Heterochromatin protein 1α (HP1α) is a crucial element of chromatin organization. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can undergo phase separation upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. Here, we combine computational and experimental approaches to elucidate the molecular interactions that drive these processes. In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged HP1α peptide ligands and disrupted by the addition of negatively charged or neutral peptides. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in HP1α LLPS where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine-tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

A variety of membraneless organelles, often termed “biological condensates”, play an important role in the regulation of cellular processes such as gene transcription, translation, and protein quality control. On the basis of experimental and theoretical investigations, liquid–liquid phase separation (LLPS) has been proposed as a possible mechanism for the origin of biological condensates. LLPS requires multivalent macromolecules that template the formation of long-range, intermolecular interaction networks and results in the formation of condensates with defined composition and material properties. Multivalent interactions driving LLPS exhibit a wide range of modes from highly stereospecific to nonspecific and involve both folded and disordered regions. Multidomain proteins serve as suitable macromolecules for promoting phase separation and achieving disparate functions due to their potential for multivalent interactions and regulation. Here, we aim to highlight the influence of the domain architecture and interdomain interactions on the phase separation of multidomain protein condensates. First, the general principles underlying these interactions are illustrated on the basis of examples of multidomain proteins that are predominantly associated with nucleic acid binding and protein quality control and contain both folded and disordered regions. Next, the examples showcase how LLPS properties of folded and disordered regions can be leveraged to engineer multidomain constructs that form condensates with the desired assembly and functional properties. Finally, we highlight the need for improvements in coarse-grained computational models that can provide molecular-level insights into multidomain protein condensates in conjunction with experimental efforts.

Recent advances in residue-level coarse-grained (CG) computational models have enabled molecular-level insights into biological condensates of intrinsically disordered proteins (IDPs), shedding light on the sequence determinants of their phase separation. The existing CG models that treat protein chains as flexible molecules connected via harmonic bonds cannot populate common secondary-structure elements. Here, we present a CG dihedral angle potential between four neighboring beads centered at Cα atoms to faithfully capture the transient helical structures of IDPs. In order to parameterize and validate our new model, we propose Cα-based helix assignment rules based on dihedral angles that succeed in reproducing the atomistic helicity results of a polyalanine peptide and folded proteins. We then introduce sequence-dependent dihedral angle potential parameters (εd) and use experimentally available helical propensities of naturally occurring 20 amino acids to find their optimal values. The single-chain helical propensities from the CG simulations for commonly studied prion-like IDPs are in excellent agreement with the NMR-based α-helix fraction, demonstrating that the new HPS-SS model can accurately produce structural features of IDPs. Furthermore, this model can be easily implemented for large-scale assembly simulations due to its simplicity.

Heterochromatin protein 1α (HP1α) is a crucial component for the proper maintenance of chromatin structure and function. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to sequester and compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can form phase separated liquid droplets upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. While it is known that LLPS requires homodimerization of HP1α and that it involves interactions between the positively charged hinge region of HP1α and the negatively charged phosphorylated NTE or nucleic acid, the precise molecular details of this process and its regulation are still unclear. Here, we combine computational modeling and experimental approaches to elucidate the phase separation properties of HP1α under phosphorylation-driven and DNA-driven LLPS conditions. We also tune these properties using peptides from four HP1α binding partners (Sgo1, CAF-1, LBR, and H3). In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged peptide ligands such as Sgo1 and H3 and disrupted by the addition of negatively charged or neutral peptides such as LBR and CAF-1. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in the LLPS of HP1α where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

The heterochromatin protein 1 (HP1) family is a crucial component of heterochromatin with diverse functions in gene regulation, cell cycle control, and cell differentiation. In humans, there are three paralogs, HP1α, HP1β, and HP1γ, which exhibit remarkable similarities in their domain architecture and sequence properties. Nevertheless, these paralogs display distinct behaviors in liquid-liquid phase separation (LLPS), a process linked to heterochromatin formation. Here, we employ a coarse-grained simulation framework to uncover the sequence features responsible for the observed differences in LLPS. We highlight the significance of the net charge and charge patterning along the sequence in governing paralog LLPS propensities. We also show that both highly conserved folded and less-conserved disordered domains contribute to the observed differences. Furthermore, we explore the potential co-localization of different HP1 paralogs in multicomponent assemblies and the impact of DNA on this process. Importantly, our study reveals that DNA can significantly reshape the stability of a minimal condensate formed by HP1 paralogs due to competitive interactions of HP1α with HP1β and HP1γ versus DNA. In conclusion, our work highlights the physicochemical nature of interactions that govern the distinct phase-separation behaviors of HP1 paralogs and provides a molecular framework for understanding their role in chromatin organization.

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The formation of amyloid fibrils in Alzheimer's disease and other neurodegenerative disorders is limited by a slow nucleation step due to the entropic cost to initiate the ordered cross-β structure. While the barrier can be lowered if the molecules maintain conformational disorder, poorly ordered clusters provide a poor binding surface for new molecules. To understand these opposing factors, we used all-atom simulations to parameterize a lattice model that treats each amino acid as a binary variable with β-sheet and non-β states. We find that the optimal degree of order in a nucleus depends on protein concentration. Low concentration systems require more ordered nuclei to capture infrequent monomer attachments. The nucleation phase transitions to the elongation phase when the β-sheet core becomes large enough to overcome the initiation cost, at which point further ordering becomes favorable and the nascent fibril efficiently captures new molecules.