tetano
Editor, Senior Moderator
J Mol Biol
. 2021 Feb 5;166875.
doi: 10.1016/j.jmb.2021.166875. Online ahead of print.
The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins
Kristina V Tugaeva[SUP] 1 [/SUP], Dorothy E D P Hawkins[SUP] 2 [/SUP], Jake L R Smith[SUP] 2 [/SUP], Oliver W Bayfield[SUP] 2 [/SUP], De-Sheng Ker[SUP] 2 [/SUP], Andrey A Sysoev[SUP] 1 [/SUP], Oleg I Klychnikov[SUP] 3 [/SUP], Alfred A Antson[SUP] 4 [/SUP], Nikolai N Sluchanko[SUP] 5 [/SUP]
Affiliations
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 K[SUB]D[/SUB] 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.
Keywords: Host-pathogen interactions; nucleocytoplasmic shuttling; phosphorylation; protein-protein complex; stoichiometry.
. 2021 Feb 5;166875.
doi: 10.1016/j.jmb.2021.166875. Online ahead of print.
The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins
Kristina V Tugaeva[SUP] 1 [/SUP], Dorothy E D P Hawkins[SUP] 2 [/SUP], Jake L R Smith[SUP] 2 [/SUP], Oliver W Bayfield[SUP] 2 [/SUP], De-Sheng Ker[SUP] 2 [/SUP], Andrey A Sysoev[SUP] 1 [/SUP], Oleg I Klychnikov[SUP] 3 [/SUP], Alfred A Antson[SUP] 4 [/SUP], Nikolai N Sluchanko[SUP] 5 [/SUP]
Affiliations
- PMID: 33556408
- PMCID: PMC7863765
- DOI: 10.1016/j.jmb.2021.166875
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 K[SUB]D[/SUB] 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.
Keywords: Host-pathogen interactions; nucleocytoplasmic shuttling; phosphorylation; protein-protein complex; stoichiometry.