RESUMEN
Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.
Asunto(s)
Ceramidas/química , Ceramidas/metabolismo , Receptores de Adiponectina/química , Receptores de Adiponectina/metabolismo , Adiponectina/metabolismo , Adiponectina/farmacología , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Ácidos Grasos no Esterificados/química , Ácidos Grasos no Esterificados/metabolismo , Humanos , Hidrólisis/efectos de los fármacos , Hidróxidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Zinc/metabolismoRESUMEN
Viruses have impacted the biosphere in numerous ways since the dawn of life. However, the evolution, genetic, structural, and taxonomic diversity of viruses remain poorly understood, in part because sparse sampling of the virosphere has concentrated mostly on exploring the abundance and diversity of dsDNA viruses. Furthermore, viral genomes are highly diverse, and using only the current sequence-based methods for classifying viruses and studying their phylogeny is complicated. Here we describe a virus, FLiP (Flavobacterium-infecting, lipid-containing phage), with a circular ssDNA genome and an internal lipid membrane enclosed in the icosahedral capsid. The 9,174-nt-long genome showed limited sequence similarity to other known viruses. The genetic data imply that this virus might use replication mechanisms similar to those found in other ssDNA replicons. However, the structure of the viral major capsid protein, elucidated at near-atomic resolution using cryo-electron microscopy, is strikingly similar to that observed in dsDNA viruses of the PRD1-adenovirus lineage, characterized by a major capsid protein bearing two ß-barrels. The strong similarity between FLiP and another member of the structural lineage, bacteriophage PM2, extends to the capsid organization (pseudo T = 21 dextro) despite the difference in the genetic material packaged and the lack of significant sequence similarity.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus ADN/genética , Flavobacterium/virología , Genoma Viral/genética , Bacteriófago PRD1/genética , Cápside , Virus ADN/clasificación , Virus ADN/aislamiento & purificación , ADN de Cadena Simple/genética , Lagos/virología , Conformación ProteicaRESUMEN
We report here the protocol adopted to build the atomic model of the newly discovered virus FLiP (Flavobacterium infecting, lipid-containing phage) into 3.9â¯Å cryo-electron microscopy (cryo-EM) maps. In particular, this report discusses the combination of density modification procedures, automatic model building and bioinformatics tools applied to guide the tracing of the major capsid protein (MCP) of this virus. The protocol outlined here may serve as a reference for future structural determination by cryo-EM of viruses lacking detectable structural homologues.
Asunto(s)
Proteínas de la Cápside/química , Microscopía por Crioelectrón/métodos , Virus ADN/química , Modelos Moleculares , Conformación Proteica , Algoritmos , Biología Computacional/métodos , Virus ADN/genética , Virus ADN/metabolismo , ADN Circular/química , ADN Circular/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Genoma Viral/genéticaRESUMEN
The replication of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), which are the major cause of hand, foot and mouth disease (HFMD) in children, can be inhibited by the capsid binder GPP3. Here, we present the crystal structure of CVA16 in complex with GPP3, which clarifies the role of the key residues involved in interactions with the inhibitor. Based on this model, in silico docking was performed to investigate the interactions with the two next-generation capsid binders NLD and ALD, which we show to be potent inhibitors of a panel of enteroviruses with potentially interesting pharmacological properties. A meta-analysis was performed using the available structural information to obtain a deeper insight into those structural features required for capsid binders to interact effectively and also those that confer broad-spectrum anti-enterovirus activity.
Asunto(s)
Antivirales/farmacología , Enterovirus Humano A/química , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/ultraestructura , Modelos Moleculares , Animales , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Línea Celular , Infecciones por Coxsackievirus/prevención & control , Cristalografía por Rayos X , HumanosRESUMEN
Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin ß-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P(3); K(D) â¼100 µM) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P(3) as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P(2) on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P(3) is affected by the presence of PO(4) ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.
