Hidden pathway for cytokine receptor activation: Structural insights into a marine sponge-derived lectin that activates the thrombopoietin receptor via recognition of the fucose moiety

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism remains unstudied because no external agonist has been discovered. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC). ThC-induced activation persists due to limited receptor internalization. Strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. We show that MPL is subject to sugar-mediated activation and that lectin-mediated activation kinetics differ from cytokine-mediated activation kinetics. Our data demonstrated the potential of lectins to provide deeper insight into human pathogenesis.

Modeling the Heterodimer Interfaces of Melatonin Receptors

Melatonin receptors are Class A G protein-coupled receptors (GPCRs) that regulate a plethora of physiological activities in response to the rhythmic secretion of melatonin from the pineal gland. Melatonin is a key regulator in the control of circadian rhythm and has multiple functional roles in retinal physiology, memory, immunomodulation and tumorigenesis. The two subtypes of human melatonin receptors, termed MT1 and MT2, utilize overlapping signaling pathways although biased signaling properties have been reported in some cellular systems. With the emerging concept of GPCR dimerization, melatonin receptor heterodimers have been proposed to participate in system-biased signaling. Here, we used computational approaches to map the dimerization interfaces of known heterodimers of melatonin receptors, including MT1/MT2, MT1/GPR50, MT2/GPR50, and MT2/5-HT2C. By homology modeling and membrane protein docking analyses, we have identified putative preferred interface interactions within the different pairs of melatonin receptor dimers and provided plausible structural explanations for some of the unique pharmacological features of specific heterodimers previously reported. A thorough understanding of the molecular basis of melatonin receptor heterodimers may enable the development of new therapeutic approaches against aliments involving these heterodimeric receptors.

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well-defined complexes with CheA and CheW and promoted a CheA kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar electron-spin resonance spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo–electron microscopy. The kinase-off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. Together, these data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognise and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2-CLD3. Comparison with a cryo-EM structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1 complex indicates conformational changes within a clade-specific CLD3 loop adjacent to co-receptor. Our observations indicate RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognises a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualise linear arrays of RETECD-GDNF-GFRα1 suggesting a conserved contact stabilises higher-order species. Our study reveals ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.HighlightsCrystal structure of zebrafish RET cadherin-like module reveals conformational flexibility at the calcium-dependent CLD2-CLD3 interfaceComparison of X-ray and cryo-EM structures indicate conformational differences between unliganded and liganded RET involving a clade-specific CLD3 loopStrict spatial separation of RETECD C-termini is imposed by each cysteine-rich domain interaction with GFL dimerDifferences in co-receptor engagement and higher-order ligand-bound RET complexes indicate potentially divergent signalling mechanisms

A DNA Aptamer That Inhibits the Formation of Unliganded Receptor Dimer and Ligand-Independent Signaling in Cancer Cells

<p>Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, in cancer cells, the overexpression of receptors often causes the formation of unliganded receptor dimers, which can be activated in a ligand-independent manner. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that inhibits ligand-independent receptor activation via preventing the formation of unliganded receptor dimer. By biasing the receptor monomer–dimer equilibrium to the monomer, this aptamer inhibited aberrant cell signaling caused by the unliganded receptor dimer. This work presents a new possibility of oligonucleotide-based therapeutics for cancer.</p>

A DNA Aptamer That Inhibits the Formation of Unliganded Receptor Dimer and Ligand-Independent Signaling in Cancer Cells

<p>Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, in cancer cells, the overexpression of receptors often causes the formation of unliganded receptor dimers, which can be activated in a ligand-independent manner. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that inhibits ligand-independent receptor activation via preventing the formation of unliganded receptor dimer. By biasing the receptor monomer–dimer equilibrium to the monomer, this aptamer inhibited aberrant cell signaling caused by the unliganded receptor dimer. This work presents a new possibility of oligonucleotide-based therapeutics for cancer.</p>

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a “dipped conformation” that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.