RESUMEN
A 1.7 Å structure is presented for an active form of the virulence factor ScpB, the C5a peptidase from Streptococcus agalactiae. The previously reported structure of the ScpB active site mutant exhibited a large separation (~20 Å) between the catalytic His and Ser residues. Significant differences are observed in the catalytic domain between the current and mutant ScpB structures resulting with a high RMSDCα (4.6 Å). The fold of the active form of ScpB is nearly identical to ScpA (RMSDCα 0.2 Å), the C5a-peptidase from Streptococcus pyogenes. Both ScpA and ScpB have comparable activity against human C5a, indicating neither enzyme require host proteins for C5a-ase activity. These studies are a first step in resolving reported differences in the specificities of these enzymes.
Asunto(s)
Endopeptidasas , Streptococcus agalactiae , Humanos , Streptococcus agalactiae/metabolismo , Dominio Catalítico , Endopeptidasas/química , Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Catálisis , Streptococcus pyogenesRESUMEN
Guanine quadruplexes (G4s) are nucleic acid structures exhibiting a complex structural behavior and exerting crucial biological functions in both cells and viruses. The specific interactions of peptides with G4s, as well as an understanding of the factors driving the specific recognition are important for the rational design of both therapeutic and diagnostic agents. In this review, we examine the most important studies dealing with the interactions between G4s and peptides, highlighting the strengths and limitations of current analytic approaches. We also show how the combined use of high-level molecular simulation techniques and experimental spectroscopy is the best avenue to design specifically tuned and selective peptides, thus leading to the control of important biological functions.
Asunto(s)
G-Cuádruplex , PéptidosRESUMEN
We investigated the mechanisms leading to the specific recognition of Guanine Guadruplex (G4) by DARPins peptides, which can lead to the design of G4â s specific sensors. To this end we carried out all-atom molecular dynamic simulations to unravel the interactions between specific nucleic acids, including human-telomeric (h-telo), Bcl-2, and c-Myc, with different peptides, forming a DARPin/G4 complex. By comparing the sequences of DARPin with that of a peptide known for its high affinity for c-Myc, we show that the recognition cannot be ascribed to sequence similarity but, instead, depends on the complementarity between the three-dimensional arrangement of the molecular fragments involved: the α-helix/loops domain of DARPin and the G4 backbone. Our results reveal that DARPins tertiary structure presents a charged hollow region in which G4 can be hosted, thus the more complementary the structural shapes, the more stable the interaction.
Asunto(s)
G-Cuádruplex , Ácidos Nucleicos , Sitios de Unión de Anticuerpos , Proteínas de Repetición de Anquirina Diseñadas , Epítopos , Guanina/química , Humanos , Péptidos/química , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
The stimulator of interferon genes (STING) protein is a cornerstone of the human immune response. Its activation by cGAMP in the presence of cytosolic DNA stimulates the production of type I interferons and inflammatory cytokines. In the human population, several STING variants exist and exhibit dramatic differences in their activity, impacting the efficiency of the host defense against infections. Understanding the molecular mechanisms of these variants opens perspectives for personalized medicine treatments against diseases such as viral infections, cancers, or autoinflammatory diseases. Through microsecond-scale molecular modeling simulations, contact analyses, and machine learning techniques, we reveal the dynamic behavior of four STING variants (wild type, G230A, R293Q, and G230A/R293Q) and rationalize the variability of efficiency observed experimentally. Our results show that the decrease in STING activity is linked to a stiffening of key structural elements of the binding cavity together with changes in the interaction patterns within the protein.
Asunto(s)
Inmunidad Innata , Proteínas de la Membrana , Humanos , Interferones , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
DNA integrity is an important factor that assures genome stability and, more generally, the viability of cells and organisms. In the presence of DNA damage, the normal cell cycle is perturbed when cells activate their repair processes. Although efficient, the repair system is not always able to ensure complete restoration of gene integrity. In these cases, mutations not only may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold that induces apoptosis and programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiation are the most toxic due to the inherent difficultly of repair, which may lead to genomic instability. In this article we show, by using classical molecular simulation techniques, that compared to canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show remarkable structural stability, even in the presence of two strand breaks. Since G4-DNA is recognized for its regulatory roles in cell senescence and gene expression, including oncogenes, this stability may be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.
Asunto(s)
Reparación del ADN , G-Cuádruplex , ADN/efectos de la radiación , Daño del ADN , Inestabilidad Genómica , HumanosRESUMEN
Human telomeric DNA, in G-quadruplex (G4) conformation, is characterized by a remarkable structural stability that confers it the capacity to resist to oxidative stress producing one or even clustered 8-oxoguanine (8oxoG) lesions. We present a combined experimental/computational investigation, by using circular dichroism in aqueous solutions, cellular immunofluorescence assays and molecular dynamics simulations, that identifies the crucial role of the stability of G4s to oxidative lesions, related also to their biological role as inhibitors of telomerase, an enzyme overexpressed in most cancers associated to oxidative stress.
