Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development.

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.

Analysis of Coxsackievirus B5 Infections in the Central Nervous System in Brazil: Insights into Molecular Epidemiology and Genetic Diversity.

Coxsackievirus B5 (CVB5) is one of the most prevalent enteroviruses types in humans and causes annual epidemics worldwide. In the present study, we explored viral genetic diversity, molecular and epidemiological aspects of CVB5 obtained from cerebrospinal fluid and stool samples of patients with aseptic meningitis or acute flaccid paralysis, information that is still scarce in Brazil. From 2005 to 2018, 57 isolates of CVB5 were identified in the scope of the Brazilian Poliomyelitis Surveillance Program. Phylogenetic analyses of VP1 sequences revealed the circulation of two CVB5 genogroups, with genogroup B circulating until 2017, further replaced by genogroup A. Network analysis based on deduced amino acid sequences showed important substitutions in residues known to play critical roles in viral host tropism, cell entry, and viral antigenicity. Amino acid substitutions were investigated by the Protein Variation Effect Analyzer (PROVEAN) tool, which revealed two deleterious substitutions: T130N and T130A. To the best of our knowledge, this is the first report to use in silico approaches to determine the putative impact of amino acid substitutions on the CVB5 capsid structure. This work provides valuable information on CVB5 diversity associated with central nervous system (CNS) infections, highlighting the importance of evaluating the biological impact of certain amino acids substitutions associated with epidemiological and structural analyses.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom.

DM64 is a toxin-neutralizing serum glycoprotein isolated from Didelphis aurita, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A2, which account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor's regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.

Molecular characterization and epidemiological aspects of non-polio enteroviruses isolated from acute flaccid paralysis in Brazil: a historical series (2005-2017).

Due to the advanced stage of polio eradication, the possible role of non-polio enteroviruses (NPEVs) associated to acute flaccid paralysis (AFP) cases has been highlighted. In this study, we described epidemiological aspects of NPEVs infections associated to AFP and explore the viral genetic diversity, information still scarce in Brazil. From 2005 to 2017, 6707 stool samples were collected in the scope of the Brazilian Poliomyelitis Surveillance Program. NPEVs were isolated in 359 samples (5.3%) and 341 (94.9%) were genotyped. About 46 different NPEV types were identified with the following detection pattern EV-B > EV-A > EV-C. The major EV-types were CVA2, CV4, EV-A71, CVB3, CVB5, E6, E7, E11, CVA13 and EV-C99, which corresponds to 51.6% of the total. Uncommon types, such as CVA12, EV-90 and CVA11, were also identified. Different E6 genogroups were observed, prevailing the GenIII, despite periods of co-circulation, and replacement of genogroups along time. CVA2 sequences were classified as genotype C and data suggested its dispersion in South-American countries. CVA13 viruses belonged to cluster B and Venezuelan viruses composed a new putative cluster. This study provides extensive information on enterovirus diversity associated with AFP, reinforcing the need of tailoring current surveillance strategies to timely monitor emergence/re-emergence of NPEVs.

The interaction between the natural metalloendopeptidase inhibitor BJ46a and its target toxin jararhagin analyzed by structural mass spectrometry and molecular modeling.

Snakebite envenoming affects millions of people worldwide, being officially considered a neglected tropical disease by the World Health Organization. The antivenom is effective in neutralizing the systemic effects of envenomation, but local effects are poorly neutralized, often leading to permanent disability. The natural resistance of the South American pit viper Bothrops jararaca to its venom is partly attributed to BJ46a, a natural snake venom metalloendopeptidase inhibitor. Upon complex formation, BJ46a binds non-covalently to the metalloendopeptidase, rendering it unable to exert its proteolytic activity. However, the structural features that govern this interaction are largely unknown. In this work, we applied structural mass spectrometry techniques (cross-linking-MS and hydrogen-deuterium exchange MS) and in silico analyses (molecular modeling, docking, and dynamics simulations) to understand the interaction between BJ46a and jararhagin, a metalloendopeptidase from B. jararaca venom. We explored the distance restraints generated from XL-MS experiments to guide the modeling of BJ46a and jararhagin, as well as the protein-protein docking simulations. HDX-MS data pinpointed regions of protection/deprotection at the interface of the BJ46a-jararhagin complex which, in addition to the molecular dynamics simulation data, reinforced our proposed interaction model. Ultimately, the structural understanding of snake venom metalloendopeptidases inhibition by BJ46a could lead to the rational design of drugs to improve anti-snake venom therapeutics, alleviating the high morbidity rates currently observed.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Natural Inhibitors of Snake Venom Metalloendopeptidases: History and Current Challenges.

