The Mechanism of SARS-CoV-2 Nucleocapsid Protein Recognition by the Human 14-3-3 Proteins

• Published in: Journal of molecular biology Vol. 433; no. 8; p. 166875
• Main Authors: Tugaeva, Kristina V., Hawkins, Dorothy E.D.P., Smith, Jake L.R., Bayfield, Oliver W., Ker, De-Sheng, Sysoev, Andrey A., Klychnikov, Oleg I., Antson, Alfred A., Sluchanko, Nikolai N.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 16-04-2021; Elsevier
• Subjects: 14-3-3 Proteins - chemistry; 14-3-3 Proteins - metabolism; Amino Acid Sequence; Binding Sites - genetics; Coronavirus Nucleocapsid Proteins - chemistry; Coronavirus Nucleocapsid Proteins - genetics; Coronavirus Nucleocapsid Proteins - metabolism; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Escherichia coli; Fast Track; host-pathogen interactions; Humans; Mutation; nucleocytoplasmic shuttling; Phosphopeptides - chemistry; Phosphopeptides - metabolism; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation; Phosphoserine - metabolism; Protein Binding; Protein Isoforms - chemistry; Protein Isoforms - metabolism; protein–protein complex; RNA, Viral - metabolism; stoichiometry; Substrate Specificity; nucleocytoplasmic shuttling; protein–protein complex; stoichiometry; host-pathogen interactions; phosphorylation
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2021.166875

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•SARS-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.
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.