RESUMEN
X-ray crystallography and cryo-electron microscopy have enabled the determination of structures of numerous viruses at high resolution and have greatly advanced the field of structural virology. These structures represent only a subset of snapshot end-state conformations, without describing all conformational transitions that virus particles undergo. Allostery plays a critical role in relaying the effects of varied perturbations both on the surface through environmental changes and protein (receptor/antibody) interactions into the genomic core of the virus. Correspondingly, allostery carries implications for communicating changes in genome packaging to the overall stability of the virus particle. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) of whole viruses is a powerful probe for uncovering virus allostery. Here we critically discuss advancements in understanding virus dynamics by HDXMS with single particle cryo-EM and computational approaches.
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Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Virión , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Virión/química , Virión/metabolismo , Regulación Alostérica , Amidas/química , Virus/química , Virus/metabolismo , Microscopía por Crioelectrón/métodos , Espectrometría de Masas/métodos , Medición de Intercambio de DeuterioRESUMEN
Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3'5'cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5'AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5'AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5'AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.
RESUMEN
The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.
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Mamíferos , Polifosfatos , Animales , Especificidad por Sustrato , Fosforilación , Dominio Catalítico , DimerizaciónRESUMEN
Flaviviruses are enveloped viruses which include human pathogens that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making vaccine-based routes of fighting infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it has the potential to diminish the effects of ADE. We examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of raft systems that represent a substantial portion of the flaviviral envelope. We utilised a benzene-mapping approach that led to a discovery of shared hotspots and conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited strain-specific characteristics. An alternative conserved cryptic site at the E protein domain interfaces showed a consistent dynamic behaviour across flaviviruses and contained a conserved cluster of ionisable residues. Constant-pH simulations revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer.
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Flavivirus , Animales , Humanos , Simulación de Dinámica Molecular , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Proteínas del Envoltorio Viral/metabolismoRESUMEN
SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.
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COVID-19 , Humanos , SARS-CoV-2/genética , Amidas , Evolución BiológicaRESUMEN
Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognized by Toll-like receptor 4, mediating proinflammatory effects. We previously reported that LPS directly interacts with SARS-CoV-2 spike (S) protein and enhances proinflammatory activities. Using native gel electrophoresis and hydrogen-deuterium exchange mass spectrometry, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the S protein. Molecular simulations validated by a microscale thermophoresis binding assay revealed that LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting a role as an intermediate in LPS transfer. Congruently, nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells is strongly boosted by S2. Using NF-κB reporter mice followed by bioimaging, a boosting effect was observed for both S1 and S2, with the former potentially facilitated by proteolysis. The Omicron S variant binds to LPS, but with reduced affinity and LPS boosting in vitro and in vivo. Taken together, the data provide a molecular mechanism by which S protein augments LPS-mediated hyperinflammation.
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COVID-19 , FN-kappa B , Humanos , Ratones , Animales , FN-kappa B/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus , Lipopolisacáridos , SARS-CoV-2/metabolismoRESUMEN
Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry. Additionally, we explored the effect of this interaction on infectivity and movement of the virus during infection was explored using plaque assay and fluorescence-based imaging and single particle tracking. GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a 10-fold and 7-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems, respectively. We determined that the interaction of the virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection. Collectively, our results suggest that GM1a functions as a coreceptor/attachment factor for DENV during infection in mammalian systems.
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Virus del Dengue , Dengue , Flavivirus , Animales , Humanos , Virus del Dengue/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Gangliósidos/metabolismo , Flavivirus/metabolismo , Mamíferos/metabolismoRESUMEN
Nuclear receptors function as ligand-regulated transcription factors whose ability to regulate diverse physiological processes is closely linked with conformational changes induced upon ligand binding. Understanding how conformational populations of nuclear receptors are shifted by various ligands could illuminate strategies for the design of synthetic modulators to regulate specific transcriptional programs. Here, we investigate ligand-induced conformational changes using a reconstructed, ancestral nuclear receptor. By making substitutions at a key position, we engineer receptor variants with altered ligand specificities. We combine cellular and biophysical experiments to characterize transcriptional activity, as well as elucidate mechanisms underlying altered transcription in receptor variants. We then use atomistic molecular dynamics (MD) simulations with enhanced sampling to generate ensembles of wildtype and engineered receptors in combination with multiple ligands, followed by conformational analysis and correlation of MD-based predictions with functional ligand profiles. We determine that conformational ensembles accurately describe ligand responses based on observed population shifts. These studies provide a platform which will allow structural characterization of physiologically-relevant conformational ensembles, as well as provide the ability to design and predict transcriptional responses in novel ligands.