The research on natural snake venom metalloendopeptidase inhibitors (SVMPIs) began in the 18th century with the pioneering work of Fontana on the resistance that vipers exhibited to their own venom. During the past 40 years, SVMPIs have been isolated mainly from the sera of resistant animals, and characterized to different extents. They are acidic oligomeric glycoproteins that remain biologically active over a wide range of pH and temperature values. Based on primary structure determination, mammalian plasmatic SVMPIs are classified as members of the immunoglobulin (Ig) supergene protein family, while the one isolated from muscle belongs to the ficolin/opsonin P35 family. On the other hand, SVMPIs from snake plasma have been placed in the cystatin superfamily. These natural antitoxins constitute the first line of defense against snake venoms, inhibiting the catalytic activities of snake venom metalloendopeptidases through the establishment of high-affinity, non-covalent interactions. This review presents a historical account of the field of natural resistance, summarizing its main discoveries and current challenges, which are mostly related to the limitations that preclude three-dimensional structural determinations of these inhibitors using "gold-standard" methods; perspectives on how to circumvent such limitations are presented. Potential applications of these SVMPIs in medicine are also highlighted.

Processing of Snake Venom Metalloproteinases: Generation of Toxin Diversity and Enzyme Inactivation.

Snake venom metalloproteinases (SVMPs) are abundant in the venoms of vipers and rattlesnakes, playing important roles for the snake adaptation to different environments, and are related to most of the pathological effects of these venoms in human victims. The effectiveness of SVMPs is greatly due to their functional diversity, targeting important physiological proteins or receptors in different tissues and in the coagulation system. Functional diversity is often related to the genetic diversification of the snake venom. In this review, we discuss some published evidence that posit that processing and post-translational modifications are great contributors for the generation of functional diversity and for maintaining latency or inactivation of enzymes belonging to this relevant family of venom toxins.

Dissection of the water cavity of yeast thioredoxin 1: the effect of a hydrophobic residue in the cavity.

The water cavity of yeast thioredoxin 1 (yTrx1) is an ancestral, conserved structural element that is poorly understood. We recently demonstrated that the water cavity is involved in the complex protein dynamics that are responsible for the catalytically relevant event of coupling hydration, proton exchange, and motion at the interacting loops. Its main feature is the presence of the conserved polar residue, Asp24, which is buried in a hydrophobic cavity. Here, we evaluated the role of the solvation of Asp24 as the main element that is responsible for the formation of the water cavity in thioredoxins. We showed that the substitution of Asp24 with a hydrophobic residue (D24A) was not sufficient to completely close the cavity. The dynamics of the D24A mutant of yTrx1 at multiple time scales revealed that the D24A mutant presents motions at different time scales near the active site, interaction loops, and water cavity, revealing the existence of a smaller dissected cavity. Molecular dynamics simulation, along with experimental molecular dynamics, allowed a detailed description of the water cavity in wild-type yTrx1 and D24A. The cavity connects the interacting loops, the central ß-sheet, and α-helices 2 and 4. It is formed by three contiguous lobes, which we call lobes A-C. Lobe A is hydrophilic and the most superficial. It is formed primarily by the conserved Lys54. Lobe B is the central lobe formed by the catalytically important residues Cys33 and Asp24, which are strategically positioned. Lobe C is the most hydrophobic and is formed by the conserved cis-Pro73. The central lobe B is closed upon introduction of the D24A mutation, revealing that independent forces other than solvation of Asp24 maintain lobes A and C in the open configuration. These data allow us to better understand the properties of this enzyme.

Hydration and conformational equilibrium in yeast thioredoxin 1: implication for H(+) exchange.

