Estelle, A. B. | Search bioRxiv

bioRxiv2023

RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1000nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with different RNA structures support a model where multiple weak interactions, such as with the CTD or the NTDs secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N. SignificanceThe nucleocapsid protein of the SARS-CoV-2 coronavirus binds to viral RNA, both to protect and condense it inside the viral particle and to facilitate viral transcription inside infected host cells. Evidence suggests that variations in RNA structure impact how and where it binds to the nucleocapsid, but these differences are not well understood at a structural level. Using nuclear magnetic resonance spectroscopy, we examine the interactions between each folded domain of the nucleocapsid and different RNA structures. Binding affinities and NMR chemical shift profiles demonstrate that binding between the N-terminal domain and single stranded RNA is driven by strong interactions at a specific site, while multiple weak nonspecific interactions at newly discovered sites lead to phase separation and RNA condensation.

biophysics↗

bioRxiv2022

Quantifying cooperative multisite binding through Bayesian inference

Multistep protein-protein interactions underlie most biological processes, but their characterization through methods such as isothermal titration calorimetry (ITC) is largely confined to simple models that provide little information on the intermediate, individual steps. In this study, we primarily examine the essential hub protein LC8, a small dimer that binds disordered regions of 100+ client proteins in two symmetrical grooves at the dimer interface. Mechanistic details of LC8 binding have remained elusive, hampered in part by ITC data analyses employing simple models that treat bivalent binding as a single event with a single binding affinity. We build on existing Bayesian ITC approaches to quantify thermodynamic parameters for multi-site binding interactions impacted by significant uncertainty in protein concentration. Using a two-site binding model, we model LC8 binding and identify positive cooperativity with high confidence for multiple client peptides. Application of an identical model to two-site binding between the coiled-coil dimer NudE and the intermediate chain of dynein reveals little evidence of cooperativity, in contrast to LC8. We propose that cooperativity in the LC8 system drives the formation of saturated 2:2 bound states, which play a functional role in many LC8 complexes. In addition to these system-specific findings, our work advances general ITC analysis in two ways. First, we describe a previously unrecognized mathematical ambiguity in concentrations in standard binding models and clarify how it impacts the precision with which binding parameters can be determined in cases of high uncertainty in analyte concentrations. Second, building on observations in the LC8 system, we develop a system-agnostic heat map of practical parameter identifiability calculated from synthetic data which demonstrates that certain binding parameters intrinsically inflate parameter uncertainty in ITC analysis, independent of experimental uncertainties. Author SummaryMulti-site protein-protein interactions govern many protein functions throughout the cell. Precise determination of thermodynamic constants of multi-site binding is a significant biophysical challenge, however. The application of complex models to multi-step interactions is difficult and hampered further by complications arising from uncertainty in analyte concentrations. To address these issues, we utilize Bayesian statistical techniques which calculate the likelihood of parameters giving rise to experimental observations to build probability density distributions for thermodynamic parameters of binding. To demonstrate the method and improve our understanding how the hub protein LC8 promotes dimerization of its 100+ binding partners, we test the pipeline on several of these partners and demonstrate that LC8 can bind clients cooperatively, driving interactions towards a fully bound functional state. We additionally examine an interaction between the dimer NudE and the intermediate chain of dynein, which does not appear to bind with cooperativity. Our work provides a solid foundation for future analysis of more complicated binding interactions, including oligomeric complexes formed between LC8 and clients with multiple LC8-binding sites.

biophysics↗