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

Abstract

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.

Keywords: Phase separation; functional amyloid; native mass spectrometry; stickers and spacers-model.

Figure 1

Sequence dependence of NT2RepCT LLPS: (a) Conversion of spidroins from droplets to fibers is accompanied by a decrease in pH and an increase in ion concentration, predominantly phosphate and bicarbonate. (b) Structure of the NT2RepCT mini-spidroin, with NTD (blue) and CTD (yellow), as well as two repeat regions (green) with the sequence of the first repeat of WT, Y to F, and Y to F, R to L shown below. (c) Fluorescence microscopy of all three variants in 0.5 M potassium phosphate shows increased droplet size and fluidity of the Y to F variant. Scale bars are 10 μM. (d) Schematic view of high-affinity stickers and hydrophobic residues illustrates how the balance between sticker affinity and hydrophobicity can control spidroin LLPS.

Figure 2

Kosmotropic ions promote LLPS and destabilize the dimeric state of NT2RepCT^YF. (a) NT2RepCT^YF droplet formation shows a strict dependence on salt concentration, requiring higher concentrations of less kosmotropic ions. Open circles show the highest concentration at which almost no droplets were observed directly upon dilution in the respective buffer. (b) Native IMMS of NT2RepCT^YF below (upper panel) and above (lower panel) ammonium acetate concentrations that induce LLPS. Compared to 0.1 M ammonium acetate, we find an increase in lowly charged monomers around the 11+ ion, and a decrease in compact as well as in highly charged dimers (around the 14+ and 31+ charge states, respectively) In 1 M ammonium acetate. Inserts: Arrival time distributions for the 14+ NT2RepCT^YF dimer reveal a slight increase in drift time at high ammonium acetate concentrations. Dashed lines indicate the arrival time centroid of the native protein in 0.1 M ammonium acetate.

Figure 3

Disordered linker primes the CTD for assembly during LLPS. (a) Native mass spectra show dissociation of the dimeric CTD when the ammonium acetate concentration is raised from 0.1 to 1 M. (b) Overlay of the top 20 NMR structures of the MiSp1 CTD dimer (PDB ID 2MFZ) show part of the disordered linker (red) and the folded core (orange). The linker is rich in residues associated with LLPS and is predicted by the FuzDrop server to undergo LLPS, with the 0.6 threshold shown as dashed line. Yellow boxes denote regions with high propensity for β-sheet aggregation predicted by Aggrescan3D. (c) Truncated CTD without linker domain that is exclusively dimeric in native MS and does not dissociate in response to increased ammonium acetate concentration. (d) Top: ThT fluorescence curves for the full-length CTD in 0.05 or 0.5 M potassium phosphate buffer at pH 8 and 5. Low pH and high phosphate concentration causes lag-free formation of ThT-positive aggregates (blue curve). Bottom: ThT fluorescence curves for the truncated CTD show no aggregation at pH 8, and inhibition of aggregation by 0.5 M phosphate at pH 5 (blue curve). Curves are averages of n = 5 repeats., error bars indicate standard deviation.

Figure 4

Role of LLPS in spider silk assembly. Kosmotropic ions promote liquid–liquid phase separation through a salting out-mechanism and simultaneously destabilize the CTD dimer by acting on its linker region (insert). Lower pH then triggers full CTD unfolding and aggregation. Both processes this generate droplets of aggregation-competent spidroins. Shear force, which is not studied here, is likely to then mediate the final assembly into fibers.