Asunto(s)
G-Cuádruplex , Telomerasa , Dicroismo Circular , ADN/metabolismo , Humanos , Conformación de Ácido Nucleico , Estrés Oxidativo , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
The identification of chemical compounds able to bind specific sites of the human/viral proteins involved in the SARS-CoV-2 infection cycle is a prerequisite to design effective antiviral drugs. Here we conduct a molecular dynamics study with the aim to assess the interactions of ivermectin, an antiparasitic drug with broad-spectrum antiviral activity, with the human Angiotensin-Converting Enzyme 2 (ACE2), the viral 3CLpro and PLpro proteases, and the viral SARS Unique Domain (SUD). The drug/target interactions have been characterized in silico by describing the nature of the non-covalent interactions found and by measuring the extent of their time duration along the MD simulation. Results reveal that the ACE2 protein and the ACE2/RBD aggregates form the most persistent interactions with ivermectin, while the binding with the remaining viral proteins is more limited and unspecific.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/metabolismo , Proteasas 3C de Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Ivermectina/metabolismo , Enzima Convertidora de Angiotensina 2/química , Antivirales/química , Sitios de Unión , Proteasas 3C de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/química , G-Cuádruplex , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ivermectina/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , ARN/genética , ARN/metabolismo , SARS-CoV-2RESUMEN
The emergence in late 2019 of the coronavirus SARS-CoV-2 has resulted in the breakthrough of the COVID-19 pandemic that is presently affecting a growing number of countries. The development of the pandemic has also prompted an unprecedented effort of the scientific community to understand the molecular bases of the virus infection and to propose rational drug design strategies able to alleviate the serious COVID-19 morbidity. In this context, a strong synergy between the structural biophysics and molecular modeling and simulation communities has emerged, resolving at the atomistic level the crucial protein apparatus of the virus and revealing the dynamic aspects of key viral processes. In this Review, we focus on how in silico studies have contributed to the understanding of the SARS-CoV-2 infection mechanism and the proposal of novel and original agents to inhibit the viral key functioning. This Review deals with the SARS-CoV-2 spike protein, including the mode of action that this structural protein uses to entry human cells, as well as with nonstructural viral proteins, focusing the attention on the most studied proteases and also proposing alternative mechanisms involving some of its domains, such as the SARS unique domain. We demonstrate that molecular modeling and simulation represent an effective approach to gather information on key biological processes and thus guide rational molecular design strategies.
Asunto(s)
Antivirales , Infecciones por Coronavirus , Diseño de Fármacos , Simulación del Acoplamiento Molecular , Pandemias , Neumonía Viral , Glicoproteína de la Espiga del Coronavirus , Betacoronavirus , COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Humanos , Simulación de Dinámica Molecular , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Internalización del VirusRESUMEN
The genome of SARS-CoV-2 coronavirus is made up of a single-stranded RNA fragment that can assume a specific secondary structure, whose stability can influence the virus's ability to reproduce. Recent studies have identified putative guanine quadruplex sequences in SARS-CoV-2 genome fragments that are involved in coding for both structural and non-structural proteins. In this contribution, we focus on a specific G-rich sequence referred to as RG-2, which codes for the non-structural protein 10 (Nsp10) and assumes a guanine-quadruplex (G4) arrangement. We provide the secondary structure of RG-2 G4 at atomistic resolution by molecular modeling and simulation, validated by the superposition of experimental and calculated electronic circular dichroism spectra. Through both experimental and simulation approaches, we have demonstrated that pyridostatin (PDS), a widely recognized G4 binder, can bind to and stabilize RG-2 G4 more strongly than RG-1, another G4 forming sequence that was previously proposed as a potential target for antiviral drug candidates. Overall, this study highlights RG-2 as a valuable target to inhibit the translation and replication of SARS-CoV-2, paving the way towards original therapeutic approaches against emerging RNA viruses.
RESUMEN
The Transmembrane Protease Serine 2 (TMPRSS2) is a human enzyme which is involved in the maturation and post-translation of different proteins. In addition to being overexpressed in cancer cells, TMPRSS2 plays a further fundamental role in favoring viral infections by allowing the fusion of the virus envelope with the cellular membrane, notably in SARS-CoV-2. In this contribution, we resort to multiscale molecular modeling to unravel the structural and dynamical features of TMPRSS2 and its interaction with a model lipid bilayer. Furthermore, we shed light on the mechanism of action of a potential inhibitor (nafamostat), determining the free-energy profile associated with the inhibition reaction and showing the facile poisoning of the enzyme. Our study, while providing the first atomistically resolved mechanism of TMPRSS2 inhibition, is also fundamental in furnishing a solid framework for further rational design targeting transmembrane proteases in a host-directed antiviral strategy.
