Atomic Resolution Interactions Regulating Partitioning of a FUS Folded RRM Domain into Model CAPRIN1 Condensates.

Biomolecular condensates enrich specific client molecules while excluding others, often modulating conformational landscapes, and hence functions, of molecules dissolved within them. NMR-based atomic resolution studies have focused on interactions between scaffold proteins and the unfolded states of client proteins to understand the factors that influence client partitioning into condensed phases. However, characterization of interactions involving the folded client conformer is required to obtain a complete picture of how dissolution within the condensed phase affects the client energy landscape.

Client-scaffold interactions suppress aggregation of a client protein in model condensates.

Many studies have shown that sequestration of client proteins into condensates locally increases their concentrations and/or modulates their conformational landscapes to promote aberrant aggregation. Far fewer examples have emerged where the proteinaceous condensed phase environment protects clients from aggregation. Here, we show that a condensate scaffolded by the C-terminal disordered region of Cell Cycle Associated Protein 1 (CAPRIN1) suppresses aggregation of the Fused in Sarcoma (FUS) RNA Recognition Motif (RRM) client, both components of stress granules.

Disorder meets its match.

Designed protein pockets recognize intrinsically disordered protein regions.

Biological insights from integrative modeling of intrinsically disordered protein systems.

Intrinsically disordered proteins and regions are increasingly appreciated for their abundance in the proteome and the many functional roles they play in the cell. In this short review, we describe a variety of approaches used to obtain biological insight from the structural ensembles of disordered proteins, regions, and complexes and the integrative biology challenges that arise from combining diverse experiments and computational models. Importantly, we highlight findings regarding structural and dynamic characterization of disordered regions involved in binding and phase separation, as well as drug targeting of disordered regions, using a broad framework of integrative modeling approaches.

Essential lipids enrich membrane-associated condensates to rescue synaptic morpho-functional deficits in a mouse model of autism.

Synaptic proteins form intracellular condensates with their scaffolds, but it is unknown whether and how essential lipids transform dynamic cytosolic condensates into stable, functional macromolecular assemblies at the membrane. We show that docosahexaenoic acid (DHA), independent of canonical fatty acid receptor 4 signaling, facilitates the re-localization of cytosolic "full-droplet" condensates composed of the key synaptic elements PSD95 and Kv1.2 to the plasma membrane as "half-droplets.

Protein interactions, calcium, phosphorylation, and cholesterol modulate CFTR cluster formation on membranes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel whose dysfunction leads to intracellular accumulation of chloride ions, dehydration of cell surfaces, and subsequent damage to airway and ductal organs. Beyond its function as a chloride channel, interactions between CFTR, epithelium sodium channel, and solute carrier (SLC) transporter family membrane proteins and cytoplasmic proteins, including calmodulin and Na+/H+ exchanger regulatory factor-1 (NHERF-1), coregulate ion homeostasis. CFTR has also been observed to form mesoscale membrane clusters.

Deep Learning of Proteins with Local and Global Regions of Disorder.

Although machine learning has transformed protein structure prediction of folded protein ground states with remarkable accuracy, intrinsically disordered proteins and regions (IDPs/IDRs) are defined by diverse and dynamical structural ensembles that are predicted with low confidence by algorithms such as AlphaFold. We present a new machine learning method, IDPForge (Intrinsically Disordered Protein, FOlded and disordered Region GEnerator), that exploits a transformer protein language diffusion model to create all-atom IDP ensembles and IDR disordered ensembles that maintains the folded domains. IDPForge does not require sequence-specific training, back transformations from coarse-grained representations, nor ensemble reweighting, as in general the created IDP/IDR conformational ensembles show good agreement with solution experimental data, and options for biasing with experimental restraints are provided if desired.

Electrostatics of salt-dependent reentrant phase behaviors highlights diverse roles of ATP in biomolecular condensates.

Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs.

PARP1 condensates differentially partition DNA repair proteins and enhance DNA ligation.

Poly(ADP-ribose) polymerase 1 (PARP1) is one of the first responders to DNA damage and plays crucial roles in recruiting DNA repair proteins through its activity - poly(ADP-ribosyl)ation (PARylation). The enrichment of DNA repair proteins at sites of DNA damage has been described as the formation of a biomolecular condensate. However, it remains unclear how exactly PARP1 and PARylation contribute to the formation and organization of DNA repair condensates.

Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates.

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function.

Phase Separation Modulates the Thermodynamics and Kinetics of RNA Hybridization.

Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'--methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a ∼270-fold decrease and a concomitant ∼15-fold increase in the on- and off-rates for duplex formation, respectively.

Chromosome compaction is triggered by an autonomous DNA-binding module within condensin.

The compaction of chromatin into mitotic chromosomes is essential for faithful transmission of the genome during cell division. In eukaryotes, chromosome morphogenesis is regulated by the condensin complex, though the exact mechanism used to target condensin to chromatin and initiate condensation is not understood. Here, we reveal that condensin contains an intrinsically disordered region (IDR) that modulates its association with chromatin in early mitosis and exhibits phase separation.

Intrinsic disorder: A term to define the specific physicochemical characteristic of protein conformational heterogeneity.

In his commentary in this issue of Molecular Cell, Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications.

PARP1 condensates differentially partition DNA repair proteins and enhance DNA ligation.

