Multivalent Interactions Between the Picornavirus 3C(D) Main Protease and RNA Oligonucleotides Induce Liquid–Liquid Phase Separation

Figures

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  Figure 1. RNA binding leads to chemical shift changes for two oppositely faced residue clusters in PV-3C. (A) 1H-15N SOFAST-HMQC NMR spectra were compared with free wild-type PV-3C (blue) and RNA-1 (GGCGGCGUACUCCGG)-bound PV-3C (red) in 1:0.5 stoichiometry. Residues with substantial chemical shift perturbation (CSP) are labeled in the spectra; zoomed-in resonances represent CSP change for the corresponding residues. (B) NMR CSPs for each residue is plotted, with residues labeled that show a considerable change in CSP after RNA-1 addition. Note that the R13 peak was missing after the addition of RNA-1. NMR CSPs were calculated using the following equation: Δδcombined = (ΔδH2 + (ΔδN/5)2)0.5, where ΔδH and ΔδN are the chemical shift differences between 3C with and without RNA for the backbone amide proton and nitrogen, respectively. Resonances with substantial CSPs have Δδcombined greater than 0.02 ppm (blue dashed line). NMR assignment of 3C is depicted from BioMagResBank database (https://bmrb.io/ (accessed on 28 October 2025)) under BMRB accession number 15222, by Amero CD et al. [14]. (C) Residues showing substantial CSPs in the presence of RNA are represented in blue on the PV-3C X-ray…

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  Figure 2. Interactions between PV-3C and RNA lead to multimeric complexes and liquid–liquid phase separation. (A) AUC of PV-3C with RNA-1, with PV-3C and RNA-1 forming a complex (green) at 1:1 molar stoichiometry. Upon increasing the RNA concentration (1:2 3C/RNA-1, red; 1:20 3C/RNA-1, blue), various multimolar ratio complexes appear as indicated in red and blue, respectively. (B) Using DIC microscopy, LLPS is not apparent with 1:1 3C/RNA-1 complex. (C,D) However, at higher ratios (panel (C), 1:2 3C/RNA-1; panel (D), 1:20 3C/RNA-1), DIC microscopy affirms the formation of LLPS. The PV-3C protein was at a concentration of 25 µM. RNA was added according to protein stoichiometry. The objective magnification was 40×. Both protein and RNA were in a buffer containing 10 mM HEPES and 50 mM NaCl, at pH 7.5. Experiments were conducted at 298 K.

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  Figure 3. Interactions between PV-3CD and RNA lead to LLPS. DIC microscopy shows evidence for LLPS under all 3CD to RNA ratios tested (panel (A), 1:0.5; panel (B), 1:1; panel (C), 1:2). The PV-3CD protein was at a concentration of 25 µM. RNA was added according to protein stoichiometry. The PV-3C protein was at a concentration of 25 µM. The objective magnification was 40×. Both protein and RNA were in a buffer containing 10 mM HEPES, 50 mM NaCl, and at pH 7.5. Experiments were conducted at 298 K.

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  Figure 4. Two interaction sites between PV-3C protein and RNA-1 by MD simulations. (A) Proximity probabilities (Pprox) of residues in 3C. (B) Distance map between the residues of RNA-1 and 3C. The color bar represents the color-coded distances underlying the distance map. The vertical axis shows the RNA residues of the two chains (each with 15 nucleotides). (C) Simulation snapshot showing two chains of RNA-1 interacting with the residues in Cluster-I and Cluster-II of the 3C protein (as also observed from NMR experiments). The protein and RNA chains are shown in green and red, respectively. The residues colored blue have proximity probabilities greater than 0.5.

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  Figure 5. Interactions between PV-3CD protein and RNA-1 from MD simulations. (A) Proximity probabilities (Pprox) of residues in 3CD. (B) Distance map between the residues of RNA-1 and 3CD. The color bar represents the color-coded distances underlying the distance map. The vertical axis shows the RNA residues of the two chains (each with 15 nucleotides). (C) Simulation snapshot showing two chains of RNA-1 interacting with the residues in Cluster-I and Cluster-II of the 3CD protein. The former also interacts with positively charged residues in the 3D domain. The protein and RNA chains are shown in green and red, respectively. The residues colored blue have proximity probabilities greater than 0.5. (D) Electrostatic map of the PV-3CD protein surface. (E) Time evolution of the radius of gyration (Rg) of PV-3CD obtained from MD simulations. Representative snapshots of the protein structures in these two distinct states are presented along with the Rg time trace (green represents protein and red represents RNA with the dashed line representing a transition between the two distinct states.).

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  Figure 6. Multivalent interactions between 3C (or 3CD) and RNA oligonucleotides lead to liquid–liquid phase separation.