A conserved oligomerization domain in the disordered linker of coronavirus nucleocapsid proteins

Abstract

The nucleocapsid (N-)protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a key role in viral assembly and scaffolding of the viral RNA. It promotes liquid-liquid phase separation (LLPS), forming dense droplets that support the assembly of ribonucleoprotein particles with as-of-yet unknown macromolecular architecture. Combining biophysical experiments, molecular dynamics simulations, and analysis of the mutational landscape, we describe a heretofore unknown oligomerization site that contributes to LLPS, is required for the assembly of higher-order protein-nucleic acid complexes, and is coupled to large-scale conformational changes of N-protein upon nucleic acid binding. The self-association interface is located in a leucine-rich sequence of the intrinsically disordered linker between N-protein folded domains and formed by transient helices assembling into trimeric coiled-coils. Critical residues stabilizing hydrophobic and electrostatic interactions between adjacent helices are highly protected against mutations in viable SARS-CoV-2 genomes, and the oligomerization motif is conserved across related coronaviruses, thus presenting a target for antiviral therapeutics.

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid

“…In previous work we have studied N-protein dimer in mixtures with sufficiently short oligonucleotides (T10) to eliminate the possibility of multi-valent binding, which allowed us to focus on the proteinprotein interactions that arise for NA-liganded N-protein through allosteric stabilization of LRS helices and their cooperative coiled-coil oligomerization (17), as schematically indicated in Figure 1C. Occupation of the NA binding site (presumably in the NTD) causes a conformational change and improves the effective KD* for oligomerization by approximately three orders of magnitude from ≈1 mM to low µM.…”

“…To shed light on this question we studied N-protein binding to T40 oligonucleotides, which are twice the length required for cross-linking N-protein (19), and four times the size of the NTD binding epitope (20). As we have shown previously (17) it is conveniently possible to reduce or eliminate LRS oligomerization through LRS mutations that suppress (L222P) or completely disrupt (L222P/R226P) helix formation, rendering N-protein capable only of presenting protein/NA interfaces (besides the obligatory highaffinity dimerization of the CTD). Thus, as a starting point to probe the binding capacity of T40 for Nprotein we carried out experiments in a concentration series with different molar ratios of mutant and wild-type (WT) N-protein binding to T40.…”

“…Ultra-weak protein-protein interactions allow N-protein to undergo liquidliquid phase separation (LLPS), enhanced in the presence of NA, to form droplets of high macromolecular density (8)(9)(10)(11), which are thought to facilitate the assembly of vRNPs (9,(11)(12)(13). IDRs also play important roles in N-protein NA interactions (14)(15)(16)(17). For example, the N-arm undergoes conformational changes upon NA binding to NTD, significantly increasing the affinity of the NTD for RNA (15).…”

“…Similar to the N-arm, an SR-rich portion of the linker IDR flanking the NTD is also perturbed by NA binding (14), as is an L-rich sequence of the linker IDR (LRS) further downstream. Recently, we have shown that highly conserved transient helices in the LRS can assemble cooperatively into coiled-coil trimers, tetramers, and higher oligomers, in a process that is allosterically enhanced by NA binding (17,18) (Figure 1C). We hypothesized that this mechanism might initiate assembly by providing additional protein-protein interfaces essential for stabilizing three-dimensional structures of the vRNPs (17,18).…”

A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding

“…Indeed mini-replicon studies have measured 10-fold increased mRNA delivery and protein expression for commonly found mutants, and a reverse genetics model revealed 50-fold more virus production for S202R and R203M mutations (50). Helix H1 has also been linked to assembly, with a recent study using analytical ultra-centrifugation, circular dichroism combined with molecular dynamics simulation, suggesting that the motif promotes higher order assembly (51).…”