Asunto(s)
Antivirales , COVID-19 , Humanos , Antivirales/farmacología , Antivirales/química , SARS-CoV-2 , Membrana Celular , SerinaRESUMEN
Guanine quadruplexes (G4s) play essential protective and regulatory roles within cells, influencing gene expression. In several gene-promoter regions, multiple G4-forming sequences are in close proximity and may form three-dimensional arrangements. We analyze the interplay among the three neighboring G4s in the c-KIT proto-oncogene promoter (WK1, WSP, and WK2). We highlight that the three G4s are structurally linked and their cross-talk favors the formation of a parallel structure for WSP. Relying on all-atom molecular dynamic simulations exceeding the µs time scale and using enhanced sampling methods, we provide the first computationally resolved structure of a well-organized G4 cluster in the promoter of a crucial gene involved in cancer development. Our results indicate that neighboring G4s influence their mutual three-dimensional arrangement and provide a powerful tool to predict and interpret complex DNA structures that can ultimately be used as a starting point for drug discovery.
Asunto(s)
G-Cuádruplex , Regiones Promotoras Genéticas/genética , ADN/química , Proto-OncogenesRESUMEN
2'-5'-Oligoadenylate synthetase 1 (OAS1) is one of the key enzymes driving the innate immune system response to SARS-CoV-2 infection whose activity has been related to COVID-19 severity. OAS1 is a sensor of endogenous RNA that triggers the 2'-5'-oligoadenylate/RNase L pathway. Upon SARS-CoV-2 infection, OAS1 is responsible for the recognition of viral RNA and has been shown to possess a particularly high sensitivity for the 5'-untranslated (5'-UTR) RNA region, which is organized in a double-strand stem loop motif (SL1). Here we report the structure of the SL1/OAS1 complex also rationalizing the high affinity for OAS1.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Inmunidad Innata , ARN Viral/metabolismo , SARS-CoV-2/genética , Regiones no Traducidas 5' , Secuencia de Bases , Sitios de Unión , COVID-19/patología , COVID-19/virología , Humanos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , SARS-CoV-2/aislamiento & purificaciónRESUMEN
In the last few years, the sudden outbreak of COVID-19 caused by SARS-CoV-2 proved the crucial importance of understanding how emerging viruses work and proliferate, in order to avoid the repetition of such a dramatic sanitary situation with unprecedented social and economic costs. West Nile Virus is a mosquito-borne pathogen that can spread to humans and induce severe neurological problems. This RNA virus caused recent remarkable outbreaks, notably in Europe, highlighting the need to investigate the molecular mechanisms of its infection process in order to design and propose efficient antivirals. Here, we resort to all-atom Molecular Dynamics simulations to characterize the structure of the 5'-untranslated region of the West Nile Virus genome and its specific recognition by the human innate immune system via oligoadenylate synthetase. Our simulations allowed us to map the interaction network between the viral RNA and the host protein, which drives its specific recognition and triggers the host immune response. These results may provide fundamental knowledge that can assist further antivirals' design, including therapeutic RNA strategies.
Asunto(s)
COVID-19 , Fiebre del Nilo Occidental , Virus del Nilo Occidental , Regiones no Traducidas 5' , Animales , Antivirales , Humanos , Sistema Inmunológico , SARS-CoV-2/genética , Virus del Nilo Occidental/fisiologíaRESUMEN
Guanine quadruplex (G4) structures in the viral genome have a key role in modulating viruses' biological activity. While several DNA G4 structures have been experimentally resolved, RNA G4s are definitely less explored. We report the first calculated G4 structure of the RG-1 RNA sequence of SARS-CoV-2 genome, obtained by using a multiscale approach combining quantum and classical molecular modeling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of the RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.
Asunto(s)
COVID-19/genética , G-Cuádruplex , Genoma Viral , ARN Viral/química , ARN Viral/genética , SARS-CoV-2/genética , COVID-19/virología , Humanos , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
Since the end of 2019, the coronavirus SARS-CoV-2 has caused more than 1000000 deaths all over the world and still lacks a medical treatment despite the attention of the whole scientific community. Human angiotensin-converting enzyme 2 (ACE2) was recently recognized as the transmembrane protein that serves as the point of entry of SARS-CoV-2 into cells, thus constituting the first biomolecular event leading to COVID-19 disease. Here, by means of a state-of-the-art computational approach, we propose a rational evaluation of the molecular mechanisms behind the formation of the protein complex. Moreover, the free energy of binding between ACE2 and the active receptor binding domain of the SARS-CoV-2 spike protein is evaluated quantitatively, providing for the first time the thermodynamics of virus-receptor recognition. Furthermore, the action of different ACE2 ligands is also examined in particular in their capacity to disrupt SARS-CoV-2 recognition, also providing via a free energy profile the quantification of the ligand-induced decreased affinity. These results improve our knowledge on molecular grounds of the SARS-CoV-2 infection and allow us to suggest rationales that could be useful for the subsequent wise molecular design for the treatment of COVID-19 cases.
Asunto(s)
Betacoronavirus/metabolismo , Ligandos , Peptidil-Dipeptidasa A/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2 , Sitios de Unión , COVID-19 , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Diosmina/química , Diosmina/metabolismo , Humanos , Simulación de Dinámica Molecular , Pandemias , Peptidil-Dipeptidasa A/química , Plicamicina/química , Plicamicina/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , Unión Proteica , Dominios Proteicos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , TermodinámicaRESUMEN
Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible for the global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting the SARS viruses to bypass the host protective response. This work also constitutes a first step in the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.