Oncogenic mutations at the EGFR ectodomain structurally converge to remove a steric hindrance on a kinase-coupled cryptic epitope
Laura OrellanaAmy Haseley ThorneRafael LemaJohan GustavssonAlison D. ParisianAdam HospitalTiago N. CordeiroPau BernadóAndrew M. ScottIsabelle Brun‐HeathErik LindahlWebster K. CaveneeFrank B. FurnariModesto Orozco
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Significance EGFR cancer mutations display an astonishing tissue-specific asymmetry: in lung cancer, mutations target the intracellular kinase (KD), while in glioblastomas (GBMs), a variety of missense clusters and deletions concentrate at the ectodomain (ECD). Intriguingly, GBM-activating mutations share a paradoxical preference for inhibitors that bind the inactive kinase. By integrating simulations, small-angle X-ray scattering, and GBM models, we demonstrate that ECD mutants converge to a transition state characterized by a cryptic epitope, allosterically coupled to an intermediate kinase, and synergistically blocked by antibodies and inhibitors. Our findings indicate that apparently heterogeneous aberrations remove a similar steric restrain on KD activation. The diversity of structural tricks in ECD mutants to achieve the same conformational state constitutes a potent example of molecular mimicry and convergence.Keywords:
Ectodomain
Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) cell entry. The gD ectodomain consists of 316 residues and has no sequence homology to any other proteins of known structure. Two fragments of the HSV-1 gD ectodomain (gD22–260: residues 22–260 and gD285: residues 1–285) have been crystallized in two crystal forms. The complex between gD285 and the ectodomain of HveA, a gD cellular receptor member of the tumor necrosis factor (TNFR) superfamily, has also been crystallized. Moreover, insect-cell-expressed selenomethionine-substituted gD285 has been purified and crystallized alone and in complex with HveA.
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Abstract The extracellular domain of several membrane‐anchored proteins is released from the cell surface as soluble proteins through a regulated proteolytic mechanism called ectodomain shedding. Cells use ectodomain shedding to actively regulate the expression and function of surface molecules, and modulate a wide variety of cellular and physiological processes. Ectodomain shedding rapidly converts membrane‐associated proteins into soluble effectors and, at the same time, rapidly reduces the level of cell surface expression. For some proteins, ectodomain shedding is also a prerequisite for intramembrane proteolysis, which liberates the cytoplasmic domain of the affected molecule and associated signaling factors to regulate transcription. Ectodomain shedding is a process that is highly regulated by specific agonists, antagonists, and intracellular signaling pathways. Moreover, only about 2% of cell surface proteins are released from the surface by ectodomain shedding, indicating that cells selectively shed their protein ectodomains. This review will describe the molecular and cellular mechanisms of ectodomain shedding, and discuss its major functions in lung development and disease. Anat Rec, 293:925–937, 2010. © 2010 Wiley–Liss, Inc.
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The renin-angiotensin system (RAS) plays a major role in regulating the cardiovascular, renal, respiratory and central nervous systems. Angiotensin-converting enzyme (ACE) and a newly identified homologue of ACE, angiotensinconverting enzyme 2 (ACE2), are regulators of the RAS. These enzymes, however, have distinct substrate specificity and physiological roles. There is evidence that a balance between ACE and ACE2 expression and activities plays an important role in maintaining normal organ physiology and also in the pathogenesis of various diseases. Thus understanding regulation of the two enzymes could have therapeutic implications. Both ACE and ACE2 are type I transmembrane glycoproteins in which the catalytic domains are located in their ectodomains. These ectoenzymes undergo proteolytic cleavage from the cell surface and this cellular process is known as ectodomain shedding. Ectodomain shedding results in release of the enzymatically active segments into the interstitial space and circulation. Both ACE and ACE2 also function as cell surface receptors that initiate intracellular signaling that can influence gene expression. Thus ectodomain shedding could influence various functions of ACE and ACE2. Recent evidence indicates that ectodomain shedding of these proteins is a highly regulated and versatile process. This review focuses on ectodomain shedding of ACE and ACE2 and summarizes what is known about the molecular and cellular mechanisms, the related clinical findings and potential therapeutic implications.
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Ectodomain shedding is a form of limited proteolysis in which a protease cleaves a transmembrane protein, releasing the extracellular domain from the cell surface. Cells use this process to regulate a wide variety of biological events. Typically, immunological detection methods are employed for the analysis of ectodomains secreted into the cultured media. In this paper, we describe a new strategy using an affinity-based protein-labeling fluorescent probe to study ectodomain shedding. We analyzed the ectodomain shedding of cell surface carbonic anhydrases (CAIX and CAXII), which are important biomarkers for tumor hypoxia. Using both chemical and genetic approaches, we identified that the ADAM17 metalloprotease is responsible for the shedding of carbonic anhydrases. Compared to current immunological methods, this protein-labeling approach not only detects ectodomain released into the culture media but also allows real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, thereby providing a more detailed insight into the mechanism of ectodomain shedding as well as protein lifetime on the cell surface.
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The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.
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Abstract The importance of the widely spread leucine-rich repeat (LRR) motif has been studied considering TLRs, the LRR-containing protein involved in animal immune response. The protein connects intracellular signalling with a chain of molecular interaction through the presence of LRRs in the ectodomain and TIR in the endodomain. Domain analyses with human TLR1-9 reported ectodomain with tandem repeats, transmembrane domain and TIR domain. The repeat number varied across members of TLRs and remains characteristic to a particular member. Analysis of gene structure revealed absence of codon interruption with TLR3 and TLR4 as exceptions. Extensive study with TLR4 from metazoans confirmed the presence of 23 LRRs in tandem. Distinct clade formation using coding and amino acid sequence of individual repeats illustrated independent evolution. Although ectodomain and endodomain exhibited differential selection pressure, however, within the ectodomain, the individual repeats displayed positive, negative and neutral selection pressure depending on their structural and functional significance.
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