Biology subjects

Bignon, E.

Publications and source records attributed to Bignon, E..

31 records · Page 2Linked to original sources

bioRxiv2022

Hijacking of Cellular Functions by SARS-CoV-2. Permeabilization and Polarization of the Host Lipid Membrane by Viroporins.

As all viral infections, SARS-CoV-2 acts at multiple levels hijacking fundamental cellular functions and assuring its replication and immune system evasion. In particular, it has been observed that the viral 3 Open Reading Frame (ORF3a) codes for a hydrophobic protein which embeds in the cellular membrane, where it acts as an ion viroporin and is related to strong inflammatory response. Here we report equilibrium and enhanced sampling molecular dynamic simulation of the SARS-CoV-2 ORF3a in a model lipid bilayer, showing how the protein permeabilizes the lipid membrane, via the formation of a water channel, which in turn assures ion transport. We report the free energy profile for both K+ and Cl- transfer from the cytosol to the extracellular domain. The important role of ORF3a in the viral cycle, and its highly conservation among coronaviruses, may also make it a target of choice for future antiviral development, further justifying the elucidation of its mechanism at the atomistic level. TOC GRAPHICS O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2022

Unraveling the Enzymatic Mechanism of the SARS-CoV-2 RNA-Dependent-RNA-Polymerase. An Unusual Active Site Leading to High Replication Rates.

Viral infection relies on the hijacking of cellular machineries to enforce the reproduction of the infecting virus and its subsequent diffusion. In this context the replication of the viral genome is a key step performed by specific enzymes, i.e. polymerases. The replication of SARS-CoV-2, the causative agent of the COVID-19 pandemics, is based on the duplication of its RNA genome, an action performed by the viral RNA-dependent-RNA polymerase. In this contribution, for the first time and by using two-dimensional enhanced sampling quantum mechanics/ molecular mechanics, we have determined the chemical mechanisms leading to the inclusion of a nucleotide in the nascent viral RNA strand. We prove the high efficiency of the polymerase, which lowers the activation free energy to less than 10 kcal/mol. Furthermore, the SARS-CoV-2 polymerase active site is slightly different from those found usually found in other similar enzymes, and particularly it lacks the possibility to enforce a proton shuttle via a nearby histidine. Our simulations show that this absence is partially compensate by lysine, whose proton assist the reaction opening up an alternative, but highly efficient, reactive channel. Our results present the first mechanistic resolution of SARS-CoV-2 genome replication and shed light on unusual enzymatic reactivity paving the way for future rational design of antivirals targeting emerging RNA viruses.

biophysics↗

bioRxiv2022

Autophagy and evasion of immune system by SARS-CoV-2. Structural features of the Non-structural protein 6 from Wild Type and Omicron viral strains interacting with a model lipid bilayer.

The viral cycle of SARS-CoV-2 is based on a complex interplay with the cellular machinery, which is mediated by specific proteins eluding or hijacking the cellular defense mechanisms. Among the complex pathways called by the viral infection autophagy is particularly crucial and is strongly influenced by the action of the non-structural protein 6 (Nsp6) interacting with the endoplasmic reticulum membrane. Importantly, differently from other non-structural proteins Nsp6 is mutated in the recently emerged Omicron variant, suggesting a possible different role of autophagy. In this contribution we explore, for the first time, the structural property of Nsp6 thanks to long-time scale molecular dynamic simulations and machine learning analysis, identifying the interaction patterns with the lipid membrane. We also show how the mutation brought by the Omicron variant may indeed modify some of the specific interactions, and more particularly help anchoring the viral protein to the lipid bilayer interface. Electronic Supplementary Information (ESI) availableAnalysis protein of the secondary structure and of the specific lipid/amino acid interactions. RMSF per amino acid. Distribution of the distance between the center of mass of the 89 to 99 -helix and the center of the lipid bilayer. Analysis of the behavior of the 195 to 207 -helix. See DOI: 10.1039/x0xx00000x

biophysics↗

bioRxiv2021

Structure of the 5' untranslated region in SARS-CoV-2 genome and its specific recognition by innate immune system via the human oligoadenylate synthase 1

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. In particular, OAS1 is a sensor of endogenous RNA that triggers the 2-5 oligoadenylate/RNase L pathway in response to viral infections, ultimately activating the RNA-Lyase which cleaves endogenous and exogenous RNA impeding the viral maturation. 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 (SLA). Since the structure of the nucleic acid/protein complex has not been resolved, here we report its structure obtained by molecular modeling, including enhanced sampling approaches. We also pinpoint that the SL1 region enhances the interaction network with the enzyme, promoting specific hydrogen bonds, absent in normal double strand RNA fragments, hence rationalizing the high affinity for OAS1.

