Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein

Abstract

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α-helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing.

Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

“…To investigate if the N protein of SARS-CoV-2 (further termed N SARS-CoV-2 ; Figure 1A) phase separates in the absence of other viral proteins, we measured the turbidity of N SARS-CoV-2 solutions at different protein concentrations. Up to 50 µM, the protein solution retained low absorbance ( Figure 1B), despite its tendency to oligomerize (Ye et al, 2020). Next we tested LLPS of N SARS-CoV-2 in the presence of RNA.…”

Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein

“…2a; Extended Data Fig. 1): (1) the 44-aa N-terminal extension (NTE), a poorly conserved prion-like sequence with a basic cluster that contributes to RNA binding 28 ; (2) the 31-aa SR region, a basic segment implicated in RNA binding, oligomerization 44 , and phosphorylation 5,6,45-50 ; (3) the 55-aa C-terminal extension (CTE), implicated in oligomerization 35,[42][43][44] ; and (4) the CTD basic patch (CBP), a 33-aa basic region that forms the RNA-binding groove on the surface of the CTD [33][34][35] , which can be deleted without affecting CTD dimer structure 36 .…”

“…The NTD is an RNA-binding domain [29][30][31][32] . The CTD forms a tightly-linked dimer with a large RNA-binding groove [33][34][35][36] , and the fundamental unit of N protein structure is a dimer 37,38 . Under some conditions, the dimer selfassociates to form oligomers that depend on multiple protein regions 33,35,[39][40][41][42][43][44] .…”

SARS-CoV-2 Nucleocapsid protein attenuates stress granule formation and alters gene expression via direct interaction with host mRNAs

“…Furthermore, the N protein is produced at high levels in infected cells 4 , where it appears to enhance the efficiency of sub-genomic viral RNA transcription and modulate host cell metabolism reviewed by 5 . The SARS-CoV-2 N protein has two distinct RNA-binding domains, an N-terminal domain [NTD] and a C-terminal domain [CTD], connected by a linker region (LKR) containing a serine/arginine-rich (SR-rich) domain (SRD) 7–9 . Several critical residues of the N protein have been shown to bind viral genomic RNA and modulate infectivity 7 .…”