The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

Tugaeva, K. V.; Hawkins, D. E. D. P.; Smith, J. L. R.; Bayfield, O. W.; Ker, D.-S.; Sysoev, A. A.; Klychnikov, O. I.; Antson, A. A.; Sluchanko, N. N.

doi:10.1101/2020.12.26.424450

bioRxiv · 10.1101/2020.12.26.424450

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

Tugaeva, K. V.·Hawkins, D. E. D. P.·Smith, J. L. R.·Bayfield, O. W.·Ker, D.-S.·Sysoev, A. A.·Klychnikov, O. I.·Antson, A. A.·Sluchanko, N. N.

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Abstract

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 M range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

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Tugaeva, K. V., Hawkins, D. E. D. P., Smith, J. L. R., Bayfield, O. W., Ker, D.-S., Sysoev, A. A., Klychnikov, O. I., Antson, A. A., Sluchanko, N. N.. 2020-12-27. The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins. https://doi.org/10.1101/2020.12.26.424450

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