Arginine multivalency stabilizes protein/RNA condensates

Abstract

Biomolecular condensates assembled through liquid–liquid phase separation (LLPS) of proteins and RNAs are currently recognized to play an important role in cellular organization. Their assembly depends on the formation of a network of transient, multivalent interactions between flexible scaffold biomolecules. Understanding how protein and RNA sequences determine these interactions and ultimately regulate the phase separation is an open key challenge. Recent in vitro studies have revealed that arginine and lysine residues, which are enriched in most cellular condensates, have markedly distinct propensities to drive the LLPS of protein/RNA mixtures. Here, we employ explicit‐solvent atomistic molecular dynamics simulations to shed light on the microscopic origin of this difference by investigating mixtures of polyU oligonucleotides with either polyR/polyK peptides. In agreement with experiments, our simulations indicate that arginine has a higher affinity for polyU than lysine both in highly diluted conditions and in concentrated solutions with a biomolecular density comparable to cellular condensate. The analysis of intermolecular contacts suggests that this differential behavior is due to the propensity of arginine side chains to simultaneously form a higher number of specific interactions with oligonucleotides, including hydrogen bonds and stacking interactions. Our results provide a molecular description of how the multivalency of the guanidinium group enables the coordination of multiple RNA groups by a single arginine residue, thus ultimately stabilizing protein/RNA condensates.

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains

“…Such inversion of the charge of RNA or DNA in the presence of small polycationic species has been shown to be responsible for reentrant phase behavior. − Only when the PA and polyU charges are matched, does the inter-molecular polyU cross-linking become efficient and lead to the formation of coacervates. Complex coacervation and reentrant behavior have been extensively studied, including systems comprising peptide-RNA, polycation-RNA, and polyamines-nucleic acids. , Finally, we note that arginine-rich domains are prone for phase separation in the presence of RNA due to electrostatic interaction and the ability of the arginine side chain to simultaneously form a higher number of specific interactions with oligonucleotides . However, the SA peptide, which has the same number of arginine residues, and at the same positions, did not form coacervates with polyU, suggesting that the sequence (and not just composition) is crucial for dimer formation and the generation of transient structure for phase separation.…”

“… 47 , 48 Finally, we note that arginine-rich domains are prone for phase separation in the presence of RNA due to electrostatic interaction 49 and the ability of the arginine side chain to simultaneously form a higher number of specific interactions with oligonucleotides. 50 However, the SA peptide, which has the same number of arginine residues, and at the same positions, did not form coacervates with polyU, suggesting that the sequence (and not just composition) is crucial for dimer formation and the generation of transient structure for phase separation. Although we suspect that attainment of the α-helical structure is coupled to dimerization, this was not explicitly shown.…”

“…The concentration of the unlabeled peptide stock solutions was determined using the bicinchoninic acid assay (Thermo Fisher Scientific) and ranged from 0.8 to 1.2 mM peptide. A stock solution of 500 mM MES, pH 5.6 was used to adjust all samples to a final concentration of 50 Each sample was freshly prepared and the time to collect CW-PR specte CW-EPR data was 30−40 min. For samples forming droplets, we could observe turbidity, but within the above time, we did not observe separation into a condensed and a dilute phase.…”

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains

“…The complex coacervation and reentrant behavior has been extensively studied, including systems comprised of peptide-RNA, polycation-RNA, and polyamines-nucleic acids 47,48 . Finally, we note that arginine-rich domains are prone for phase separation in the presence of RNA due to electrostatic interaction 49 and the ability of the arginine side chain to simultaneously form a higher number of specific interactions with oligonucleotides 50 . However, the SA peptide, which has the same number of arginine residues, and at the same positions, did not form coacervates with polyU, suggesting that sequence (and not just composition) is crucial for dimer formation and the generation of transient structure for phase separation.…”

Role of NS2 specific RNA binding and phosphorylation in liquid–liquid phase separation and virus assembly

“…Using an electrophoretic mobility shift assay (EMSA) specific RNA binding was mapped to Arg 6 and 7 with no apparent role for lysines in the central and C-terminal domains ( 47 ). The guanidinium group in arginine residues can simultaneously form multivalent co-ordinations with many RNA groups (through H-bonds and pi stacking), which stabilize protein-RNA complexes ( 48 ) and induce phase separation ( 49 ). LLPS is exceptionally sensitive to even minor changes in the charge or conformation of macromolecules ( 50 ) and it seems probable that RNA binding to the stable N-terminal structure of NS2 initiates conformational change in the disordered central domain may facilitate further intermolecular interactions resulting in phase separation (reviewed in ( 44 )).…”