The disordered N-terminal tail of SARS-CoV-2 Nucleocapsid protein forms a dynamic complex with RNA

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

The nucleation mechanisms of biological protein phase separation are poorly understood. Here, the authors perform time-resolved SAXS experiments with the low-complexity domain (LCD) of hnRNPA1 and uncover multiple kinetic regimes on the micro- to millisecond timescale. Initially, individual proteins collapse. Nucleation then occurs via two steps distinguished by their protein cluster size distributions

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer\u27s disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein

Steady-state NTPase activity of Dengue virus NS3: number of catalytic sites, nucleotide specificity and activation by ssRNA.

Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (kcat /KM )- was GTP[Formula: see text]ATP[Formula: see text]CTP [Formula: see text] UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat ) and a 100 fold decrease in the apparent affinity (KM ) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and [Formula: see text]20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA

Competition plots for ATP and a second nucleotide.

<p>Initial rates of phosphate release (<i>vi</i>) were measured in reaction mixtures containing two nucleotides at a time. The value of abscissa <i>x</i> is the proportion of [ATP] relative to the reference concentration [ATP]0 (see text). All experiments were performed using 51 nM NS3f and 100 mM KCl while other components were as indicated in Materials and Methods. <i>k_cat</i> and <i>K_M</i> values obtained from substrate curves in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058508#pone-0058508-g001" target="_blank">Figure 1</a> were used to simulate the prediction of the single-active-site model (continuous line) and the two-independent-active-sites model (dashed line) according to the following equations: and . The former model shows the better correspondence with these results.</p

Parameters of the steady-state NTPase activity of NS3f in the absence of RNA.

<p><i>k_cat</i>, <i>K_M</i> and <i>k_cat</i>/<i>K_M</i> values (SE) are for the substrate curves shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058508#pone-0058508-g001" target="_blank">Figure 1</a>. These parameters corresponds to a single hyperbolic function.</p

Effect of RNA on the kinetic parameters of the ATPase activity of NS3h.

<p><i>k_cat</i> (a), <i>K_M</i> (b) and <i>k_cat/K_M</i> (c) values were obtained from substrate curves in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058508#pone-0058508-g003" target="_blank">Figure 3</a>. In the absence of RNA the values of <i>k_cat, K_M</i> and <i>k_cat/K_M</i> were 2.91 (0.03) s-1, 0.018 (0.001) mM and 16 (1) 10⁴ M^-1s^-1, respectively. The limit saturating values of <i>k_cat, K_M</i> and <i>k_cat/K_M</i> were respectively 28 (1) s^-1, 0.33 (0.03) mM and 8.4 (0.4) 10⁴M^-1s^-1 for poly(A) and 16.6 (0.3) s^-1, 0.30 (0.02) mM and 5.6 (0.2) 10⁴M^-1s^-1 or poly(C). Continuous lines are the best fitting curves to a rational equation and are only intended to guide the eye. Error bars are the standard error of the parameters fitted to the experimental results by nonlinear regression analysis.</p

Steady-state NTPase activity of NS3f as a function of substrate concentration.

<p>Initial rates of NTP hydrolysis were obtained for ATP (empty diamond), GTP (filled circle), CTP (filled square) and UTP (filled diamond).The experiments were performed using 50 nM NS3f in a reaction media containing 100 mM KCl and all other reagents as indicated in Materials and Methods. Continuous lines are plots of hyperbolic functions whose parameter values (<i>k_cat</i> and <i>K_M</i>) were obtained by non-linear regression analysis and are shown in Table1.</p

Effect of RNA on the ATPase activity at different concentrations of NS3h.

<p>Initial rates of phosphate release (<i>vi</i>) were obtained in the absence (♦) and in the presence of poly(C): 0.50 M () or 3.1 M (). ATP concentration was 0.10 mM and reactions were carried out in the same reaction media as were indicated above (see Materials and Methods). Data showed in (a) as a function of [NS3h] are plotted in (b) as a function of the ratio [poly(C)]/[NS3h].</p