Poly(ADP-ribose) polymerase 1 (PARP1) is one of the first responders to DNA damage and plays crucial roles in recruiting DNA repair proteins through its activity - poly(ADP-ribosyl)ation (PARylation). The enrichment of DNA repair proteins at sites of DNA damage has been described as the formation of a biomolecular condensate. However, it is not understood how PARP1 and PARylation contribute to the formation and organization of DNA repair condensates.

Poly(ADP-ribosyl)ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates.

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3.

Local Disordered Region Sampling (LDRS) for ensemble modeling of proteins with experimentally undetermined or low confidence prediction segments.

Summary: The Local Disordered Region Sampling (LDRS, pronounced loaders) tool is a new module developed for IDPConformerGenerator, a previously validated approach to model intrinsically disordered proteins (IDPs). The IDPConformerGenerator LDRS module provides a method for generating all-atom conformations of intrinsically disordered protein regions at N- and C-termini of and in loops or linkers between folded regions of an existing protein structure. These disordered elements often lead to missing coordinates in experimental structures or low confidence in predicted structures.

The Energetic Origins of Pi-Pi Contacts in Proteins.

Accurate potential energy models of proteins must describe the many different types of noncovalent interactions that contribute to a protein's stability and structure. Pi-pi contacts are ubiquitous structural motifs in all proteins, occurring between aromatic and nonaromatic residues and play a nontrivial role in protein folding and in the formation of biomolecular condensates. Guided by a geometric criterion for isolating pi-pi contacts from classical molecular dynamics simulations of proteins, we use quantum mechanical energy decomposition analysis to determine the molecular interactions that stabilize different pi-pi contact motifs.

Systematic identification of conditionally folded intrinsically disordered regions by AlphaFold2.

The AlphaFold Protein Structure Database contains predicted structures for millions of proteins. For the majority of human proteins that contain intrinsically disordered regions (IDRs), which do not adopt a stable structure, it is generally assumed that these regions have low AlphaFold2 confidence scores that reflect low-confidence structural predictions. Here, we show that AlphaFold2 assigns confident structures to nearly 15% of human IDRs.

A FRET-Based Assay and Computational Tools to Quantify Enzymatic Rates and Explore the Mechanisms of RNA Deadenylases in Heterogeneous Environments.

We developed a medium-throughput assay that can measure the time-dependent distribution of RNA products generated as a deadenylase degrades a polyadenosine (poly(A)) RNA tract, thereby providing insight into the mechanism of deadenylation. Importantly, this assay can be performed in both homogeneous and heterogeneous environments without relying on gel electrophoresis of RNA products or coupled enzymatic reactions that indirectly report on the RNA distribution through the detection of freed adenosine monophosphate. In parallel, we have established an open-source, Python-based command-line software package, deadenylationkinetics, that can be used to numerically simulate and/or fit the datasets afforded by our assay with different deadenylation mechanisms to determine the most likely case and estimate the associated rate constants.

Delineating Structural Propensities of the 4E-BP2 Protein via Integrative Modeling and Clustering.

The intrinsically disordered 4E-BP2 protein regulates mRNA cap-dependent translation through interaction with the predominantly folded eukaryotic initiation factor 4E (eIF4E). Phosphorylation of 4E-BP2 dramatically reduces the level of eIF4E binding, in part by stabilizing a binding-incompatible folded domain. Here, we used a Rosetta-based sampling algorithm optimized for IDRs to generate initial ensembles for two phospho forms of 4E-BP2, non- and 5-fold phosphorylated (NP and 5P, respectively), with the 5P folded domain flanked by N- and C-terminal IDRs (N-IDR and C-IDR, respectively).

Local Disordered Region Sampling (LDRS) for Ensemble Modeling of Proteins with Experimentally Undetermined or Low Confidence Prediction Segments.

The Local Disordered Region Sampling (LDRS, pronounced ) tool, developed for the IDPConformerGenerator platform (Teixeira 2022), provides a method for generating all-atom conformations of intrinsically disordered regions (IDRs) at N- and C-termini of and in loops or linkers between folded regions of an existing protein structure. These disordered elements often lead to missing coordinates in experimental structures or low confidence in predicted structures. Requiring only a pre-existing PDB structure of the protein with missing coordinates or with predicted confidence scores and its full-length primary sequence, LDRS will automatically generate physically meaningful conformational ensembles of the missing flexible regions to complete the full-length protein.

Learning to evolve structural ensembles of unfolded and disordered proteins using experimental solution data.

The structural characterization of proteins with a disorder requires a computational approach backed by experiments to model their diverse and dynamic structural ensembles. The selection of conformational ensembles consistent with solution experiments of disordered proteins highly depends on the initial pool of conformers, with currently available tools limited by conformational sampling. We have developed a Generative Recurrent Neural Network (GRNN) that uses supervised learning to bias the probability distributions of torsions to take advantage of experimental data types such as nuclear magnetic resonance J-couplings, nuclear Overhauser effects, and paramagnetic resonance enhancements.

Transitions in the framework of condensate biology.

As phase separation is found in an increasing variety of biological contexts, additional challenges have arisen in understanding the underlying principles of condensate formation and function. We spoke with researchers across disciplines about their views on the ever-changing landscape of biomolecular condensates.