Biology subjects

Soranno, A.

Publications and source records attributed to Soranno, A..

9 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

Accounting for fast vs slow exchange in single molecule FRET experiments reveals hidden conformational states

Proteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles remains challenging, limiting our understanding of the relationships between sequence, structure, and function. Combining single molecule Forster resonance energy transfer (smFRET) experiments with molecular dynamics simulations could provide experimentally grounded, all-atom models of a proteins structural ensemble. However, agreement between the two techniques is often insufficient to achieve this goal. Here, we explore whether accounting for important experimental details like averaging across structures sampled during a given smFRET measurement is responsible for this apparent discrepancy. We present an approach to account for this time-averaging by leveraging the kinetic information available from Markov state models of a proteins dynamics. This allows us to accurately assess which timescales are averaged during an experiment. We find this approach significantly improves agreement between simulations and experiments in proteins with varying degrees of dynamics, including the well-ordered protein T4 lysozyme, the partially disordered protein apolipoprotein E (ApoE), and a disordered amyloid protein (A{beta}40). We find evidence for hidden states that are not apparent in smFRET experiments because of time averaging with other structures, akin to states in fast exchange in NMR, and evaluate different force fields. Finally, we show how remaining discrepancies between computations and experiments can be used to guide additional simulations and build structural models for states that were previously unaccounted for. We expect our approach will enable combining simulations and experiments to understand the link between sequence, structure, and function in many settings.

biophysics↗

bioRxiv2023

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Forster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

biochemistry↗

bioRxiv2023

Conserved molecular recognition by an intrinsically disordered region in the absence of sequence conservation

Intrinsically disordered regions (IDRs) are critical for cellular function, yet often appear to lack sequence conservation when assessed by multiple sequence alignments. This raises the question of if and how function can be encoded and preserved in these regions despite massive sequence variation. To address this question, we have applied coarse-grained molecular dynamics simulations to investigate non-specific RNA binding of coronavirus nucleocapsid proteins. Coronavirus nucleocapsid proteins consist of multiple interspersed disordered and folded domains that bind RNA. We focussed here on the first two domains of coronavirus nucleocapsid proteins, the disordered N-terminal domain (NTD) followed by the folded RNA binding domain (RBD). While the NTD is highly variable across evolution, the RBD is structurally conserved. This combination makes the NTD-RBD a convenient model system to explore the interplay between an IDR adjacent to a folded domain, and how changes in IDR sequence can influence molecular recognition of a partner. Our results reveal a surprising degree of sequence-specificity encoded by both the composition and the precise order of the amino acids in the NTD. The presence of an NTD can - depending on the sequence - either suppress or enhance RNA binding. Despite this sensitivity, large-scale variation in NTD sequences is possible while certain sequence features are retained. Consequently, a conformationally-conserved fuzzy RNA:protein complex is found across nucleocapsid protein orthologs, despite large-scale changes in both NTD sequence and RBD surface chemistry. Taken together, these insights shed light on the ability of disordered regions to preserve functional characteristics despite their sequence variability.

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↗

bioRxiv2022

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

The {varepsilon}4-allele variant of Apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimers disease, though 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. 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. Our data reveal that ApoE4 is far more disordered than previously thought and retains significant conformational heterogeneity after binding lipids. In particular, the behavior of the hinge region and C-terminal domain of ApoE4 differs substantially from that proposed in previous models and provides a crucial foundation for understanding how ApoE4 differs from non-pathogenic and protective variants of the protein.

biophysics↗

bioRxiv2021

Shelterin components modulate nucleic acids condensation and phase-separation

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, upon changes in their protein composition, telomeres can cluster to form telomere-induced-foci (TIFs) 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 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.

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↗