Structural basis for SARS-CoV-2 nucleocapsid (N) protein recognition by 14-3-3 proteins.
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ABSTRACT:
SUBMITTER: Eisenreichova A
PROVIDER: S-EPMC9245327 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly-conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.
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Project description:The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.
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Project description:The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.
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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.
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Project description:The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.
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Project description:O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3?/? and ? bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.
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Project description:The main protease (Mpro) of SARS-CoV and SARS-CoV-2 is a key enzyme in viral replication and a promising target for the development of antiviral therapeutics. The understanding of this protein is based on a number of observations derived from earlier x-ray structures, which mostly consider substrates or ligands as the main reason behind modulation of the active site. This lead to the concept of substrate-induced subsite cooperativity as an initial attempt to explain the dual binding specificity of this enzyme in recognizing the cleavage sequences at its N- and C-termini, which are important processing steps in obtaining the mature protease. The presented hypothesis proposes that structural heterogeneity is a property of the enzyme, independent of the presence of a substrate or ligand. Indeed, the analysis of Mpro structures of SARS-CoV and SARS-CoV-2 reveals a conformational diversity for the catalytically competent state in ligand-free structures. Variation in the binding site appears to result from flexibility at residues lining the S1 subpocket and segments incorporating methionine 49 and glutamine 189. The structural evidence introduces "structure-based recognition" as a new paradigm in substrate proteolysis by Mpro. In this concept, the binding space in subpockets of the enzyme varies in a non-cooperative manner, causing distinct conformations, which recognize and process different cleavage sites, as the N- and C-termini. Insights into the recognition basis of the protease provide explanation to the ordered processing of cleavage sites. The hypothesis expands the conformational space of the enzyme and consequently opportunities for drug development and repurposing efforts.
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Project description:COVID-19 is increasingly affecting human health and global economy. Understanding the fundamental mechanisms of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is highly demanded to develop treatments for COVID-19. SARS-CoV and SARS-CoV-2 share 92.06% identity in their N protein RBDs' sequences, which results in very similar structures. However, the SARS-CoV-2 is more easily to spread. Utilizing multi-scale computational approaches, this work studied the fundamental mechanisms of the nucleocapsid (N) proteins of SARS-CoV and SARS-CoV-2, including their stabilities and binding strengths with RNAs at different pH values. Electrostatic potential on the surfaces of N proteins show that both the N proteins of SARS-CoV and SARS-CoV-2 have dominantly positive potential to attract RNAs. The binding forces between SARS-CoV N protein and RNAs at different distances are similar to that of SARS-CoV-2, both in directions and magnitudes. The electric filed lines between N proteins and RNAs are also similar for both SARS-CoV and SARS-CoV-2. The folding energy and binding energy dependence on pH revealed that the best environment for N proteins to perform their functions with RNAs is the weak acidic environment.
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Project description:The newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global human health crisis. The CoV nucleocapsid (N) protein plays essential roles both in the viral genomic RNA packaging and the regulation of host cellular machinery. Here, to contribute to the structural information of the N protein, we describe the 2.0 Å crystal structure of the SARS-CoV-2 N protein C-terminal domain (N-CTD). The structure indicates an extensive interaction dimer in a domain-swapped manner. The interface of this dimer was first thoroughly illustrated. Also, the SARS-CoV-2 N-CTD dimerization form was verified in solution using size-exclusion chromatography. Based on the structural comparison of the N-CTDs from alpha-, beta-, and gamma-CoVs, we demonstrate the common and specific characteristics of the SARS-CoV-2 N-CTD. Furthermore, we provide evidence that the SARS-CoV-2 N-CTD possesses the binding ability to single-stranded RNA, single-stranded DNA as well as double-stranded DNA in vitro. In conclusion, this study could potentially accelerate research to understand the complete biological functions of the new CoV N protein.
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