Principles Governing the Phase Separation of Multidomain Proteins

Abstract

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.

Protein conformation and biomolecular condensates

“…In the scaffold-client model, liquid phase separation occurs through the formation of dense networks of intermolecular interactions, in which the multivalency of each constituent is provided by folded domains and/or by IDDs (P. Li et al., 2012 ; Shin and Brangwynne, 2017 ; Kamagata et al., 2021 ; Mohanty et al., 2022 ; Feng et al., 2021 ). Interactions between folded domains are specific and stoichiometrically defined.…”

“…However, the modern concept of domain as the unit of protein structure reconciles order, disorder and biological function. In fact, if one defines IDRs as rightly domains and thinks of multidomain proteins as composed of coexisting folded and intrinsically disordered domains (IDDs), a general function for the latter is envisaged: to allow multiple, long-range, and fuzzy interactions with different ligands and partners in large biological complexes ( Tompa and Fuxreiter, 2008 ); Uversky and Dunker (2010) ; Van Der Lee et al., 2014 ; Uversky and Finkelstein (2019) ; Mohanty et al. (2022) .…”

“…However, given its vastness, we mostly focus on the protein conformation in liquid condensates. Several excellent and recent reviews on the multiple aspects of protein condensation are available, and the interested reader is directed to them for more information on specialized aspects of the subject ( Uversky, 2017 ; Banani et al., 2017 ; Holehouse and Pappu, 2018a ; Peran and Mittag, 2020 ; Choi et al., 2020 ; Alberti and Hyman, 2021 ; Lyon et al., 2021 ; Abyzov et al., 2022 ; Mohanty et al., 2022 ). In this review, we endeavored to cover the subject from a structural biology perspective and to exhaustively illustrate with examples the ideas and basic principles discussed.…”

Effect of Charge Distribution on the Dynamics of Polyampholytic Disordered Proteins

“…Intrinsically disordered proteins (IDPs) or regions (IDRs) are a driving force in the formation of membraneless organelles − that are responsible for many physiological functions, e.g., signaling, stress response, transcription, and metabolic processes. − Due to the lack of well-defined three-dimensional folded structures, IDPs display remarkable heterogeneity in terms of structural architectures, binding multiplicity, and functional diversity. Many known IDPs are polyampholytesa class of polymers that consists of both positively and negatively charged monomers in varying fractions.…”

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates

“…Biomolecular condensates formed via liquid-liquid phase separation of proteins and nucleic acids serve as on-demand membraneless organelles that are involved in the spatiotemporallycontrolled organization, compartmentalization, and regulation inside living cells. Owing to their ability to form and dissipate in response to cellular cues, regulatability, permeability, and the ability to selectively concentrate the biomolecules, these noncanonical liquid-like organelles are emerging as central players at all levels of essential cellular activity ranging from the expression and regulation of genes to the modulation of intricate signaling pathways (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). An emerging body of work has revealed that intrinsically disordered proteins/regions (IDPs/IDRs) often comprising polypeptide repeat units, low sequence complexity, and prionlike domains are ideal candidates for biological phase separation.…”