Principles Governing the Phase Separation of Multidomain Proteins.

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.

Transient interdomain interactions modulate the monomeric structural ensemble and oligomerization landscape of Huntingtin Exon 1.

Polyglutamine expansion (≥ 36 residues) within the N-terminal exon-1 of Huntingtin (Httex1) leads to Huntington's disease, a neurogenerative condition marked by the presence of intranuclear Htt inclusions. Notably, the polyglutamine tract in Httex1 is flanked by an N-terminal coiled-coil domain - N17 (17 amino acids), which undergoes self-association to promote the formation of soluble Httex1 oligomers and brings the aggregation-prone polyQ tracts in close spatial proximity. However, the mechanisms underlying the subsequent conversion of soluble oligomers into insoluble ß-rich aggregates with increasing polyQ length, remain unclear. Current knowledge suggests that expansion of the polyQ tract increases its helicity, and this favors its oligomerization and aggregation. In addition, studies utilizing conformation-specific antibodies and a stable coiled-coil heterotetrametric system fused to polyQ indicate that domain "cross-talk" (i.e., interdomain interactions) may be necessary to efficiently promote the emergence of toxic conformations (in monomers and oligomers) and fibrillar aggregation. Here, we performed extensive atomistic molecular dynamics (MD) simulations (aggregate time ∼ 0.7 ms) of N17-polyQ fragments to uncover the interplay between structural transformation and domain "cross-talk" on the monomeric structural ensemble and oligomerization landscape of Httex1. Our simulation ensembles of N17-polyQ monomers validated against 13 C NMR chemical shifts indicated that in addition to elevated α-helicity, polyQ expansion also favors transient, interdomain (N17-polyQ) interactions which result in the emergence of ß-conformations. Further, interdomain interactions decreased the overall stability of N17-mediated dimers by counteracting the stabilizing effect of increased α-helicity and promoted a heterogenous oligomerization landscape on the sub-microsecond timescale. Overall, our study uncovers the significance of domain "cross-talk" in modulating the monomeric conformational ensemble and oligomerization landscape of Httex1 to favor the formation of amyloid aggregates.

Challenges in studying the liquid-to-solid phase transitions of proteins using computer simulations.

"Membraneless organelles," also referred to as biomolecular condensates, perform a variety of cellular functions and their dysregulation is implicated in cancer and neurodegeneration. In the last two decades, liquid-liquid phase separation (LLPS) of intrinsically disordered and multidomain proteins has emerged as a plausible mechanism underlying the formation of various biomolecular condensates. Further, the occurrence of liquid-to-solid transitions within liquid-like condensates may give rise to amyloid structures, implying a biophysical link between phase separation and protein aggregation. Despite significant advances, uncovering the microscopic details of liquid-to-solid phase transitions using experiments remains a considerable challenge and presents an exciting opportunity for the development of computational models which provide valuable, complementary insights into the underlying phenomenon. In this review, we first highlight recent biophysical studies which provide new insights into the molecular mechanisms underlying liquid-to-solid (fibril) phase transitions of folded, disordered and multi-domain proteins. Next, we summarize the range of computational models used to study protein aggregation and phase separation. Finally, we discuss recent computational approaches which attempt to capture the underlying physics of liquid-to-solid transitions along with their merits and shortcomings.