In response to emergence of global COVID-19 pandemic, studies of SARS-CoV-2 have been well underway with an unprecedentedly fast phase particularly for vaccine development. While spike proteins and proteases Mpro and PLpro are getting much of attention as therapeutic drug targets against COVID-19, however, the progress in developing drugs is still lagging behind. Non-structural protein 15 (Nsp15) is another SARS-CoV-2 protein demanding researchers’ attention as a critical drug target. Nsp15 is an endoribonuclease and an essential enzyme for SARS CoV-2 with a role of interfering host immune response. It has been reported that Nsp15 is evading melanoma differentiation-associated gene 5 (MDA5) activity which is triggered by ds/ssRNA molecular pattern produced by replication-transcription complex by trimming replication produced (-) strand polyU track. We characterized Nsp15 by crystallography, biochemical, and whole-cell assays. Several structures of Nsp15: the Apo-form and several ligands bound forms are determined. Our Nsp15 structures with nucleotide-base ligands elucidated how Nsp15 recognizes uridine base specifically. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms which is mimicking that of RNaseA. We also found an uracil analog Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. These findings can be new insights for the development of uracil scaffold-based therapeutic drugs.

The PDBe-KB COVID-19 data portal, developed by the team at Protein Data Bank in Europe Knowledge Base (PDBe-KB), aggregates all the available structure data from SARS-CoV-2 structures in the PDB, to help researchers easily identify important structural features to support the development of treatments and vaccines.

Since the beginning of the COVID-19 pandemic, an unprecedented number of scientific efforts have taken place worldwide in order to help combat the SARS-CoV-2 virus. One of the biggest challenges during this fast-moving situation was to share data and findings in a coordinated way and ensure this was available to any researchers who needed it.

To support research efforts to understand more about the SARS-CoV virus and the structures of its proteins, we created dedicated PDBe-KB pages to highlight important structural features of PDB entries and allow easy download of all relevant data files. These pages highlight ligand binding sites and residues involved in protein-protein interactions through visualisation tools, including a new structure superimposition feature. These pages help researchers to easily identify common features from all the available structure data, supporting drug and vaccine development.

To view the PDBe-KB COVID-19 Data Portal, please visit PDBe.org/covid19.

3DBionotes-WS, an ELIXIR recommended interoperability resource, is a set of web services that provides multiple annotations oriented to structural biology analysis. It can be accessed through a website interface that features a fully interactive 3D viewer for macromolecular structures and functional, genomic, proteomic and structural feature annotations.

Motivated by COVID-19 pandemic, we present a new section (https://3dbionotes.cnb.csic.es/ws/covid19) dedicated to SARS-CoV-2 viral protein structures that have been provided by X-ray crystallography, cryo-EM, NMR and various modelling and structural predictions approaches.The aim of this section is collecting and providing centralized access to all available structural information on the SARS-CoV-2 viral proteins, as well as other related viruses or interacting molecules. In addition, when validation and quality information is available from PDB-REDO [1] and the Coronavirus Structural Task Force [2], special tags are incorporated for every entry, pointing to the re-refined models.

Among the new annotations added are functional mappings for ligand binding sites and protein-protein interaction sites. Functional mapping annotations allow to locate the residues that are likely to constitute binding sites between SARS-CoV-2 proteins and other viral or human proteins [3] and for multiple candidate inhibitors already identified for SARS and MERS homologous proteins. Of particular interest are ligands tested in large-scale studies searching for potential drugs, like the one performed against the SARS-CoV-2 main protease using the PanDDA method [4] at the Diamond synchrotron, Oxford (https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem).

Regarding the genomic context, SARS-CoV-2 variants compiled at the China National Center for Bioinformation (https://bigd.big.ac.cn/ncov/variation) have been summarized in a new annotation track. Also, some methods to evaluate the quality of cryo-EM maps and the fit to their atomic models was incorporated. These methods are deepRes [5], that analyse the map local resolution and FSC-Q [6] and map Q-score [7], that inform about the fit and resolvability of the built atomic model.

