NS1 from Two Zika Virus Strains Differently Interact with a Membrane: Insights to Understand Their Differential Virulence.

Zika virus (ZIKV) from Uganda (UG) expresses a phenotype related to fetal loss, whereas the variant from Brazil (BR) induces microcephaly in neonates. The differential virulence has a direct relation to biomolecular mechanisms that make one strain more aggressive than the other. The nonstructural protein 1 (NS1) is a key viral toxin to comprehend these viral discrepancies because of its versatility in many processes of the virus life cycle. Here, we aim to examine through coarse-grained models and molecular dynamics simulations the protein-membrane interactions for both NS1ZIKV-UG and NS1ZIKV-BR dimers. A first evaluation allowed us to establish that the NS1 proteins, in the membrane presence, explore new conformational spaces when compared to systems simulated without a lipid bilayer. These events derive from both differential coupling patterns and discrepant binding affinities to the membrane. The N-terminal domain, intertwined loop, and greasy finger proposed previously as binding membrane regions were also computationally confirmed by us. The anchoring sites have aromatic and ionizable residues that manage the assembly of NS1 toward the membrane, especially for the Ugandan variant. Furthermore, in the presence of the membrane, the difference in the dynamic cross-correlation of residues between the two strains is particularly pronounced in the putative epitope regions. This suggests that the protein-membrane interaction induces changes in the distal part related to putative epitopes. Taken together, these results open up new strategies for the treatment of flaviviruses that would specifically target these dynamic differences.

Both Charge-Regulation and Charge-Patch Distribution Can Drive Adsorption on the Wrong Side of the Isoelectric Point.

The mechanism of protein-polyelectrolyte complexation on the wrong side of the isoelectric point has long puzzled researchers. Two alternative explanations have been proposed in the literature: (a) the charge-patch (CP) mechanism, based on the inhomogeneous distribution of charges on the protein, and (b) the charge-regulation (CR) mechanism, based on the variable charge of weak acid and base groups, which may invert the protein charge in the presence of another highly charged object. To discern these two mechanisms, we simulated artificially constructed short peptides, containing acidic and basic residues, arranged in a blocklike or alternating sequence. Our simulations of these peptides, interacting with polyelectrolytes, showed that charge patch and charge regulation alone can both lead to adsorption on the wrong side of the pI value. Their simultaneous presence enhances adsorption, whereas their absence prevents adsorption. Our simulation results were rationalized by following the variation of the charge regulation capacity and dipole moments of these peptides with the pH. Specifically for lysozyme, we found that charge patch prevails at physiological pH, whereas charge regulation prevails near the pI, thereby explaining seemingly contradicting conclusions in the literature. By applying the same approach to other proteins, we developed a general framework for assessing the role of the CP and CR mechanisms in existing case studies and for predicting how various proteins interact with polyelectrolytes at different pH values.

Identification of Electrostatic Epitopes in Flavivirus by Computer Simulations: The PROCEEDpKa Method.

Viruses are enthusiastically studied due to the great impact that these organisms can have on human health. Computational approaches can contribute offering tools that can shed light on important molecular mechanisms that help to design new diagnostic procedures. Several cellular processes between the immune-host system and the pathogenic organism are dependent on specific intermolecular interactions. In this study, we evaluated theoretical approaches to understand some properties of the antigen-antibody interactions considering the titratable properties of all ionizable residues of the nonstructural viral protein 1 (NS1) of the West Nile virus (WNV) and the Zika virus (ZIKV). Constant-pH Monte Carlo simulations were performed to estimate electrostatic properties such as the pKa shifts (ΔpKa). We proposed an alternative criterion for the discrimination of antigenic residues based on ΔpKas. Our outcomes were analyzed by an evaluation of the sensitivity and specificity through a receiver operating characteristic (ROC). As a starting point, we used the known crystallographic structure for the complex of NS1WNV(176-352) and the specific antibody 22NS1 (PDB ID 4OII ) to differentiate the residues belonging to that interface. With an optimal threshold for the absolute value of the pKa shifts, we found that is possible to predict antigenic epitopes reproducing the interfaces as defined by the X-ray structure. After this validation, we evaluated theoretical predictions based on protein-protein (PP) complexation simulations. From them, we observe amino acids with an antigenic potential and defined the optimum threshold that was applied for two strains of ZIKV (i.e., Uganda and Brazil). Several ionizable residues with antigenic capacity were identified. This is favorably related to some studies that show the high immunogenicity of secreted NS1. This approach opens up an important discussion about what are termed here "electrostatic epitopes" and how they work as an important reference in the paratope-epitope interaction for viral systems.