Asunto(s)
Proteínas Portadoras/química , Simulación de Dinámica Molecular , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Ratones , Mutagénesis , Fosfatos/química , Fosfatos/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
The human DEAD-Box Helicase 3 X-Linked (DDX3X) is an ATP-dependent RNA helicase involved in virtually every step of RNA metabolism, ranging from transcription regulation in the nucleus to translation initiation and stress granule (SG) formation, and plays crucial roles in innate immunity, as well as tumorigenesis and viral infections. This review discusses latest advances in DDX3X biology and structure-function relationship, including the implications of the recent DDX3X crystal structure in complex with double stranded RNA for RNA metabolism, DDX3X involvement in the cross-talk between innate immune responses and cell stress adaptation, and the roles of DDX3X in controlling cell fate.
RESUMEN
The design of new therapeutic molecules can be significantly informed by studying protein-ligand interactions using biophysical approaches directly after purification of the protein-ligand complex. Well-established techniques utilized in drug discovery include isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and structure-based drug discovery which mainly rely on protein crystallography and, more recently, cryo-electron microscopy. Protein-ligand complexes are dynamic, heterogeneous, and challenging systems that are best studied with several complementary techniques. Native mass spectrometry (MS) is a versatile method used to study proteins and their non-covalently driven assemblies in a native-like folded state, providing information on binding thermodynamics and stoichiometry as well as insights on ternary and quaternary protein structure. Here, we discuss the basic principles of native mass spectrometry, the field's recent progress, how native MS is integrated into a drug discovery pipeline, and its future developments in drug discovery.
RESUMEN
During the past few years, there have been exciting developments in the field of flavoenzymology. New flavoenzymes have been discovered that are implicated in a variety of biological processes, including cell signaling, chromatin remodeling and cell development. The structures of several of these new flavoenzymes have been described, as exemplified by crystallographic analyses of MICAL, histone demethylase LSD1 and tryptophan dehalogenase. In addition, new structural information has revealed the evolutionary and mechanistic complexity of the enzymes of the riboflavin biosynthetic pathway. The integration of the enzymology data with crystallographic studies at atomic resolution is resulting in unprecedented insight into the chemical and geometric properties underlying flavoenzyme function.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Flavoproteínas/química , Flavoproteínas/metabolismo , Catálisis , Mononucleótido de Flavina/biosíntesis , Mononucleótido de Flavina/química , Flavina-Adenina Dinucleótido/biosíntesis , Flavina-Adenina Dinucleótido/química , Histona Demetilasas , Humanos , Técnicas In Vitro , Modelos Moleculares , Estructura Molecular , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Oxígeno/metabolismo , Conformación ProteicaRESUMEN
Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.
Asunto(s)
Virus de Archaea/fisiología , Ensamble de Virus/fisiología , Virus de Archaea/química , Virus de Archaea/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/ultraestructura , Modelos MolecularesRESUMEN
Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3(1)21 (or P3(2)21), with unit-cell parameters a = 88.6, c = 138.6 A, and exhibit a diffraction limit of 2.3 A.
Asunto(s)
Flavivirus/enzimología , ARN Helicasas/química , Proteínas Virales/química , Cristalización , Estructura Terciaria de Proteína , ARN Helicasas/aislamiento & purificación , Proteínas Virales/aislamiento & purificaciónRESUMEN
Type A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABAARs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABAAR construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABAAR potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABAARs, a process of broad relevance to pentameric ligand-gated ion channels.
Asunto(s)
Neurotransmisores/química , Neurotransmisores/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot and mouth disease (HFMD), for which there are currently no licenced treatments. Here, the acquisition of resistance towards two novel capsid-binding compounds, NLD and ALD, was studied and compared to the analogous compound GPP3. During serial passage, EV71 rapidly became resistant to each compound and mutations at residues I113 and V123 in VP1 were identified. A mutation at residue 113 was also identified in CVA16 after passage with GPP3. The mutations were associated with reduced thermostability and were rapidly lost in the absence of inhibitors. In silico modelling suggested that the mutations prevented the compounds from binding the VP1 pocket in the capsid. Although both viruses developed resistance to these potent pocket-binding compounds, the acquired mutations were associated with large fitness costs and reverted to WT phenotype and sequence rapidly in the absence of inhibitors. The most effective inhibitor, NLD, had a very large selectivity index, showing interesting pharmacological properties as a novel anti-EV71 agent.
