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

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

Single-molecule Spectroscopy of the SARS-CoV-2 Nucleocapsid Protein

The COVID pandemic has affected over 760,000,000 individuals worldwide since late 2019. Understanding how SARS-CoV-2, the virus responsible for the disease, functions at a mechanistic level is essential to develop therapeutics and vaccines. SARS-CoV-2 utilizes four structural proteins that work together during the viral life cycle to ensure the spread of infections: spike (S), envelope (E), membrane (M) and nucleocapsid (N). Though much work has focused on the S protein for the purpose of vaccines, the N protein plays a key function in the viral life cycle as well. Nucleocapsid is responsible for packaging the viral genome and incorporating the viral ribonucleocapsid into the virion. In SARS-CoV-2, the packaged viral genome adopts a more “beads on a string” organization than the helical configuration previously observed in other coronaviruses. Little is known about how N protein controls the packaging of the viral genome. N protein is composed of five domains: a folded RNA binding domain, a folded dimerization domain, and three flanking intrinsically disordered regions that were proposed to modulate interaction with RNA. Despite their potential role in modulating genome compaction, properties of corresponding disordered regions in nucleocapsid proteins from other coronaviruses remains largely understudied. At the beginning of the COVID pandemic, there was no insight on whether predicted disordered regions in SARS-CoV-2 remain disordered in the context of the full-length protein and how they modulated protein-RNA interactions. In my thesis work, I made use of single-molecule confocal fluorescence spectroscopy, and in particular, single-molecule Förster Resonance Energy Transfer (FRET) to close this knowledge gap and investigate conformations, dynamics, and interactions of the disordered regions within the SARS-CoV-2 nucleocapsid protein. I first determined that the three predicted disordered regions of N protein are disordered in the context of full-length protein. The combination of single-molecule FRET experiments and all-atom Monte Carlo simulations revealed that the monomeric full-length protein is flexible and dynamic. In addition, we observed that the protein undergoes phase separation when mixed with RNA. Having characterized the monomeric form of the protein, I next investigated the protein-protein interactions that lead to dimerization. I found that the dimerization domain is partially disordered and flexible when N protein is monomeric. I further determined the concentration under which dimerization occurs (KD = 11 ± 3 nM) to be in good agreement with previous AUC experiments and found that even in the dimeric state, N protein retains some of the dynamic nature of the monomer. I also quantified that dimer formation does not alter the conformations of the disordered NTD and folded RBD, but causes an expansion of the disordered linker and CTD. These observations were consistent with my previous determination of interactions of the linker and CTD with the dimerization domain. As a next step, I started to investigate the interactions of the N protein with RNA. To understand the role of a disordered region in aiding the recruitment of RNA, I started to investigate whether the NTD enhances the affinity of RNA to the RBD. For this, I focused on using truncations of the NTD-RBD and RBD in isolation. My experiments showed that the presence of the NTD enhances the affinity by over 50-fold compared to the RBD in isolation. Furthermore, when in complex with RNA, the NTD forms a dynamic fuzzy complex, as seen also in coarse-grained simulations. Comparison of single- and double-stranded RNA provided evidence that the NTD-RBD preferentially binds to single-stranded RNA. Finally, I examined how a crowded environment (mimicked by polyethylene glycol molecules) can modify binding properties of the NTD-RBD to RNA and found that the NTD binding is sensitive to the solution environment. Comparison of Omicron and wildtype (Wuhan-Hu-1) variants revealed that significant differences in binding affinity observed in absence of crowding are equalized in presence of the crowders. In conclusion, single-molecule fluorescence spectroscopy has offered a powerful toolbox for investigating protein conformations and interactions of disordered regions. The work has provided new insights on the molecular interactions encoded in the SARS-CoV-2 N protein and paves the way to quantitative studies of interactions with other binding partners, viral genome RNA, and small molecules