RESUMEN
Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.
Asunto(s)
Receptores de Trombopoyetina , Trombopoyetina , Animales , Humanos , Ratones , Ciclo Celular , Microscopía por Crioelectrón , Receptores de Trombopoyetina/genética , Trombopoyesis , Metilación de ADNRESUMEN
Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.
Asunto(s)
Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Factores Inmunológicos/inmunología , Receptores Acoplados a Proteínas G/inmunología , Proteínas Virales/inmunología , Animales , Sitios de Unión/inmunología , Línea Celular , Quimiocinas/inmunología , Microscopía por Crioelectrón/métodos , Infecciones por Virus de Epstein-Barr/virología , Células HEK293 , Humanos , Unión Proteica/inmunología , Células Sf9 , Transducción de Señal/inmunologíaRESUMEN
The IL-17 family of cytokines and receptors have central roles in host defence against infection and development of inflammatory diseases1. The compositions and structures of functional IL-17 family ligand-receptor signalling assemblies remain unclear. IL-17E (also known as IL-25) is a key regulator of type 2 immune responses and driver of inflammatory diseases, such as allergic asthma, and requires both IL-17 receptor A (IL-17RA) and IL-17RB to elicit functional responses2. Here we studied IL-25-IL-17RB binary and IL-25-IL-17RB-IL-17RA ternary complexes using a combination of cryo-electron microscopy, single-molecule imaging and cell-based signalling approaches. The IL-25-IL-17RB-IL-17RA ternary signalling assembly is a C2-symmetric complex in which the IL-25-IL-17RB homodimer is flanked by two 'wing-like' IL-17RA co-receptors through a 'tip-to-tip' geometry that is the key receptor-receptor interaction required for initiation of signal transduction. IL-25 interacts solely with IL-17RB to allosterically promote the formation of the IL-17RB-IL-17RA tip-to-tip interface. The resulting large separation between the receptors at the membrane-proximal level may reflect proximity constraints imposed by the intracellular domains for signalling. Cryo-electron microscopy structures of IL-17A-IL-17RA and IL-17A-IL-17RA-IL-17RC complexes reveal that this tip-to-tip architecture is a key organizing principle of the IL-17 receptor family. Furthermore, these studies reveal dual actions for IL-17RA sharing among IL-17 cytokine complexes, by either directly engaging IL-17 cytokines or alternatively functioning as a co-receptor.
Asunto(s)
Interleucina-17 , Receptores de Interleucina-17 , Microscopía por Crioelectrón , Interleucina-17/química , Interleucina-17/metabolismo , Ligandos , Dominios Proteicos , Multimerización de Proteína , Receptores de Interleucina-17/química , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/ultraestructura , Transducción de Señal , Imagen Individual de MoléculaRESUMEN
Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate ß-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker "grafts" were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.
Asunto(s)
Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Proteínas Wnt , Proteínas Wnt/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Transducción de Señal , Receptores Frizzled/metabolismo , Membrana Celular/metabolismo , beta Catenina/metabolismo , Vía de Señalización WntRESUMEN
The cytokine interferon-γ (IFNγ) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNγ pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNγ receptor IFNγR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNγ-IFNγR1-IFNγR2 signalling complex at 3.25 Šresolution. The structure reveals the mechanism underlying deficits in IFNγ responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNγR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNγ variants to tune IFNγ receptor signalling output. Unexpectedly, we found that several partial IFNγ agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNγ for therapeutic applications.
Asunto(s)
Diseño de Fármacos , Interferón gamma/agonistas , Interferón gamma/inmunología , Receptores de Interferón/química , Receptores de Interferón/metabolismo , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/genética , Línea Celular Tumoral , Agonismo Parcial de Drogas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Interferón gamma/química , Interferón gamma/genética , Ligandos , Modelos Moleculares , Mutación , Infecciones por Mycobacterium/genética , Infecciones por Mycobacterium/inmunología , Estabilidad Proteica , Receptores de Interferón/genética , Transducción de Señal , Relación Estructura-Actividad , Receptor de Interferón gammaRESUMEN
Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.
