Hydrogen bond guidance and aromatic stacking drive liquid-liquid phase separation of intrinsically disordered histidine-rich peptides

Bartosz Gabryelczyk,Stefan Salentinig,Xiangyan Shi,Ali Miserez,Konstantin Pervushin,Piet J M Swinkels,Hao Cai,Yue Sun

doi:10.1038/s41467-019-13469-8

Abstract

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues. Despite growing understanding of LLPS, molecular-level mechanisms behind this process are still not fully established. Here, we use histidine-rich squid beak proteins (HBPs) as model IDPs to shed light on molecular interactions governing LLPS. We show that LLPS of HBPs is mediated though specific modular repeats. The morphology of separated phases (liquid-like versus hydrogels) correlates with the repeats’ hydrophobicity. Solution-state NMR indicates that LLPS is a multistep process initiated by deprotonation of histidine residues, followed by transient hydrogen bonding with tyrosine, and eventually by hydrophobic interactions. The microdroplets are stabilized by aromatic clustering of tyrosine residues exhibiting restricted molecular mobility in the nano-to-microsecond timescale according to solid-state NMR experiments. Our findings provide guidelines to rationally design pH-responsive peptides with LLPS ability for various applications, including bioinspired protocells and smart drug-delivery systems.

Highlights

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues
Recent studies of proteins involved in LLPS have revealed that such proteins usually belong to the family of intrinsically disordered proteins (IDPs) or contain intrinsically disordered regions (IDRs)
A combined solution and solid-state NMR study on elastin-like peptides (ELPs) that exhibit LLPS through hydrophobic interactions triggered by temperature changes established a model by which the final biomaterial structure is self-assembled[23]

Summary

Introduction

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues. While coacervation studies were initiated in the field of biopolymeric colloids, in recent years LLPS has attracted considerable interest from life scientists[5,6] with numerous studies showing its role in organizing biomolecules in living cells via formation of membraneless organelles[7,8,9,10,11] Another less recognized but increasingly appreciated biological role of LLPS is associated with the assembly of extracellular, load-bearing structures[12]. IDPs that drive LLPS are typically characterized by conformational heterogeneity at equilibrium and by molecular motions that span timescales from ns to ms[15] They usually exhibit a low sequence complexity with a modular organization of their primary structure[6,16,17]. A combined solution and solid-state NMR study on elastin-like peptides (ELPs) that exhibit LLPS through hydrophobic interactions triggered by temperature changes established a model by which the final biomaterial structure is self-assembled[23]. Solid-state NMR experiments have been used to study the low complexity domain of the FUS24 and TDP-43 (ref. 25) RNA binding proteins, which undergo LLPS and in the pathological state may lead to the formation of insoluble fibril-like structures[26]

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Results

Conclusion