Asunto(s)
Antivirales/farmacología , Enterovirus Humano A/efectos de los fármacos , Enterovirus/efectos de los fármacos , Enfermedad de Boca, Mano y Pie/virología , Mutación/efectos de los fármacos , Animales , Cápside/efectos de los fármacos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Cristalografía por Rayos X , Farmacorresistencia Viral , Enterovirus/genética , Enterovirus Humano A/genética , Enfermedad de Boca, Mano y Pie/tratamiento farmacológico , Humanos , Modelos Moleculares , Conformación Proteica , Células VeroRESUMEN
Enterovirus 71 (HEV71) epidemics in children and infants result mainly in mild symptoms; however, especially in the Asia-Pacific region, infection can be fatal. At present, no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics-enhanced ligand docking and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (with IC50 of 25 pM) is an order of magnitude more potent than the best previously reported inhibitor and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections.
Asunto(s)
Proteínas de la Cápside/química , Cápside/química , Enterovirus Humano A/efectos de los fármacos , Imidazoles/química , Antivirales/química , Sitios de Unión , Química Farmacéutica , Diseño de Fármacos , Infecciones por Enterovirus/tratamiento farmacológico , Infecciones por Enterovirus/virología , Concentración 50 Inhibidora , Ligandos , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Cytoplasmic terminal uridylyl transferases comprise a conserved family of enzymes that negatively regulate the stability or biological activity of a variety of eukaryotic RNAs, including mRNAs and tumor-suppressor let-7 microRNAs. Here we describe crystal structures of the Schizosaccharomyces pombe cytoplasmic terminal uridylyl transferase Cid1 in two apo conformers and bound to UTP. We demonstrate that a single histidine residue, conserved in mammalian Cid1 orthologs, is responsible for discrimination between UTP and ATP. We also describe a new high-affinity RNA substrate-binding mechanism of Cid1, which is essential for enzymatic activity and is mediated by three basic patches across the surface of the enzyme. Overall, our structures provide a basis for understanding the activity of Cid1 and a mechanism of UTP selectivity conserved in its human orthologs, suggesting potential implications for anticancer drug design.
Asunto(s)
Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Secuencia de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Secuencia de Bases , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Cartilla de ADN/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nucleotidiltransferasas/genética , Conformación Proteica , ARN de Hongos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Uridina Trifosfato/metabolismoRESUMEN
Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.
Asunto(s)
Proteínas Citotóxicas Formadoras de Poros/química , Esfingomielinas/química , Toxinas Biológicas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Evolución Molecular , Humanos , Células Jurkat , Modelos Moleculares , Datos de Secuencia Molecular , Oligoquetos , Fosforilcolina/química , Proteínas Citotóxicas Formadoras de Poros/farmacología , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Toxinas Biológicas/farmacologíaRESUMEN
The three-dimensional structure of recombinant human monoamine oxidase A (hMAO A) as its clorgyline-inhibited adduct is described. Although the chain-fold of hMAO A is similar to that of rat MAO A and human MAO B (hMAO B), hMAO A is unique in that it crystallizes as a monomer and exhibits the solution hydrodynamic behavior of a monomeric form rather than the dimeric form of hMAO B and rat MAO A. hMAO A's active site consists of a single hydrophobic cavity of approximately 550 A3, which is smaller than that determined from the structure of deprenyl-inhibited hMAO B (approximately 700 A3) but larger than that of rat MAO A (approximately 450 A3). An important component of the active site structure of hMAO A is the loop conformation of residues 210-216, which differs from that of hMAO B and rat MAO A. The origin of this structural alteration is suggested to result from long-range interactions in the monomeric form of the enzyme. In addition to serving as a basis for the development of hMAO A specific inhibitors, these data support the proposal that hMAO A involves a change from the dimeric to the monomeric form through a Glu-151 --> Lys mutation that is specific of hMAO A [Andrès, A. M., Soldevila, M., Navarro, A., Kidd, K. K., Oliva, B. & Bertranpetit, J. (2004) Hum. Genet. 115, 377-386]. These considerations put into question the use of MAO A from nonhuman sources in drug development for use in humans.