhibitors (TKIs) are used to treat non-small cell lung cancers (NSCLC) driven by epidermal growth factor receptor (EGFR) mutations in the tyrosine kinase domain (TKD). TKI responses ...vary across tumors driven by the heterogeneous group of exon 19 deletions and mutations, but the molecular basis for these differences is not understood. Using purified TKDs, we compared kinetic properties of several exon 19 variants. Although unaltered for the second generation TKI afatinib, sensitivity varied significantly for both the first and third generation TKIs erlotinib and osimertinib. The most sensitive variants showed reduced ATP-binding affinity, whereas those associated with primary resistance retained wild type ATP-binding characteristics (and low K
). Through crystallographic and hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies, we identify possible origins for the altered ATP-binding affinity underlying TKI sensitivity and resistance, and propose a basis for classifying uncommon exon 19 variants that may have predictive clinical value.
The proto-oncogene ALK encodes anaplastic lymphoma kinase, a receptor tyrosine kinase that is expressed primarily in the developing nervous system. After development, ALK activity is associated with ...learning and memory
and controls energy expenditure, and inhibition of ALK can prevent diet-induced obesity
. Aberrant ALK signalling causes numerous cancers
. In particular, full-length ALK is an important driver in paediatric neuroblastoma
, in which it is either mutated
or activated by ligand
. Here we report crystal structures of the extracellular glycine-rich domain (GRD) of ALK, which regulates receptor activity by binding to activating peptides
. Fusing the ALK GRD to its ligand enabled us to capture a dimeric receptor complex that reveals how ALK responds to its regulatory ligands. We show that repetitive glycines in the GRD form rigid helices that separate the major ligand-binding site from a distal polyglycine extension loop (PXL) that mediates ALK dimerization. The PXL of one receptor acts as a sensor for the complex by interacting with a ligand-bound second receptor. ALK activation can be abolished through PXL mutation or with PXL-targeting antibodies. Together, these results explain how ALK uses its atypical architecture for its regulation, and suggest new therapeutic opportunities for ALK-expressing cancers such as paediatric neuroblastoma.
The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza ...drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.
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.
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•The EGFRvIII extracellular region adopts the conformation seen in tethered EGFR•Domain III of EGFRvIII is accessible for ligand or antibody binding•Domain II is largely disordered, but the extracellular region is compact•A nanobody/VHH selective for EGFRvIII recognized an epitope on domain IV
The EGFRvIII truncation variant is frequently found in glioblastoma multiforme but its role and potential as a therapeutic target remain controversial. Bagchi, Stayrook et al. determined the EGFRvIII extracellular region structure. They also describe a VHH/nanobody recognizing an unexpected epitope in domain IV that is occluded in unliganded intact EGFR.
New technologies for efficient solar-to-fuel energy conversion will help facilitate a global shift from dependence on fossil fuels to renewable energy. Nature uses photosynthetic reaction centers to ...convert photon energy into a cascade of electron-transfer reactions that eventually produce chemical fuel. The design of new reaction centers
de novo
deepens our understanding of photosynthetic charge separation and may one day allow production of biofuels with higher thermodynamic efficiency than natural photosystems. Recently, we described the multi-step electron-transfer activity of a designed reaction center maquette protein (the RC maquette), which can assemble metal ions, tyrosine, a Zn tetrapyrrole, and heme into an electron-transport chain. Here, we detail our modular strategy for rational protein design and show that the intended RC maquette design agrees with crystal structures in various states of assembly. A flexible, dynamic apo-state collapses by design into a more ordered holo-state upon cofactor binding. Crystal structures illustrate the structural transitions upon binding of different cofactors. Spectroscopic assays demonstrate that the RC maquette binds various electron donors, pigments, and electron acceptors with high affinity. We close with a critique of the present RC maquette design and use electron-tunneling theory to envision a path toward a designed RC with a substantially higher thermodynamic efficiency than natural photosystems.
WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases ...(RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. Our crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also find that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, our structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.
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•WNT-binding CRDs of ROR family receptors differ structurally from those in FZDs•The WIF domain of the RYK ortholog Derailed lacks WIF-1’s lipid binding site•Drosophila WNT-5 binds ROR and RYK family receptors despite lacking an acyl chain•WNT-binding receptor tyrosine kinases recognize different WNT features than FZDs
Several receptor tyrosine kinase (RTK) family members function as WNT receptors. Shi et al. show that although RTKs bind WNTs using similar domains to those seen in Frizzleds (FZDs) and WIF-1, they bind WNTs in a distinct way that may allow a single WNT to bind both receptor types simultaneously.
Pmel17 is a transmembrane protein that mediates the early steps in the formation of melanosomes, the subcellular organelles of melanocytes in which melanin pigments are synthesized and stored. In ...melanosome precursor organelles, proteolytic fragments of Pmel17 form insoluble, amyloid-like fibrils upon which melanins are deposited during melanosome maturation. The mechanism(s) by which Pmel17 becomes competent to form amyloid are not fully understood. To better understand how amyloid formation is regulated, we have defined the domains within Pmel17 that promote fibril formation in vitro. Using purified recombinant fragments of Pmel17, we show that two regions, an N-terminal domain of unknown structure and a downstream domain with homology to a polycystic kidney disease-1 repeat, efficiently form amyloid in vitro. Analyses of fibrils formed in melanocytes confirm that the polycystic kidney disease-1 domain forms at least part of the physiological amyloid core. Interestingly, this same domain is also required for the intracellular trafficking of Pmel17 to multivesicular compartments within which fibrils begin to form. Although a domain of imperfect repeats (RPT) is required for fibril formation in vivo and is a component of fibrils in melanosomes, RPT is not necessary for fibril formation in vitro and in isolation is unable to adopt an amyloid fold in a physiologically relevant time frame. These data define the structural core of Pmel17 amyloid, imply that the RPT domain plays a regulatory role in timing amyloid conversion, and suggest that fibril formation might be physically linked with multivesicular body sorting.
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