[1] Joosten, R. P., Long F., Murshudov, G. N. & Perrakis, A. (2014). IUCrJ, 1, pp. 213–220

[2] Croll, T. I., Diederichs, K., Fischer, F, Fyfe C. D., Gao, Y., Horrell, S., Joseph, A. P, Kandler, L., Kippes O., Kirsten, F., Müller, K., Nolte, K., Payne, A. M., Reeves, M., Richardson, J.S., Santoni, G., Stäb, S., Tronrud, D. E., von Soosten, L. C., Williams C. J. & Thorn, A. (2021). Nat Struct Mol Biol 28, pp. 404–408

[3] Srinivasan, S., Cui, H., Gao, Z., Liu, M., Lu, S., Mkandawire, W., Narykov, O., Sun, M. & Korkin, D. (2020). Viruses, 12(4)

[4] Pearce, N.M., Krojer, T., Bradley, A. R., Collins, P., Nowak, R.P., Talon, R., Marsden, B.D. Kelm, S., Shi, J., Deane, C.M. & von Delft, F. (2017). Nat Commun., 8, 15123

[5] Ramírez-Aportela E., Mota J., Conesa P., Carazo J. M. & Sorzano C. O. S. (2019). IUCrJ, 6, pp. 1054-1063

[6] Ramírez-Aportela, E., Maluenda, D., Fonseca, Y. C., Conesa P., Marabini, R., Heymann, J. B., Carazo J.M. & Sorzano C.O.S. (2021) Nat Commun, 12(42)

[7] Pintilie, G., Zhang K., Su Z., Li S., Schmid M. F. & Chiu W. (2020) Nat Methods. 17(3), pp. 328-334.

We acknowledge financial support from: CSIC (PIE/COVID-19 number 202020E079), the Comunidad de Madrid through grant CAM (S2017/BMD- 3817), the Spanish Ministry of Science and Innovation through projects (SEV 2017-0712, FPU-2015/264, PID2019 104757RB-I00 / AEI / 10.13039/501100011033), the Instituto de Salud Carlos III: PT17/0009/0010 (ISCIII-SGEFI / ERDF-) and the European Union and Horizon 2020 through grant EOSC Life (INFRAEOSC-04-2018, Proposal: 824087). Contributions from the Coronavirus Structural Task Force were supported by the German Federal Ministry of Education and Research [grant no. 05K19WWA] and Deutsche Forschungsgemeinschaft [project TH2135/2-1]. The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI project.

Tailed bacteriophages are one of the most widespread biological entities on Earth. Their singular structures, such as spikes or fibers are of special interest given their potential use in a wide range of biotechnological applications. In particular, the long fibers present at the termini of the T4 phage tail have been studied in detail and are important for host recognition and adsorption. Although significant progress has been made in elucidating structural mechanisms of model phages, the high-resolution structural description of the vast population of marine phages is still unexplored.

Our group studies the marine flavobacterium Bizionia argentinensis JUB59, a psychrotolerant Gram-negative microorganism isolated from surface seawater in Potter Cove, Antarctica, and whose genome has been sequenced. This bacterium constitutes a relevant source for the discovery of new proteins showing biological activity in extreme conditions of low temperature. In recent years, we have developed a medium-throughput structural genomics project to functionally classify B. argentinensis JUB59 proteins annotated with unknown function. We set up a screening protocol based on bioinformatics analysis, NMR and crystallography to identify and characterize suitable targets for structure determination [1-4]. In this context, and amongst other members, we selected a 277-residue protein named C24, whose sequence lacks homology to proteins of known function.

