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.

Thermodynamic and mechanistic analysis of the functional properties of dengue virus NS3 helicase.

The Dengue Virus (DENV) non-structural protein 3 (NS3) is a multi-functional protein critical in the viral life cycle. The DENV NS3 is comprised of a serine protease domain and a helicase domain. The helicase domain itself acts as a molecular motor, either translocating in a unidirectional manner along single-stranded RNA or unwinding double-stranded RNA, processes fueled by the hydrolysis of nucleoside triphosphates. In this brief review, we summarize our contributions and ongoing efforts to uncover the thermodynamic and mechanistic functional properties of the DENV NS3 as an NTPase and helicase.

Susceptibility of Cu(I) transport ATPases to sodium dodecyl sulfate. Relevance of the composition of the micellar phase.

Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.

Excluded Volume and Weak Interactions in Crowded Solutions Modulate Conformations and RNA Binding of an Intrinsically Disordered Tail.

The cellular milieu is a solution crowded with a significant concentration of different components (proteins, nucleic acids, metabolites, etc.). Such a crowded environment affects protein conformations, dynamics, and interactions. Intrinsically disordered proteins and regions are particularly sensitive to these effects. Here, we investigate the impact on an intrinsically disordered tail that flanks a folded domain, the N-terminal domain, and the RNA-binding domain of the SARS-CoV-2 nucleocapsid protein. We mimic the crowded environment of the cell using polyethylene glycol (PEG) and study its impact on protein conformations using single-molecule Förster resonance energy transfer. We found that high-molecular-weight PEG induces a collapse of the disordered N-terminal tail, whereas low-molecular-weight PEG induces a chain expansion. Our data can be explained by accounting for two opposing contributions: favorable interactions between the protein and crowder molecules and screening of excluded volume interactions. We further characterized the interaction between protein and RNA in the presence of crowding agents. While for all PEG molecules tested, we observed an increase in the binding affinity, the trend is not monotonic as a function of the degree of PEG polymerization. This points to the role of nonspecific protein-PEG interactions on binding in addition to the entropic effects due to crowding. To separate the enthalpic and entropic components of the effects, we investigated the temperature dependence of the association constants in the absence and presence of crowders. Finally, we compared the effects of crowding across mutations in the disordered region and found that the threefold difference in association constants for two naturally occurring variants of the SARS-CoV-2 nucleocapsid protein is reduced to almost identical affinities in the presence of crowders. Overall, our data provide new insights into understanding and modeling the contribution of crowding effects on disordered regions, including the impact of interactions between proteins and crowders and their interplay when binding a ligand.

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's 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.

Shelterin Components Modulate Nucleic Acids Condensation and Phase Separation in the Context of Telomeric DNA.

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, telomeres can cluster together, form telomere-induced-foci or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule force spectroscopy measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

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

Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. Here, we combine rapid-mixing time-resolved small-angle X-ray scattering (SAXS) approaches to characterize the assembly kinetics of a prototypical prion-like domain with equilibrium techniques that characterize its phase boundaries and the size distribution of clusters prior to phase separation. We find two kinetic regimes on the micro- to millisecond timescale that are distinguished by the size distribution of clusters. At the nanoscale, small complexes are formed with low affinity. After initial unfavorable complex assembly, additional monomers are added with higher affinity. At the mesoscale, assembly resembles classical homogeneous nucleation. Careful multi-pronged characterization is required for the understanding of condensate assembly mechanisms and will promote understanding of how the kinetics of biological phase separation is encoded in biomolecules.

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

Valence and patterning of aromatic residues determine the phase behavior of prion-like domains.

Prion-like domains (PLDs) can drive liquid-liquid phase separation (LLPS) in cells. Using an integrative biophysical approach that includes nuclear magnetic resonance spectroscopy, small-angle x-ray scattering, and multiscale simulations, we have uncovered sequence features that determine the overall phase behavior of PLDs. We show that the numbers (valence) of aromatic residues in PLDs determine the extent of temperature-dependent compaction of individual molecules in dilute solutions. The valence of aromatic residues also determines full binodals that quantify concentrations of PLDs within coexisting dilute and dense phases as a function of temperature. We also show that uniform patterning of aromatic residues is a sequence feature that promotes LLPS while inhibiting aggregation. Our findings lead to the development of a numerical stickers-and-spacers model that enables predictions of full binodals of PLDs from their sequences.

Thermodynamic study of the effect of ions on the interaction between dengue virus NS3 helicase and single stranded RNA.

Dengue virus nonstructural protein 3 (NS3) fulfills multiple essential functions during the viral replication and constitutes a prominent drug target. NS3 is composed by a superfamily-2 RNA helicase domain joined to a serine protease domain. Quantitative fluorescence titrations employing a fluorescein-tagged RNA oligonucleotide were used to investigate the effect of salts on the interaction between NS3 and single stranded RNA (ssRNA). We found a strong dependence of the observed equilibrium binding constant, Kobs, with the salt concentration, decreasing at least 7-fold for a 1-fold increase on cation concentration. As a result of the effective neutralization of ~10 phosphate groups, binding of helicase domain of NS3 to ssRNA is accompanied by the release of 5 or 7 monovalent cations from an oligonucleotide or a polynucleotide, respectively and of 3 divalent cations from the same oligonucleotide. Such estimates are not affected by the type of cation, either monovalent (KCl, NaCl and RbCl) or divalent (MgCl2 and CaCl2), nor by the presence of the protease domain or the fluorescein label. Combined effect of mono and divalent cations was well described by a simple equilibrium binding model which allows to predict the values of Kobs at any concentration of cations.

Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA.

Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

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.