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Simulación de Dinámica Molecular , Receptores Citoplasmáticos y Nucleares , Ligandos , Conformación Molecular , Conformación Proteica , Factores de Transcripción , Activación TranscripcionalRESUMEN
The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na+ into bacterial cells. Hyperosmotic stress leads to increased intracellular K+ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K+ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K+ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.
Asunto(s)
Proteínas Bacterianas , Betaína , Corynebacterium glutamicum , Proteínas Transportadoras de GABA en la Membrana Plasmática , Presión Osmótica , Proteínas Bacterianas/química , Betaína/química , Sitios de Unión , Corynebacterium glutamicum/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Enlace de Hidrógeno , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
The COVID-19 pandemic has prompted a rapid response in vaccine and drug development. Herein, we modeled a complete membrane-embedded SARS-CoV-2 spike glycoprotein and used molecular dynamics simulations with benzene probes designed to enhance discovery of cryptic pockets. This approach recapitulated lipid and host metabolite binding sites previously characterized by cryo-electron microscopy, revealing likely ligand entry routes, and uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop. A full representation of glycan moieties was essential to accurately describe pocket dynamics. A multi-conformational behavior of the 617-628 loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry experiments, supportive of opening and closing dynamics. The pocket is the site of multiple mutations associated with increased transmissibility found in SARS-CoV-2 variants of concern including Omicron. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.
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SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Benceno , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Unión Proteica , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Rice black-streaked dwarf virus (RBSDV) is an important reovirus that infects both plants and its transmission vector small brown planthopper, causing severe crop loss. High affinity binding between RBSDV P10 and PI(3,5)P2 lipid layer was measured using biolayer interferometry (BLI). Subcellular co-localization of PI(3,5)P2 and RBSDV P10 was observed on membranous structures in insect cells with stochastic optical reconstruction microscopy (STORM) imaging. Putative interacting sites of PI(3,5)P2 lipid on a computational predicted RBSDV P10 structure were mapped to its "C-domain" (250-470 aa), using HDXMS data. The BLI and STORM results showed binding and co-localization of RBSDV P10, and PI(3,5)P2 on vesicle-like membranous structures were corroborated with the prediction of the binding interface. Understanding the lipid binding sites on viral proteins will lead to developing strategies to block viral-lipid interaction and disrupt viral pathogenesis in insect vectors and to block virus transmission and achieve disease control of crops in the field.
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Hemípteros , Oryza , Virus de Plantas , Reoviridae , Animales , Lípidos , Enfermedades de las Plantas , Virus de Plantas/genéticaRESUMEN
The human monoclonal antibody (HmAb) C10 potently cross-neutralizes Zika virus (ZIKV) and dengue virus. Analysis of antibody fragment (Fab) C10 interactions with ZIKV and dengue virus serotype 2 (DENV2) particles by cryoelectron microscopy (cryo-EM) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS) shows that Fab C10 binding decreases overall ZIKV particle dynamics, whereas with DENV2, the same Fab causes increased dynamics. Testing of different Fab C10:DENV2 E protein molar ratios revealed that, at higher Fab ratios, especially at saturated concentrations, the Fab enhanced viral dynamics (detected by HDXMS), and observation under cryo-EM showed increased numbers of distorted particles. Our results suggest that Fab C10 stabilizes ZIKV but that with DENV2 particles, high Fab C10 occupancy promotes E protein dimer conformational changes leading to overall increased particle dynamics and distortion of the viral surface. This is the first instance of a broadly neutralizing antibody eliciting virus-specific increases in whole virus particle dynamics.
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Anticuerpos Neutralizantes , Virus del Dengue , Dengue , Proteínas del Envoltorio Viral , Infección por el Virus Zika , Virus Zika , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Dengue/inmunología , Dengue/virología , Virus del Dengue/inmunología , Virus del Dengue/fisiología , Humanos , Unión Proteica , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/inmunología , Virus Zika/fisiología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems-cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG, and dual-specificity RegA-RD complexes-and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.
