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

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

doi:10.1038/s41467-019-13469-8

Cited by 137 publications

(173 citation statements)

References 57 publications

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“…Phase separation of LAF-1 RGG is hypothesized to be driven by several different modes of interaction, including electrostatic, π-π, and cation-π interactions (27). In addition, hydrogen bonds and hydrophobic contacts may play a role in phase separation for sequences containing residues capable of such interactions (16,(33)(34)(35)(36)(37). However, it is difficult to characterize these interactions using experimental techniques due to the dynamic nature of the phase-separated proteins and the high spatiotemporal resolution needed to probe the interactions (35).…”

Section: Resultsmentioning

confidence: 99%

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Schuster

Dignon

Tang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

231

302

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Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

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Section: Resultsmentioning

confidence: 99%

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Schuster

Dignon

Tang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

231

302

View full text Add to dashboard Cite

show abstract

“…Phase separation of LAF-1 RGG is hypothesized to be driven by several different modes of interaction, including electrostatic, π-π, and cation-π interactions 27 . In addition, hydrogen bonds and hydrophobic contacts may play a role in phase separation for sequences containing residues capable of such interactions 16, 33–37 . However, it is difficult to characterize these interactions using experimental techniques due to the dynamic nature of the phase-separated proteins and the high spatiotemporal resolution needed to probe the interactions 35 .…”

Section: Resultsmentioning

confidence: 99%

“…We note that arginine may be particularly prone to promoting LLPS with aromatic-rich sequences due to cooperative cation-π and sp 2 /π interactions that co-occur. Another important interaction mode is hydrogen bonding, which has also recently been demonstrated to be important to LLPS 35, 37 and is present in interactions between cationic residues and tyrosine. Our simulations suggest that the reduced LLPS propensity when mutating tyrosine to phenylalanine can be explained by the loss of sidechain hydrogen bonding, as phenylalanine lacks the hydroxyl group.…”

Section: Discussionmentioning

confidence: 99%

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

Schuster

Dignon

Tang³

et al. 2020

Preprint

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AbstractPhase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bio-inspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. By altering the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation, we designed sequences with significantly increased phase separation propensity. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that in spite of these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance a predictive framework and identify key biophysical principles of sequence features important to phase separation.Significance StatementMembraneless organelles are assemblies of highly concentrated biomolecules that form through a liquid-liquid phase separation process. These assemblies are often enriched in intrinsically disordered proteins, which play an important role in driving phase separation. Understanding the sequence-to-phase behavior relationship of these disordered proteins is important for understanding the biochemistry of membraneless organelles, as well as for designing synthetic organelles and biomaterials. In this work, we explore a model protein, the disordered N-terminal domain of LAF-1, and highlight how three key features of the sequence control the protein’s propensity to phase separate. Combining predictive simulations with experiments, we find that phase behavior of this model IDP is dictated by the presence of a short conserved domain, charge patterning, and arginine-tyrosine interactions.

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“…All kinds of weak and non-specific interactions can contribute to such adhesive contacts. Specific involvement in coacervation has been documented for cation-π contacts, between positively charged residues and π electrons in aromatic residues, π-π interactions between aromatic rings, electrostatic attractions, and dipolar forces [128,136], along with hydrogen bonding [192] and hydrophobic interactions [193]. Poly-ions, as well as RNA or ssDNA, can seed LLPS by favoring the interactions of RNA with the RNA-binding domain [130] or RNA base pairing [129].…”

Section: Appendix C Liquid-liquid Phase Separationmentioning

confidence: 99%

Relevance of Electrostatic Charges in Compactness, Aggregation, and Phase Separation of Intrinsically Disordered Proteins

Bianchi

Longhi

Grandori

et al. 2020

IJMS

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The abundance of intrinsic disorder in the protein realm and its role in a variety of physiological and pathological cellular events have strengthened the interest of the scientific community in understanding the structural and dynamical properties of intrinsically disordered proteins (IDPs) and regions (IDRs). Attempts at rationalizing the general principles underlying both conformational properties and transitions of IDPs/IDRs must consider the abundance of charged residues (Asp, Glu, Lys, and Arg) that typifies these proteins, rendering them assimilable to polyampholytes or polyelectrolytes. Their conformation strongly depends on both the charge density and distribution along the sequence (i.e., charge decoration) as highlighted by recent experimental and theoretical studies that have introduced novel descriptors. Published experimental data are revisited herein in the frame of this formalism, in a new and possibly unitary perspective. The physicochemical properties most directly affected by charge density and distribution are compaction and solubility, which can be described in a relatively simplified way by tools of polymer physics. Dissecting factors controlling such properties could contribute to better understanding complex biological phenomena, such as fibrillation and phase separation. Furthermore, this knowledge is expected to have enormous practical implications for the design, synthesis, and exploitation of bio-derived materials and the control of natural biological processes.

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

Cited by 137 publications

References 57 publications

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

Relevance of Electrostatic Charges in Compactness, Aggregation, and Phase Separation of Intrinsically Disordered Proteins

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