One of the ancestral features of thioredoxins is the presence of a water cavity. Here, we report that a largely hydrated, conserved, buried aspartic acid in the water cavity modulates the dynamics of the interacting loops of yeast thioredoxin 1 (yTrx1). It is well-established that the aspartic acid, Asp24 for yTrx1, works as a proton acceptor in the reduction of the target protein. We propose a complementary role for Asp24 of coupling hydration and conformational motion of the water cavity and interacting loops. The intimate contact between the water cavity and the interacting loops means that motion at the water cavity will affect the interacting loops and vice versa. The D24N mutation alters the conformational equilibrium for both the oxidized and reduced states, quenching the conformational motion in the water cavity. By measuring the hydration and molecular dynamics simulation of wild-type yTrx1 and the D24N mutant, we showed that Asn24 is more exposed to water than Asp24 and the water cavity is smaller in the mutant, closing the inner part of the water cavity. We discuss how the conformational equilibrium contributes to the mechanism of catalysis and H(+) exchange.

Modeling the interaction of dodecylphosphocholine micelles with the anticoccidial peptide PW2 guided by NMR data.

Antimicrobial peptides are highly dynamic entities that acquire structure upon binding to a membrane interface. To better understand the structure and the mechanism for the molecular recognition of dodecylphosphocholine (DPC) micelles by the anticoccidial peptide PW2, we performed molecular dynamics (MD) simulations guided by NMR experimental data, focusing on strategies to explore the transient nature of micelles, which rearrange on a millisecond to second timescale. We simulated the association of PW2 with a pre-built DPC micelle and with free-DPC molecules that spontaneously forms micelles in the presence of the peptide along the simulation. The simulation with spontaneous micelle formation provided the adequate environment which replicated the experimental data. The unrestrained MD simulations reproduced the NMR structure for the entire 100 ns MD simulation time. Hidden discrete conformational states could be described. Coulomb interactions are important for initial approximation and hydrogen bonds for anchoring the aromatic region at the interface, being essential for the stabilization of the interaction. Arg9 is strongly attached with phosphate. We observed a helix elongation process stabilized by the intermolecular peptide-micelle association. Full association that mimics the experimental data only happens after complete micelle re-association. Fast micelle dynamics without dissociation of surfactants leads to only superficial binding.

The disordered N-terminal region of dengue virus capsid protein contains a lipid-droplet-binding motif.

Dengue is the major arthropod-borne human viral disease, for which no vaccine or specific treatment is available. We used NMR, zeta potential measurements and atomic force microscopy to study the structural features of the interaction between dengue virus C (capsid) protein and LDs (lipid droplets), organelles crucial for infectious particle formation. C protein-binding sites to LD were mapped, revealing a new function for a conserved segment in the N-terminal disordered region and indicating that conformational selection is involved in recognition. The results suggest that the positively charged N-terminal region of C protein prompts the interaction with negatively charged LDs, after which a conformational rearrangement enables the access of the central hydrophobic patch to the LD surface. Taken together, the results allowed the design of a peptide with inhibitory activity of C protein-LD binding, paving the way for new drug development approaches against dengue.

Effect of micelle interface on the binding of anticoccidial PW2 peptide.

PW2 is an anticoccidial peptide active against Eimeria acervulina and Eimeria tenella. We determined the structure of PW2 in dodecylphosphocholine micelles. The structure showed two distinct regions: an amphipathic N-terminal 3(10) helix and an aromatic region containing WWR interface-binding motif. The aromatic region acted as a scaffold of the protein in the interface and shared the same structure in both DPC and SDS micelles. N-terminal helix interacted with DPC but not with SDS interface. Chemical shift change was slow when SDS was added to PW2 in DPC and fast when DPC was added to PW2 in SDS, indicating that interaction with DPC micelles was kinetically more stable than with SDS micelles. Also, DPC interface was able to accommodate PW2, but it maintained the conformational arrangement in the aromatic region observed for SDS micelles. This behavior, which is different from that observed for other antimicrobial peptides with WWR motif, may be associated with the absence of PW2 antibacterial activity and its selectivity for Eimeria parasites.