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Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Fosfodiesterasa/farmacología , Extractos Vegetales/farmacología , Proteínas Quinasas/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Dominio Catalítico , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Polarización de Fluorescencia , Terapia Molecular Dirigida , Complejos Multiproteicos/metabolismo , Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Especificidad por SustratoRESUMEN
By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5'-3' panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5'-3' panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.
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Proteínas de la Cápside/metabolismo , Virus del Dengue/metabolismo , Chaperonas Moleculares/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Recombinación Genética/genética , Replicación Viral/genética , Cápside/metabolismo , Proteínas de la Cápside/genética , Codón Iniciador , Ciclización/genética , Virus del Dengue/genética , Cinética , Chaperonas Moleculares/genética , Conformación de Ácido Nucleico , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Epítopos/inmunología , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Dengue/virología , Virus del Dengue/química , Virus del Dengue/metabolismo , Epítopos/metabolismo , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , SerogrupoRESUMEN
The spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface angiotensin-converting enzyme 2 (ACE2) receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat [HR]) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the prefusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Thus, protease docking sites flanking the S1/S2 cleavage site represent alternate allosteric hotspot targets for potential therapeutic development.
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Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sitio Alostérico , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Sitios de Unión , COVID-19/metabolismo , Humanos , Espectrometría de Masas/métodos , Simulación de Dinámica Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteolisis , Receptores Virales/química , Receptores Virales/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Internalización del VirusRESUMEN
A combination of X-ray crystallography, NMR, and mass spectrometry has revealed how diverse small-molecule inhibitors bind Bruton's tyrosine kinase and alter the conformation of this enzyme.
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Agammaglobulinemia Tirosina Quinasa , Cristalografía por Rayos X , Conformación MolecularRESUMEN
The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.
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Regiones no Traducidas 5'/genética , Proteínas de la Cápside/fisiología , Virus del Dengue/metabolismo , Emparejamiento Base/genética , Secuencia de Bases , Proteínas de la Cápside/metabolismo , Secuencia Conservada , Virus del Dengue/genética , Virus del Dengue/fisiología , Genoma Viral/fisiología , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/genética , ARN Circular/química , ARN Circular/genética , ARN Viral/química , ARN Viral/genética , Replicación Viral/genéticaRESUMEN
INTRODUCTION: Erectile dysfunction is the persistent or recurrent inability to achieve or maintain an erection sufficient for intercourse. Despite various treatment options, not all patients respond adequately and their usefulness is limited by adverse effects and cost. Botanical medicine and natural products have been and continue to be invaluable and untapped sources of new drugs, including potentially those to treat erectile dysfunction. OBJECTIVES: To review the current literature on botanical medicine traditionally used as aphrodisiacs and treatment of erectile dysfunction, in particular, scientific and clinical investigations that have been performed, possible active phytoconstituents, and mechanisms of action and to identify gaps in current knowledge to better guide future research efforts. METHODS: A comprehensive literature search was conducted via PubMed, Scopus, Science Direct, and Web of Science on English publications, using various keywords, for example, "herb", "natural product", combined with "erectile dysfunction", "aphrodisiac", and "sexual performance". RESULTS: 369 relevant articles studying medicinal plants used for erectile dysfunction were analyzed. A total of 718 plants from 145 families and 499 genera were reported to be used traditionally as aphrodisiacs and treatment of erectile dysfunction. Top plants used include Pausinystalia johimbe, Lepidium meyenii, and Panax ginseng. Different plant parts are used, with roots being the most common. Less than half of these plants have been evaluated scientifically, using various research methodologies. Clinical trials conducted were collated. Current scientific investigation shows mixed results about their usefulness in enhancing sexual performance. A limited number of studies have attempted to elucidate the mechanisms of action of these medicinal plants. CONCLUSION: A comprehensive literature review on botanical medicine and natural products used for treatment of erectile dysfunction was successfully conducted. Although medicinal plants serve as a potential source of lead compounds for erectile dysfunction drugs, further studies are warranted to further evaluate their efficacy and safety. SinVJ-E, Anand GS, Koh H-L. Botanical Medicine and Natural Products Used for Erectile Dysfunction. Sex Med Rev 2021;9:568-592.
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Afrodisíacos , Productos Biológicos , Disfunción Eréctil , Plantas Medicinales , Afrodisíacos/uso terapéutico , Productos Biológicos/uso terapéutico , Disfunción Eréctil/tratamiento farmacológico , Humanos , Erección PenianaRESUMEN
Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved ß-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.