biophysics↗

bioRxiv2021

Never cared for what they do. High structural stability of Guanine-quadruplexes in presence of strand-break damages

DNA integrity is an important factor to assure genome stability and, more generally, cells and organisms viability. In presence of DNA damage, the normal cell cycle is perturbed while cells activate their repair processes. Although efficient, the repair system is not always able to ensure the complete restoration of gene integrity. In these cases, not only mutations may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold which induces apoptosis and the programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiations are the most toxic, due to their inherently difficult repair, which may lead to genomic instability. In this article we show, by using classical molecular simulations techniques, that differently from the canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show a remarkable structural stability, even in presence of two strand breaks. Since G4-DNA are recognized for their regulatory roles in cell senescence and gene expression, also involving oncogene, their stability can be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.

biophysics↗

bioRxiv2021

Molecular mechanisms associated with clustered lesion-induced impairment of 8-oxoG recognition by the human glycosylase OGG1

The 8-oxo-7,8-dihydroguanine, referred to as 8-oxoG, is a highly mutagenic DNA lesion that can provoke the appearance of mismatches if it escapes the DNA Damage Response. The specific recognition of its structural signature by the hOGG1 glycosylase is the first step along the Base Excision Repair pathway, that ensures the integrity of the genome by preventing the emergence of mutations. 8-oxoG formation, structural features and repair have been the matter of extensive research and more recently this active field of research expended to the more complicated case of 8-oxoG within clustered lesions. Indeed, the presence of a second lesion within 1 or 2 helix turns can dramatically impact the repair yields of 8-oxoG by glycosylases. In this work, we use s-range molecular dynamics simulations and machine learning-based post-analysis to explore the molecular mechanisms associated with the recognition of 8-oxoG by hOGG1 when embedded in a multiple lesions site with a mismatch in 5 or 3. We delineate the stiffening of the DNA-protein interactions upon the presence of the mismatches, and rationalize the much lower repair yields reported with a 5 mismatch by describing the perturbation of 8-oxoG structural features upon addition of an adjacent lesion.

biophysics↗

bioRxiv2021

The iron maiden. Cytosolic aconitase/IRP1 conformational transition in the regulation of ferritin translation and iron hemostasis

Maintaining iron homeostasis is fundamental for almost all living being, and its deregulation correlates with severe and debilitating pathologies. The process is made more complicated by the omnipresence of iron and by its role as a fundamental component of a number of crucial metallo proteins. The response to modifications in the amount of the free iron pool is performed via the inhibition of ferritin translation by sequestering consensus messenger RNA (mRNA) sequences. In turn this is regulated by the iron-sensitive conformational equilibrium between aconitase and IRP, mediated by the presence of an iron-sulfur cluster. In this contribution we analyze by full-atom molecular dynamics simulation, the factors leading to both the interaction with mRNA, and the conformational transition. Furthermore, the role of the iron-sulfur cluster in driving the conformational transition is assessed by obtaining the related free energy profile via enhanced sampling molecular dynamics simulations.

biophysics↗

bioRxiv2021

How fragile we are. Influence of STimulator of INterferon Genes, STING, variants on pathogen recognition and immune response efficiency

The STimulator of INterferon Genes (STING) protein is a cornerstone of the human immune response. Its activation by cGAMP upon the presence of cytosolic DNA stimulates the production of type I interferons and inflammatory cytokines which are crucial for protecting cells from infections. STING signaling pathway can also influence both tumor-suppressive and tumor-promoting mechanisms, rendering it an appealing target for drug design. 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 differential molecular mechanisms exhibited by these variants is of utmost importance notably towards personalized medicine treatments against diseases such as viral infections (COVID-19, Dengue...), cancers, or auto-inflammatory diseases. Owing to micro-seconds scale molecular modeling simulations and post-processing by contacts analysis and Machine Learning techniques, we reveal the dynamical behavior of four STING variants (wild type, G230A, R293Q, and G230A-R293Q) and we rationalize the variability of efficiency observed experimentally. Our results show that the decrease of STING activity is linked to a stiffening of key-structural features of the binding cavity, together with changes of the interaction patterns within the protein.

biochemistry↗

bioRxiv2021

Structure and dynamics of RNA guanine quadruplexes in SARS-CoV-2 genome. Original strategies against emerging viruses

Guanine quadruplexes (G4) structures in 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 modelling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.

biophysics↗

bioRxiv2021

A dynamic view of histone tails interaction with clustered abasic sites in a nucleosome core particle