Asunto(s)
Receptores de Trombopoyetina/metabolismo , Trombopoyetina/metabolismo , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Epítopos/inmunología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ligandos , Megacariocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Trombopoyetina/inmunología , Receptores de Trombopoyetina/fisiología , Transducción de Señal/fisiología , Trombocitemia Esencial/metabolismo , Trombopoyetina/fisiologíaRESUMEN
Membrane receptor guanylyl cyclases play a role in many important facets of human physiology, from regulating blood pressure to intestinal fluid secretion. The structural mechanisms which influence these important physiological processes have yet to be explored. We present the 3.9 Å resolution cryo-EM structure of the human membrane receptor guanylyl cyclase GC-C in complex with Hsp90 and its co-chaperone Cdc37, providing insight into the mechanism of Cdc37 mediated binding of GC-C to the Hsp90 regulatory complex. As a membrane protein and non-kinase client of Hsp90-Cdc37, this work shows the remarkable plasticity of Cdc37 to interact with a broad array of clients with significant sequence variation. Further, this work shows how membrane receptor guanylyl cyclases hijack the regulatory mechanisms used for active kinases to facilitate their regulation. Given the known druggability of Hsp90, these insights can guide the further development of membrane receptor guanylyl cyclase-targeted therapeutics and lead to new avenues to treat hypertension, inflammatory bowel disease, and other membrane receptor guanylyl cyclase-related conditions.
RESUMEN
Membrane receptor guanylyl cyclases play a role in many important facets of human physiology, from regulating blood pressure to intestinal fluid secretion. The structural mechanisms which influence these important physiological processes have yet to be explored. We present the 3.9 Å resolution cryo-EM structure of the human membrane receptor guanylyl cyclase GC-C in complex with Hsp90 and its co-chaperone Cdc37, providing insight into the mechanism of Cdc37 mediated binding of GC-C to the Hsp90 regulatory complex. As a membrane protein and non-kinase client of Hsp90-Cdc37, this work shows the remarkable plasticity of Cdc37 to interact with a broad array of clients with significant sequence variation. Furthermore, this work shows how membrane receptor guanylyl cyclases hijack the regulatory mechanisms used for active kinases to facilitate their regulation. Given the known druggability of Hsp90, these insights can guide the further development of membrane receptor guanylyl cyclase-targeted therapeutics and lead to new avenues to treat hypertension, inflammatory bowel disease, and other membrane receptor guanylyl cyclase-related conditions.
Asunto(s)
Proteínas de Ciclo Celular , Chaperoninas , Proteínas HSP90 de Choque Térmico , Receptores Acoplados a la Guanilato-Ciclasa , Humanos , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Unión Proteica , Receptores Acoplados a la Guanilato-Ciclasa/metabolismoRESUMEN
Janus kinases (JAKs) mediate signal transduction downstream of cytokine receptors. Cytokine-dependent dimerization is conveyed across the cell membrane to drive JAK dimerization, trans-phosphorylation, and activation. Activated JAKs in turn phosphorylate receptor intracellular domains (ICDs), resulting in the recruitment, phosphorylation, and activation of signal transducer and activator of transcription (STAT)-family transcription factors. The structural arrangement of a JAK1 dimer complex with IFNλR1 ICD was recently elucidated while bound by stabilizing nanobodies. While this revealed insights into the dimerization-dependent activation of JAKs and the role of oncogenic mutations in this process, the tyrosine kinase (TK) domains were separated by a distance not compatible with the trans-phosphorylation events between the TK domains. Here, we report the cryoelectron microscopy structure of a mouse JAK1 complex in a putative trans-activation state and expand these insights to other physiologically relevant JAK complexes, providing mechanistic insight into the crucial trans-activation step of JAK signaling and allosteric mechanisms of JAK inhibition.
Asunto(s)
Proteínas de Unión al ADN , Quinasas Janus , Animales , Ratones , Quinasas Janus/metabolismo , Proteínas de Unión al ADN/metabolismo , Microscopía por Crioelectrón , Transactivadores/metabolismo , Janus Quinasa 1/metabolismo , Transducción de Señal , Fosforilación , Janus Quinasa 2/metabolismo , Janus Quinasa 3/metabolismoRESUMEN
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.