In the present work, we crystallized and solved the structure of C24 at 1.82 Å resolution by means of the single-wavelength anomalous diffraction method (manganese peak) with excellent statistics [5]. The protein folds as an 89-kDa homotrimer with a rocket shape. It bears a total length of 160 Å and a varying diameter along the particle axis, with a maximum value of 60 Å at its base. The structure of C24 closely resembles that of the receptor-binding tip from the bacteriophage T4 long tail fiber [6], although there are notorious differences in their domain organization, sequence, molecular dimension and number and type of bound structural divalent cations. We then confirmed the viral origin of C24 by bioinformatic and experimental approaches: (i) the C24 sequence is located inside a detected prophage by the ACLAME Prophinder tool, and (ii) the antibiotic mitomycin C induces the lytic cycle of a virus present in the bacterial genome, which was able to be isolated and visualized by transmission electron microscopy, revealing a morphology that is compatible with the order Caudovirales and, more importantly, these viral particles carry the nucleotide sequence of C24 in their genome.

As a general conclusion, the crystal structure of C24, together with induction and electron microscopy experiments, reveal that this protein may be the receptor-binding tip of a novel uncharacterized tailed bacteriophage present as a lysogen in B. argentinensis JUB59. These findings bring new avenues for the discovery of novel viral structures and provide valuable information to expand our current knowledge on the viral machinery prevalent in the oceans.

[1] Aran, M., Smal, C., Pellizza, L., Gallo, M., Otero, L. H., Klinke, S., Goldbaum, F. A., Ithurralde, E. R., Bercovich, A., Mac Cormack, W. P., Turjanski, A. G. & Cicero, D. O. (2014). Proteins 82, 3062-3078.

[2] Pellizza, L., Smal, C., Ithurralde, E. R., Turjanski, A. G., Cicero, D. O. & Aran, M. (2016). FEBS J. 283, 4370-4385.

[3] Cerutti, M. L., Otero, L. H., Smal, C., Pellizza, L., Goldbaum, F. A., Klinke, S. & Aran, M. (2017). J. Struct. Biol. 197, 201-209.

[4] Pellizza, L., Smal, C., Rodrigo, G. & Aran, M. (2018). Sci. Rep. 8, 10618.

[5] Pellizza, L., López, J. L., Vázquez, S., Sycz, G., Guimarães, B. G., Rinaldi, J., Goldbaum, F. A., Aran, M., Mac Cormack, W. P. & Klinke, S. (2020). J. Struct. Biol. 212, 107595.

[6] Bartual, S. G., Otero, J. M., García-Doval, C., Llamas-Saiz, A. L., Kahn, R., Fox, G. C. & van Raaij, M. J. (2010). Proc. Natl. Acad. Sci. USA 107, 20287-20292.

Keywords: Protein structure; Prophage; Mitomycin C; Flavobacteriaceae; Caudovirales

This work was supported by the Argentinian Ministry of Science and the University of Buenos Aires. We are grateful for access to the SOLEIL Synchrotron in France.

Since one and a half years the world has been fighting a COVID-19 pandemic, which caused unprecedented crisis all over the world. And, the full evolutionary potential of SARS-CoV-2 has yet to be revealed. A super-virus with features of the highly transmissible SARS-CoV-2 and the deadly SARS and MERS viruses could lead to more catastrophic loss of life. What is more, a new serious onslaughts due to (corona)viruses and other pathogens are inevitable.

Structural biology has been at the centre of the efforts of development of effective therapeutics. It helps to „see” 3D protein structures of invisible viruses and their interactions with host proteins, and potential ligands, knowing molecular mechanisms driving the viral evolution and shape biomedicinal research field [Barcena et al., 2021; Lynch et al., 2021]. Only through structural biology can we gain a deeper insight into the new variants of viruses and effect of mutations on the proteins. The ongoing outbreak has pushed numerous structural studies, using crystallography, cryoelectron microscopy, structural virology and vaccinology, structural bioinformatics, dynamics, and omics, leading to either revolutionary progress of structural biology or understanding of pandemic evolution leading to therapeutic findings. Here, we should mention about some examples.