Apurinic/apyrimidinic sites are the most common DNA damage under physiological conditions. Yet, their structural and dynamical behavior within nucleosome core particles has just begun to be investigated, and show dramatic differences with the one of abasic sites in B-DNA. Clusters of two or more abasic sites are repaired even less efficiently and hence constitute hotspots of high mutagenicity notably due to enhanced double-strand breaks formation. Based on a X-ray structure of a 146-bp DNA wrapped onto a histone core, we investigate the structural behavior of two bistranded abasic sites positioned at mutational hotspots along microsecond-range molecular dynamics simulations. Our simulations allow us to probe histone tails interactions at clustered abasic sites locations, with a definitive assignment of the key residues in-volved in the NCP-catalyzed formation of DNA-protein cross-linking in line with recent experimental findings, and pave the way towards a systematic assessment of histone tails response to DNA lesions.

biochemistry↗

bioRxiv2021

Recognition of a Tandem Lesion by DNA Glycosylases Explored Combining Molecular Dynamics and Machine Learning

The combination of several closely spaced DNA lesions, which can be induced by a single radical hit, constitutes a hallmark in the DNA damage landscape and radiation chemistry. The occurrence of such tandem base lesions give rise to a strong coupling with the double helix degrees of freedom and induce important structural deformations, in contrast to DNA strands containing a single oxidized nucleobase. Although such complex lesions are known to be refractory to repair by DNA glycosylases, there is still a lack of structural evidence to rationalize these phenomena. In this contribution, we explore, by numerical modeling and molecular simulations, the behavior of the bacterial glycosylase responsible for base excision repair (MutM), specialized in excising oxidatively-damaged defects such as 7,8-dihydro-8-oxoguanine (8-oxoG). The difference in lesion recognition between a simple damage and a tandem lesions featuring an additional abasic site is assessed at atomistic resolution owing to microsecond molecular dynamics simulation and machine learning postprocessing, allowing to extensively pinpoint crucial differences in the interaction patterns of the damaged bases. This work advocates for the use of such high throughput numerical simulations for exploring the complex combinatorial chemistry of tandem DNA lesions repair and more generally multiple damaged sites of the utmost significance in radiation chemistry.

biochemistry↗

bioRxiv2020

Impact of the nucleosome histone core on the structure and dynamics of DNA containing pyrimidine-pyrimidone (6-4) photoproduct

The pyrimidine-pyrimidone (6-4) photoproduct (64-PP) is an important photoinduced DNA lesion, which constitutes a mutational signature for melanoma. The structural impact of 64-PP on DNA complexed with compaction proteins, and notably histones, affects the mechanism of its mutagenicity and repair but remains poorly understood. Here we investigate the conformational dynamics of DNA containing 64-PP lesions within the nucleosome core particle by atomic-resolution molecular dynamics simulations at the multi-microsecond time scale. We demonstrate that the histone core exerts important mechanical restraints that largely decrease global DNA structural fluctuations. However, we also show that local DNA flexibility at the damaged site is enhanced, due to imperfect structural adaptation to restraints imposed by the histone core. In particular, if 64-PP faces the histone core and is therefore not directly accessible by the repair protein, the complementary strand facing the solvent exhibits higher flexibility than the corresponding strand in a naked, undamaged DNA. This may serve as an initial recognition signal for repair. Our simulations also pinpoint the structural role of proximal residues from the truncated histone tails.

biophysics↗

bioRxiv2020

Nucleosomal embedding reshapes the dynamics of abasic sites

Apurinic/apyrimidinic (AP) sites are the most common DNA lesions, which benefit from a most efficient repair by the base excision pathway. The impact of losing a nucleobase on the conformation and dynamics of B-DNA is well characterized. Yet AP sites seem to present an entirely different chemistry in nucleosomal DNA, with lifetimes reduced up to 100-fold, and the much increased formation of covalent DNA-protein cross-links, refractory to repair. We report microsecond range, all-atom molecular dynamics simulations that capture the conformational dynamics of AP sites and their tetrahydrofuran analogs at two symmetrical positions within a nucleosome core particle, starting from a recent crystal structure. Different behaviours between the deoxyribo-based and tetrahydrofuran-type abasic sites are evidenced. The two solvent-exposed lesion sites present contrasted extrahelicities, revealing the crucial role of the position of a defect around the histone core. Our all-atom simulations also identify and quantify the occurrence of several spontaneous, non-covalent interactions between AP and positively-charged residues from the histones H2A and H2B tails that prefigure DNA-protein cross-links. This study paves the way towards an in silico mapping of DNA-protein cross-links.

biochemistry↗