Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks

Abstract

Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.

Fig. 1.. Structures of LARKS (B-F) compared to a steric zipper (A).

All structures composed of two mating β-sheets, one purple and the other yellow. The left-hand column shows the trace of the backbones of mating sheets to highlight kinks in the backbones of LARKS and the pleating of the classical β-sheets in steric zippers. The second column shows the atomic structures of mating sheets viewed down the fibril axes. The third column shows cartoons of the mating β-sheets viewed nearly perpendicular to the fibril axes. Each interface is characterized by the shape complementarity score (Sc=1.0 for perfect complementarity) and buried solvent-accessible surface area (Ab) in Ų between the mated sheets. Carbon atoms are colored purple or yellow, Nitrogen is blue, and Oxygen is red. Five layers of β-sheets are shown of the hundreds of thousands in the crystals. The kinked structures of LARKS are rare among mating β-sheets; dozens of other paired β-sheets form steric zippers (35).

Fig. 2.. Synthetic LARKS construct forms a labile hydrogel.

A synthetic LARKS construct with the sequence SYSGYSGDTSYSSYGQSNGPSTGGYG forms a labile hydrogel when dissolved in water at 50mg/ml and left overnight at 4°C. The hydrogel melts upon heating the sample to 60°C for two hours. A bubble (blue arrow) was introduced to the sample to show the difference between the liquid state (bubble rises) and hydrogel state (bubble does not rise). Electron microscopy confirms that fibrils were indeed melted. The hydrogel-forming property of this triple-LARKS sequence suggests that it is the multiple LARKS found in many LCDs that endow their unusual property of forming hydrogels. Scale bars are equal to 200nm.

Fig. 3.. 3D profiling to identify LARKS in LC domains of human proteins.

(A) Method: Sidechains are removed from the backbones of one of our atomic structures of a LARKS. Then the sequence of interest (hnRNPA1 shown) is threaded through the six residue template by placing the query sidechains on the template backbone. Sidechains are repacked and a Rosetta energy function is used to estimate if the structure is favorable for the threaded sequence. The sequence then advances through the template by one residue increments, producing successive models. (B) The frequency of the number of LARKS in 1725 human proteins predicted to house at least two LARKS. Proteins having two or more LARKS are predicted to have the capacity to form networks and possibly gels. (C) The annotated functions of the 400 proteins with the most predicted LARKS.

Fig. 4.

Functions of proteins among the 400 proteins most enriched in LARKS and dynamic intracellular bodies they are known to be a part of.