Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures

Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context

coreMYC

Molecular dynamics simulation trajectories of peptide derivatives from c-MYC.</p

Stability of Bcl-X_L structure in PME simulations.

<p>MD trajectories of (A) percentage helical content and (B) RMSD profiles shown for the four simulations that used PME to treat the long-range interactions. Percentage helical content of each MD simulated structure was calculated as described in the Methods section. RMSD was calculated by considering the Cα atoms of all stable helical segments H1 to H6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054397#pone-0054397-t002" target="_blank">Table 2</a>).</p

Average solvent accessible surface area (SASA in Ų)^a of loop LB hydrophobic residues.

a<p>SASA was calculated for a given residue using the g_sas tool available in GROMACS version 3.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054397#pone.0054397-vanderSpoel1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054397#pone.0054397-Lindahl1" target="_blank">[40]</a>.</p>b<p>Residues shown in bold participate in stable interactions with at least one of the BH3 peptide ligands in Bcl-X_L complex simulations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054397#pone.0054397-Lama1" target="_blank">[30]</a>.</p>c<p>For each residue, the top and bottom numbers represent respectively the average SASA values calculated for the first and the last 5 ns of production runs. If the residues are buried during the simulation, then the values are shown in italics and underlined. SASA values of exposed residues are shown in bold.</p

Average minimum distance (in Å) for residue pairs involved in interactions^a.

a<p>These interactions are stable in apo/holo Bcl-X_L simulations but not in the Bcl-X_L complex simulations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054397#pone.0054397-Lama1" target="_blank">[30]</a>.</p>b<p>Residue shown in bold participate in stable interactions with the BH3 peptide ligands in Bcl-X_L complex simulations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054397#pone.0054397-Lama1" target="_blank">[30]</a>.</p>c<p>The numbers in bracket represent the percentage time in which the minimum distance between the residue pairs was less than 4.0 Å during the analysis period.</p

Stable helical regions in Bcl-X_L apo/holo twin-range cut-off simulations.

a<p>The residue numbering is according to the Bcl-X_L sequence in UniProt database with the accession code Q64373.</p>b<p>The helical regions common to the two Bcl-X_L crystal structures (PDB ID: 1PQ0 and 1PQ1) are reported. The definition of helical segment in the protein crystal structure is given in the Methods section.</p>c<p>The loop region between H1 and H2 is not resolved in the experimentally determined crystal structures. The loop was built by homology modeling procedure with another Bcl-XL structure (PDB ID: 1G5J) as the template. In this model, the residue 44 is covalently linked to residue 85.</p>d<p>These helical segments are stable in at least two out of four twin-range cut-off simulations.</p

Interactions of hydrophobic residues in the hydrophobic groove and their accessible surface areas.

<p>Interactions among the hydrophobic residues in the hydrophobic groove are shown for (A) Apo-I and (B) Holo-I simulations. Helices and side-chains of hydrophobic residues are displayed in ribbon and stick representation respectively. Surface and ribbon representations of helices H2, H3, H4, H5 and loop LD (cyan) along with the hydrophobic residues from these regions (yellow) are shown for (C and E) Apo-I and (D and F) Holo-I simulations without loop LB (C and D) and with loop LB (E and F). Loop LB surface is represented in purple color in (E) and (F). The Bcl-X_L structures shown in this figures were saved at the end of 55 ns production runs from Apo-I and Holo-I simulations.</p

Stability of Bcl-X_L structure in twin-range cut-off simulations.

<p>MD trajectories of (A) percentage helical content and (B) RMSD profiles shown for the four simulations that used twin-range cut-off to evaluate the long-range interactions. Percentage helical content of each MD simulated structure was calculated as described in the Methods section. RMSD was calculated by considering the Cα atoms of all stable helical segments H1 to H6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054397#pone-0054397-t002" target="_blank">Table 2</a>).</p