Combined Experimental and Molecular Simulation Study of Insulin-Chitosan Complexation Driven by Electrostatic Interactions.

Protein-polysaccharide complexes constructed via self-assembly methods are often used to develop novel biomaterials for a wide range of applications in biomedicine, food, and biotechnology. The objective of this work was to investigate theoretically and to demonstrate via constant-pH Monte Carlo simulations that the complexation phenomenon between insulin (INS) and the cationic polyelectrolyte chitosan (CS) is mainly driven by an electrostatic mechanism. Experimental results obtained from FTIR spectra and ζ-potential determinations allowed us to complement the conclusions. The characteristic absorption bands for the complexes could be assigned to a combination of signals from CS amide I and INS amide II. The second peak corresponds to the interaction between the polymer and the protein at the level of amide II. INS-CS complexation processes not expected when INS is in its monomeric form, but for both tetrameric and hexameric forms, incipient complexation due to charge regulation mechanism took place at pH 5. The complexation range was observed to be 5.5 < pH < 6.5. In general, when the number of INS units increases in the simulation process, the solution pH at which the complexation can occur shifts toward acidic conditions. CS's chain interacts more efficiently, i.e. in a wider pH range, with INS aggregates formed by the highest monomer number. The charge regulation mechanism can be considered as a previous phase toward complexation (incipient complexation) caused by weak interactions of a Coulombic nature.

Towards an optimal monoclonal antibody with higher binding affinity to the receptor-binding domain of SARS-CoV-2 spike proteins from different variants.

A highly efficient and robust multiple scales in silico protocol, consisting of atomistic Molecular Dynamics (MD), coarse-grain (CG) MD, and constant-pH CG Monte Carlo (MC), has been developed and used to study the binding affinities of selected antigen-binding fragments of the monoclonal antibody (mAbs) CR3022 and several of its here optimized versions against 11 SARS-CoV-2 variants including the wild type. Totally 235,000 mAbs structures were initially generated using the RosettaAntibodyDesign software, resulting in top 10 scored CR3022-like-RBD complexes with critical mutations and compared to the native one, all having the potential to block virus-host cell interaction. Of these 10 finalists, two candidates were further identified in the CG simulations to be the best against all SARS-CoV-2 variants. Surprisingly, all 10 candidates and the native CR3022 exhibited a higher affinity for the Omicron variant despite its highest number of mutations. The multiscale protocol gives us a powerful rational tool to design efficient mAbs. The electrostatic interactions play a crucial role and appear to be controlling the affinity and complex building. Studied mAbs carrying a more negative total net charge show a higher affinity. Structural determinants could be identified in atomistic simulations and their roles are discussed in detail to further hint at a strategy for designing the best RBD binder. Although the SARS-CoV-2 was specifically targeted in this work, our approach is generally suitable for many diseases and viral and bacterial pathogens, leukemia, cancer, multiple sclerosis, rheumatoid, arthritis, lupus, and more.

Self-association features of NS1 proteins from different flaviviruses.

Flaviviruses comprise a large group of arboviral species that are distributed in several countries of the tropics, neotropics, and some temperate zones. Since they can produce neurological pathologies or vascular damage, there has been intense research seeking better diagnosis and treatments for their infections in the last decades. The flavivirus NS1 protein is a relevant clinical target because it is involved in viral replication, immune evasion, and virulence. Being a key factor in endothelial and tissue-specific modulation, NS1 has been largely studied to understand the molecular mechanisms exploited by the virus to reprogram host cells. A central part of the viral maturation processes is the NS1 oligomerization because many stages rely on these protein-protein assemblies. In the present study, the self-associations of NS1 proteins from Zika, Dengue, and West Nile viruses are examined through constant-pH coarse-grained biophysical simulations. Free energies of interactions were estimated for different oligomeric states and pH conditions. Our results show that these proteins can form both dimers and tetramers under conditions near physiological pH even without the presence of lipids. Moreover, pH plays an important role mainly controlling the regimes where van der Waals interactions govern their association. Finally, despite the similarity at the sequence level, we found that each flavivirus has a well-characteristic protein-protein interaction profile. These specific features can provide new hints for the development of binders both for better diagnostic tools and the formulation of new therapeutic drugs.