Asunto(s)
Microbioma Gastrointestinal , Oxalatos , Animales , Oxalatos/química , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Bacterias/metabolismoRESUMEN
Herpesviruses are ancient large DNA viruses that have exploited gene capture as part of their strategy to escape immune surveillance, promote virus spreading, or reprogram host cells to benefit their survival. Most acquired genes are transmembrane proteins and cytokines, such as viral G protein-coupled receptors (vGPCRs), chemokines, and chemokine-binding proteins. This review focuses on the vGPCRs encoded by the human ß- and γ-herpesviruses. These include receptors from human cytomegalovirus, which encodes four vGPCRs: US27, US28, UL33, and UL78; human herpesvirus 6 and 7 with two receptors: U12 and U51; Epstein-Barr virus with one: BILF1; and Kaposi's sarcoma-associated herpesvirus with one: open reading frame 74, ORF74. We discuss ligand binding, signaling, and structures of the vGPCRs in light of robust differences from endogenous receptors. Finally, we briefly discuss the therapeutic targeting of vGPCRs as future treatment of acute and chronic herpesvirus infections.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesviridae , Quimiocinas/metabolismo , Herpesviridae/genética , Herpesvirus Humano 4/genética , Humanos , Ligandos , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores Virales/metabolismoRESUMEN
Interleukin 27 (IL-27) is a heterodimeric cytokine that functions to constrain T cell-mediated inflammation and plays an important role in immune homeostasis. Binding of IL-27 to cell surface receptors, IL-27Rα and gp130, results in activation of receptor-associated Janus Kinases and nuclear translocation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT3 transcription factors. Despite the emerging therapeutic importance of this cytokine axis in cancer and autoimmunity, a molecular blueprint of the IL-27 receptor signaling complex, and its relation to other gp130/IL-12 family cytokines, is currently unclear. We used cryogenic-electron microscopy to determine the quaternary structure of IL-27, composed of p28 and Epstein-Barr Virus-Induced 3 (Ebi3) subunits, bound to receptors, IL-27Rα and gp130. The resulting 3.47 Å resolution structure revealed a three-site assembly mechanism nucleated by the central p28 subunit of the cytokine. The overall topology and molecular details of this binding are reminiscent of IL-6 but distinct from related heterodimeric cytokines IL-12 and IL-23. These results indicate distinct receptor assembly mechanisms used by heterodimeric cytokines with important consequences for targeted agonism and antagonism of IL-27 signaling.
Asunto(s)
Receptor gp130 de Citocinas , Interleucina-27 , Receptores de Interleucina , Receptor gp130 de Citocinas/química , Humanos , Interleucina-12 , Interleucina-27/química , Estructura Cuaternaria de Proteína , Receptores de Interleucina/químicaRESUMEN
Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus kinases (JAKs). We report the 3.6-angstrom-resolution cryo-electron microscopy structure of full-length JAK1 complexed with a cytokine receptor intracellular domain Box1 and Box2 regions captured as an activated homodimer bearing the valineâphenylalanine (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit, the dimerization of which is mediated by close-packing of the pseudokinase (PK) domains from the monomeric subunits. The oncogenic VF mutation lies within the core of the JAK1 PK interdimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The carboxy-terminal tyrosine kinase domains are poised for transactivation and to phosphorylate the receptor STAT (signal transducer and activator of transcription)-recruiting motifs projecting from the overhanging FERM (four-point-one, ezrin, radixin, moesin)-SH2 (Src homology 2)-domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals opportunities for selective therapeutic targeting of oncogenic JAK signaling.
Asunto(s)
Janus Quinasa 1 , Receptores de Citocinas , Dominios Homologos src , Regulación Alostérica , Microscopía por Crioelectrón , Activación Enzimática , Humanos , Janus Quinasa 1/química , Janus Quinasa 1/metabolismo , Mutación , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Fosforilación , Multimerización de Proteína , Receptores de Citocinas/química , Factores de Transcripción STAT/metabolismoRESUMEN
Human cytomegalovirus (HCMV) encodes G protein-coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo-electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The "orphan" GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate-bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR-G protein coupling for signaling. Our collective results shed new insight into unique G protein-mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity.
