The Mechanism of SARS-CoV-2 Nucleocapsid Protein Recognition by the Human 14-3-3 Proteins
| Autor: | Oleg I. Klychnikov, Jake L. R. Smith, Oliver W. Bayfield, Dorothy E. D. P. Hawkins, Andrey Sysoev, Kristina V. Tugaeva, Alfred A. Antson, Nikolai N. Sluchanko, De Sheng Ker |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Nervous system
Phosphopeptides Gene isoform Mutant Plasma protein binding medicine.disease_cause Substrate Specificity Phosphoserine 03 medical and health sciences Viral genome packaging 0302 clinical medicine Structural Biology Escherichia coli medicine Coronavirus Nucleocapsid Proteins Humans Protein Isoforms Amino Acid Sequence Protein kinase A Molecular Biology Peptide sequence 030304 developmental biology Coronavirus 0303 health sciences Binding Sites Host-pathogen interactions phosphorylation Chemistry Phosphoproteins Cyclic AMP-Dependent Protein Kinases stoichiometry Cell biology medicine.anatomical_structure 14-3-3 Proteins Cytoplasm Mutation Fast Track RNA Viral Phosphorylation protein-protein complex 030217 neurology & neurosurgery nucleocytoplasmic shuttling Protein Binding |
| Zdroj: | Journal of Molecular Biology |
| ISSN: | 0022-2836 |
| Popis: | 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 can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.HighlightsSARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a μM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction. |
| Databáze: | OpenAIRE |
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