Electrostatic Features for the Receptor Binding Domain of SARS-COV-2 Wildtype and Its Variants. Compass to the Severity of the Future Variants with the Charge-Rule.

Electrostatic intermolecular interactions are important in many aspects of biology. We have studied the main electrostatic features involved in the interaction of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with the human receptor Angiotensin-converting enzyme 2 (ACE2). As the principal computational tool, we have used the FORTE approach, capable to model proton fluctuations and computing free energies for a very large number of protein-protein systems under different physical-chemical conditions, here focusing on the RBD-ACE2 interactions. Both the wild-type and all critical variants are included in this study. From our large ensemble of extensive simulations, we obtain, as a function of pH, the binding affinities, charges of the proteins, their charge regulation capacities, and their dipole moments. In addition, we have calculated the pKas for all ionizable residues and mapped the electrostatic coupling between them. We are able to present a simple predictor for the RBD-ACE2 binding based on the data obtained for Alpha, Beta, Gamma, Delta, and Omicron variants, as a linear correlation between the total charge of the RBD and the corresponding binding affinity. This "RBD charge rule" should work as a quick test of the degree of severity of the coming SARS-CoV-2 variants in the future.

VLP-Based COVID-19 Vaccines: An Adaptable Technology against the Threat of New Variants.

Virus-like particles (VLPs) are a versatile, safe, and highly immunogenic vaccine platform. Recently, there are developmental vaccines targeting SARS-CoV-2, the causative agent of COVID-19. The COVID-19 pandemic affected humanity worldwide, bringing out incomputable human and financial losses. The race for better, more efficacious vaccines is happening almost simultaneously as the virus increasingly produces variants of concern (VOCs). The VOCs Alpha, Beta, Gamma, and Delta share common mutations mainly in the spike receptor-binding domain (RBD), demonstrating convergent evolution, associated with increased transmissibility and immune evasion. Thus, the identification and understanding of these mutations is crucial for the production of new, optimized vaccines. The use of a very flexible vaccine platform in COVID-19 vaccine development is an important feature that cannot be ignored. Incorporating the spike protein and its variations into VLP vaccines is a desirable strategy as the morphology and size of VLPs allows for better presentation of several different antigens. Furthermore, VLPs elicit robust humoral and cellular immune responses, which are safe, and have been studied not only against SARS-CoV-2 but against other coronaviruses as well. Here, we describe the recent advances and improvements in vaccine development using VLP technology.

On the interactions of the receptor-binding domain of SARS-CoV-1 and SARS-CoV-2 spike proteins with monoclonal antibodies and the receptor ACE2.

A new betacoronavirus named SARS-CoV-2 has emerged as a new threat to global health and economy. A promising target for both diagnosis and therapeutics treatments of the new disease named COVID-19 is the coronavirus (CoV) spike (S) glycoprotein. By constant-pH Monte Carlo simulations and the PROCEEDpKa method, we have mapped the electrostatic epitopes for four monoclonal antibodies and the angiotensin-converting enzyme 2 (ACE2) on both SARS-CoV-1 and the new SARS-CoV-2 S receptor binding domain (RBD) proteins. We also calculated free energy of interactions and shown that the S RBD proteins from both SARS viruses binds to ACE2 with similar affinities. However, the affinity between the S RBD protein from the new SARS-CoV-2 and ACE2 is higher than for any studied antibody previously found complexed with SARS-CoV-1. Based on physical chemical analysis and free energies estimates, we can shed some light on the involved molecular recognition processes, their clinical aspects, the implications for drug developments, and suggest structural modifications on the CR3022 antibody that would improve its binding affinities for SARS-CoV-2 and contribute to address the ongoing international health crisis.

Occurrence of Biased Conformations as Precursors of Assembly States in Fibril Elongation of Amyloid-ß Fibril Variants: An In Silico Study.

We investigate the prevalence, and so the role in the amyloidogenesis, of biased conformations in large ensembles of monomeric forms for Aß42 and Aß40 that can trigger the formation and growth of fibrils described by a dock-lock mechanism. We model such biased conformations as the structural monomeric units that constitute the Protein Data Bank fibrils 2beg, 2mxu, and 2lmn. These units were employed as templates to search for similar structures in statistical conformational ensembles of Aß peptides generated by molecular dynamics with an accurate force field in explicit solvation, whose high quality is revealed by comparison with residual dipolar coupling (RDC) experiments. The conformational ensembles generated by these intrinsically disordered peptides do not contain conformations highly similar to the amyloidogenic templates. This is a consequence of the low thermodynamic stability exhibited by the template-like conformations. A further constant-pH Monte Carlo study has revealed that this stability can be increased by suitable pH conditions, which helps to trigger the fibril elongation. Moreover, our analyses on the free energy landscapes, hydrogen bond prevalences, and principal component analysis distributions emphasize the relevance of many-body long-range cooperative interactions, likely acting over the infrequent preexisting structurally biased conformations, to explain the fibrils' emergence.