Asunto(s)
Citomegalovirus , Receptores de Quimiocina , Animales , Microscopía por Crioelectrón , Citomegalovirus/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Mamíferos/metabolismo , Receptores de Quimiocina/metabolismo , Proteínas Virales/químicaRESUMEN
Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rß, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rß binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.
Asunto(s)
Interleucina-10/química , Interleucina-10/metabolismo , Animales , Sitios de Unión , Linfocitos T CD8-positivos/inmunología , Línea Celular , Microscopía por Crioelectrón , Citocinas/metabolismo , Evolución Molecular Dirigida , Humanos , Inflamación , Interleucina-10/agonistas , Subunidad alfa del Receptor de Interleucina-10/química , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Subunidad beta del Receptor de Interleucina-10/química , Subunidad beta del Receptor de Interleucina-10/metabolismo , Activación de Macrófagos , Ratones , Modelos Moleculares , Monocitos/inmunología , Monocitos/metabolismo , Células Mieloides/inmunología , Células Mieloides/metabolismo , Unión Proteica , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Factor de Transcripción STAT3/metabolismo , Sepsis/inmunología , Transducción de SeñalRESUMEN
This paper reports the self-assembling and electrochemical nature of hexaazatriphenylene-based electron-deficient heteroaromatics with an expanded π-electron system. The tri(phenanthro)hexaazatriphenylenes (TPHAT-Cs) and tri(phenanthrolino)hexaazatriphenylenes (TPHAT-Ns) were prepared by condensation reactions of the corresponding phenanthrenequinones and phenanthrolinediones, respectively, with hexaaminobenzene. Their electron affinity was indicated from cyclic voltammetry measurements, in which the first reduction potentials were evaluated at around -1.7 V (vs Fc/Fc(+)) in dichloromethane. In nonpolar and polar solvents and in the film state, the TPHAT-Cs and TPHAT-Ns formed one-dimensional aggregates with an H-type parallel stacking mode. In the MALDI-TOF mass spectra, significant peaks were seen at several multiples of the parent ion up to tetramer aggregates. The (1)H NMR spectra indicated a line-broadening effect due to the aggregation. The UV-vis and fluorescence spectra showed a concentration dependence, which is attributed to a dynamic exchange between the monomer and aggregate species. The order of the aggregative nature was estimated from the concentration dependence and the fluorescence quantum yield. By replacement of the peripheral aromatic moieties instead of the phenanthrene (TPHAT-Cs) with the phenanthroline (TPHAT-Ns), the aggregative nature was enhanced.
Asunto(s)
Compuestos Aza/síntesis química , Crisenos/síntesis química , Compuestos Aza/química , Crisenos/química , Electroquímica , Electrones , Microscopía de Fuerza Atómica , Estructura Molecular , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/ß-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/ß-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.
Asunto(s)
Receptores Frizzled/ultraestructura , Vía de Señalización Wnt , Microscopía por Crioelectrón , Receptores Frizzled/fisiología , HumanosRESUMEN
Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators.
Asunto(s)
Venenos Elapídicos/química , Receptor Muscarínico M1/química , Receptor Muscarínico M1/genética , Animales , Atropina/química , Cristalografía por Rayos X , Ingeniería Genética , Antagonistas Muscarínicos/química , Conformación Proteica , Receptor Muscarínico M1/antagonistas & inhibidores , Células Sf9RESUMEN
Uncontrolled secretion of mature interleukin (IL)-1ß and IL-18 is responsible for severe autoinflammatory or autoimmune disorders and various allergic diseases. Here we report an intramolecular interaction between IL-18 and its propeptide, which is proteolytically removed from its precursor proIL-18 during maturation. The intramolecular interaction was recapitulated intermolecularly using recombinant propeptide. These results suggest the possibility of developing a novel class of peptide-based IL-18 inhibitors that could serve as therapeutic agents for IL-18-related inflammatory diseases.
Asunto(s)
Interleucina-18/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Humanos , Interleucina-18/química , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Precursores de Proteínas/química , Estabilidad Proteica , ProteolisisRESUMEN
MyD88 (myeloid differentiation factor 88) is an important protein in innate immunity. Two structural domains of MyD88 have been well characterized separately, but the global architecture of full-length MyD88 remained unclear. Here, we propose an autosuppressive mechanism of MyD88 regulated by the intramolecular interaction between the two domains.