Biology subjects

Cubuk, J.

Publications and source records attributed to Cubuk, J..

5 recordsLinked to original sources

bioRxiv2024

Phosphorylation Toggles the SARS-CoV-2 Nucleocapsid Protein Between Two Membrane-Associated Condensate States

The Nucleocapsid protein (N) of SARS-CoV-2 plays a critical role in the viral lifecycle by regulating RNA replication and packaging the viral genome. N and RNA phase separate to form condensates that may be important for these functions. Both functions occur at membrane surfaces, but how N toggles between these two membrane-associated functional states is unclear. Here, we reveal that phosphorylation switches how N condensates interact with membranes, partly by modulating condensate material properties. Phosphorylated N forms liquid condensates that wet membranes, reminiscent of Ns role in RNA replication. In contrast, unmodified N forms viscoelastic condensates that can be engulfed by membranes, evoking viral genome packaging. These results suggest that phosphorylation serves as a regulatory switch within the viral replication cycle by modulating Ns association with membranes. We gained mechanistic insight through structural analysis and molecular simulations, which suggest phosphorylation induces a conformational change that softens condensate material properties. Our studies also show that phosphorylation alters Ns interaction with viral membrane proteins. Together, our findings uncover a novel aspect of SARS-CoV-2 biology by identifying membrane association as a key feature of N condensates and providing mechanistic insights into the regulatory role of phosphorylation. Understanding this mechanism suggests potential therapeutic targets for COVID infection.

biophysics↗

bioRxiv2024

The dimerization domain of SARS CoV 2 Nucleocapsid protein is partially disordered as a monomer and forms a high affinity dynamic complex.

The SARS-CoV-2 Nucleocapsid (N) is a 419 amino acids protein that drives the compaction and packaging of the viral genome. This compaction is aided not only by protein-RNA interactions, but also by protein-protein interactions that contribute to increasing the valence of the nucleocapsid protein. Here, we focused on quantifying the mechanisms that control dimer formation. Single-molecule Forster Resonance Energy Transfer enabled us to investigate the conformations of the dimerization domain in the context of the full-length protein as well as the energetics associated with dimerization. Under monomeric conditions, we observed significantly expanded configurations of the dimerization domain (compared to the folded dimer structure), which are consistent with a dynamic conformational ensemble. The addition of unlabeled protein stabilizes a folded dimer configuration with a high mean transfer efficiency, in agreement with predictions based on known structures. Dimerization is characterized by a dissociation constant of [~] 12 nM at 23 OC and is driven by strong enthalpic interactions between the two protein subunits, which originate from the coupled folding and binding. Interestingly, the dimer structure retains some of the conformational heterogeneity of the monomeric units, and the addition of denaturant reveals that the dimer domain can significantly expand before being completely destabilized. Our findings suggest that the inherent flexibility of the monomer form is required to adopt the specific fold of the dimer domain, where the two subunits interlock with one another. We proposed that the retained flexibility of the dimer form may favor the capture and interactions with RNA, and that the temperature dependence of dimerization may explain some of the previous observations regarding the phase separation propensity of the N protein.

biophysics↗

bioRxiv2024

Structural dynamics of the intrinsically disordered linker region of cardiac troponin T

The cardiac troponin complex, composed of troponins I, T, and C, plays a central role in regulating the calcium-dependent interactions between myosin and the thin filament. Mutations in troponin can cause cardiomyopathies; however, it is still a major challenge to connect how changes in sequence affect troponins function. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of troponin, but there remain large, unresolved segments of troponin, including the troponin-T linker region that is a hotspot for cardiomyopathy mutations. This linker region is predicted to be intrinsically disordered, with behaviors that are not well described by traditional structural approaches; however, this proposal has not been experimentally verified. Here, we used a combination of single-molecule Forster resonance energy transfer (FRET), molecular dynamics simulations, and functional reconstitution assays to investigate the troponin-T linker region. We show that in the context of both isolated troponin and the fully regulated troponin complex, the linker behaves as a dynamic, intrinsically disordered region. This region undergoes polyampholyte expansion in the presence of high salt and distinct conformational changes during the assembly of the troponin complex. We also examine the {Delta}E160 hypertrophic cardiomyopathy mutation in the linker and demonstrate that it does not affect the conformational dynamics of the linker, rather it allosterically affects interactions with other troponin complex subunits, leading to increased molecular contractility. Taken together, our data clearly demonstrate the importance of disorder within the troponin-T linker and provide new insights into the molecular mechanisms driving the pathogenesis of cardiomyopathies. SIGNIFICANCE STATEMENTTroponin plays a central role in regulating heart contraction, and mutations in troponin can cause human cardiomyopathies. There are several functionally-significant regions of troponin that have not been structurally resolved, including the troponin T linker region that contains multiple cardiomyopathy mutations. In these unresolved regions, it is not possible to understand how changes in sequence affect function. We used computational and experimental techniques to demonstrate that this linker is dynamic and intrinsically disordered both in isolation and in the fully regulated thin filament. Moreover, we show how a cardiomyopathy mutation in this region affects function via allosteric disruption of intermolecular interactions. Our results highlight the need to consider how key mutations affect troponin disorder rather than the structure-function relationship.

biophysics↗

bioRxiv2023

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 Forster 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.

biophysics↗

bioRxiv2020

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.

biophysics↗