A revised order of subunits in mammalian septin complexes.

Septins are GTP binding proteins considered to be novel components of the cytoskeleton. They polymerize into filaments based on hexameric or octameric core particles in which two copies of either three or four different septins, respectively, assemble into a specific sequence. Viable combinations of the 13 human septins are believed to obey substitution rules in which the different septins involved must come from distinct subgroups. The hexameric assembly, for example, has been reported to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Here, we have replaced SEPT2 by SEPT5 according to the substitution rules and used transmission electron microscopy to demonstrate that the resulting recombinant complex assembles into hexameric particles which are inverted with respect that predicted previously. MBP-SEPT5 constructs and immunostaining show that SEPT5 occupies the terminal positions of the hexamer. We further show that this is also true for the assembly including SEPT2, in direct contradiction with that reported previously. Consequently, both complexes expose an NC interface, as reported for yeast, which we show to be more susceptible to high salt concentrations. The correct assembly for the canonical combination of septins 2-6-7 is therefore established to be SEPT2-SEPT6-SEPT7-SEPT7-SEPT6-SEPT2, implying the need for revision of the mechanisms involved in filament assembly.

Insights into the ZIKV NS1 Virology from Different Strains through a Fine Analysis of Physicochemical Properties.

The flavivirus genus has several organisms responsible for generating various diseases in humans. Recently, especially in tropical regions, Zika virus (ZIKV) has raised great health concerns due to the high number of cases affecting the area during the last years that has been accompanied by a rise in the cases of the Guillain-Barré syndrome and fetal and neonatal microcephaly. Diagnosis is still difficult since the clinical symptoms between ZIKV and other flaviviruses (e.g., dengue and yellow fever) are highly similar. The understanding of their common physicochemical properties that are pH-dependent and biomolecular interaction features and their differences sheds light on the relation strain-virulence and might suggest alternative strategies toward differential serological diagnostics and therapeutic intervention. Due to their immunogenicity, the primary focus of this study was on the ZIKV nonstructural proteins 1 (NS1). By means of computational studies and semiquantitative theoretical analyses, we calculated the main physicochemical properties of this protein from different strains that are directly responsible for the biomolecular interactions and, therefore, can be related to the differential infectivity of the strains. We also mapped the electrostatic differences at both the sequence and structural levels for the strains from Uganda to Brazil, which could suggest possible molecular mechanisms for the increase of the virulence of ZIKV in Brazil. Exploring the interfaces used by NS1 to self-associate in some different oligomeric states and interact with membranes and the antibody, we could map the strategy used by the ZIKV during its evolutionary process. This indicates possible molecular mechanisms that can be correlated with the different immunological responses. By comparing with the known antibody structure available for the West Nile virus, we demonstrated that this antibody would have difficulties to neutralize the NS1 from the Brazilian strain. The present study also opens up perspectives to computationally design high-specificity antibodies.

A combined experimental and molecular simulation study of factors influencing interaction of quinoa proteins-carrageenan.

The interaction between quinoa proteins isolate (QP isolate) and the negatively charged polysaccharide ι-Carragennan (Carr) as a function of pH was studied. Experimental measurements as turbidity, hydrophobic surface, ζ-potential, and hydrodynamic size were carried out. Associative interaction between QP and Carr was found in the pH range between 1 and 2.9. When both molecules are negatively charged (pH>5,5), a pure Coulombic repulsion regime is observed and the self-association of QP due to the Carr exclusion is proposed. In the intermediate pH range, the experimental data suggests that the charge regulation mechanism can overcome the electrostatic repulsion that may take place (and an attraction between QP and Carr can still be observed). Computational simulations by means of free energy derivatives using the Monte Carlo method were carried out to better understand the interaction mechanism between QP and Carr. QP was modeled as a single protein using one of the major proteins, Chenopodin (Ch), and Carr was modeled as a negatively charged polyelectrolyte (NCP) chain, both in the cell model framework. Simulation results showed attractive interactions in agreement with the experimental data.