The basic biomolecules, amino acids, and short peptides, as constituents of proteins associated with RNA, are crucial elements in the pandemic`s puzzles. Amino acid variations in the spike of SARS-CoV-2 affects the shape, binding, and function of the protein. The virus can escape neutralizing antibodies through only a single amino acid replacement. Thanks to structural studies, we found evidence how amino acids and short peptides drive mutations, which is critical in predicting emergent strains and their deadly potential in the context of designing effective pan-vaccines and preventing the spread of disease [In preparation]. Some studies have found evidence that coevolving amino acids play a pivotal role in increasing the affinity of the spike protein against ACE2, leading to more successful infection, with some of these amino acids under more evolutionary pressure than others [Priya et al, 2021]. On the other hand, other viral proteins, such as the non-structural protein 1, contribute to immune evasion. The coevolution impact on interaction patterns of proteins among a growing number of variants should not be neglected.

Short peptides are naturally suited to treating infectious disease as they can disrupt protein-protein interactions [Apostolopous et al, 2021; Bojarska et al., 2021]. Notably, these inter-contacts are the heart of most important cellular processes and primary targets for smart drug discovery but are ”undruggable” by small-molecules due to the large and flat contact surfaces characteristic of protein-protein interactions. Notably, there are a plethora of disease-relevant protein-protein interactions, but most of them have so far been unexplored. More specifically, viral proteins take over cellular host functions through short peptide interaction motifs (in unstructured regions) that bind to defined pockets on globular host domains. These motifs evolve by mutation, enabling viruses to interact with novel host factors. An understanding of these peptide-mediated protein-protein interactions can predict viral tropism and molecular processes within host cells. They could be targets for novel antiviral inhibitors, such as integrin-targeted drugs in controlling COVID-19 [Kruse et al, 2021].

The design of suitable antiviral drugs became possible by thorough knowing the composition of the binding site pocket of virus (SARS-CoV-2) main protease [Dai et al, 2020].

Structural mapping a protein network, that enables studying suitable protein interactions with other proteins, can identify repurposed drugs that could target disease relevant processes. This new tactic has identified cyclic depsipeptide (PF-07321332), a known anticancer drug, which is more potent than remdesivir in COVID-19. This idea could be used for other pathogens [White et al, 2021].

The concept of smart vaccines using machine learning, structural modelling, to precisely predict the binding between viral peptides and host proteins from the adaptive immune system or other evolutionary peptides, leading to an increase in the speed of vaccine development in future health crises [Alam et al, 2021].

Thus, advanced structural biology is a valuable tool to gather information helpful in controlling current and next outbreaks of deadly pathogens as well as rapid progress in the development of drugs and vaccines.

We will discuss key cutting-edge approaches in detail, highlighting their strengths and weaknesses, and indicating the important gaps as well as the further advances in bio-informatics methodology needed to fill them.

References

* A. Alam, A. Khan, N. Imam et al. Briefings in Bioinformatics, 22, 1309 (2021)

* V. Apostolopoulos, J. Bojarska, T.T. Chai et al. Molecules 26, 430 (2021)

* M. Bárcena, C.O. Barnes, M. Beck et al. Nat. Struct. Mol. Biol., 28, 2 (2021)

* J. Bojarska, R. New, P. Borowiecki et al. Front. Chem. 9, 679776 (2021)

* W. Dai, B. Zhang, X.M. Jiang et al. Science, 368, 1331 (2020)

* T. Kruse, C. Benz, D.H. Garvanska et al. bioRxiv 19, 2021. 10.1101/2021.04.19.440086

* M.L. Lynch, E.H. Snell, S.E.J. Bowman. IUCrJ 8, 335 (2021)

* P. Priya, A. Shanker. Infect, Genet. & Evol. 87, 104646 (2021)

* K.M. White, R. Rosales, S. Yildiz et al. Science, 371, 926 (2021)