Specific protein-RNA interactions are mostly preserved in biomolecular condensates.

Many biomolecular condensates are enriched in and depend on RNAs and RNA binding proteins (RBPs). So far, only a few studies have addressed the characterization of the intermolecular interactions responsible for liquid-liquid phase separation (LLPS) and the impact of condensation on RBPs and RNAs. Here, we present an approach to study protein-RNA interactions inside biomolecular condensates by applying cross-linking of isotope labeled RNA and tandem mass spectrometry to phase-separating systems (LLPS-CLIR-MS). LLPS-CLIR-MS enables the characterization of intermolecular interactions present within biomolecular condensates at residue-specific resolution and allows a comparison with the same complexes in the dispersed phase. We observe that sequence-specific RBP-RNA interactions present in the dispersed phase are generally maintained inside condensates. In addition, LLPS-CLIR-MS identifies structural alterations at the protein-RNA interfaces, including additional unspecific contacts in the condensed phase. Our approach offers a procedure to derive structural information of protein-RNA complexes within biomolecular condensates that could be critical for integrative structural modeling of ribonucleoproteins (RNPs) in this form.

A solid beta-sheet structure is formed at the surface of FUS droplets during aging.

Phase transitions are important to understand cell dynamics, and the maturation of liquid droplets is relevant to neurodegenerative disorders. We combined NMR and Raman spectroscopies with microscopy to follow, over a period of days to months, droplet maturation of the protein fused in sarcoma (FUS). Our study reveals that the surface of the droplets plays a critical role in this process, while RNA binding prevents it. The maturation kinetics are faster in an agarose-stabilized biphasic sample compared with a monophasic condensed sample, owing to the larger surface-to-volume ratio. In addition, Raman spectroscopy reports structural differences upon maturation between the inside and the surface of droplets, which is comprised of ß-sheet content, as revealed by solid-state NMR. In agreement with these observations, a solid crust-like shell is observed at the surface using microaspiration. Ultimately, matured droplets were converted into fibrils involving the prion-like domain as well as the first RGG motif.

Ensemble structure of the N-terminal domain (1-267) of FUS in a biomolecular condensate.

Solutions of some proteins phase separate into a condensed state of high protein concentration and a dispersed state of low concentration. Such behavior is observed in living cells for a number of RNA-binding proteins that feature intrinsically disordered domains. It is relevant for cell function via the formation of membraneless organelles and transcriptional condensates. On a basic level, the process can be studied in vitro on protein domains that are necessary and sufficient for liquid-liquid phase separation (LLPS). We have performed distance distribution measurements by electron paramagnetic resonance for 13 sections in an N-terminal domain (NTD) construct of the protein fused in sarcoma (FUS), consisting of the QGSY-rich domain and the RGG1 domain, in the denatured, dispersed, and condensed state. Using 10 distance distribution restraints for ensemble modeling and three such restraints for model validation, we have found that FUS NTD behaves as a random-coil polymer under good-solvent conditions in both the dispersed and condensed state. Conformation distribution in the biomolecular condensate is virtually indistinguishable from the one in an unrestrained ensemble, with the latter one being based on only residue-specific Ramachandran angle distributions. Over its whole length, FUS NTD is slightly more compact in the condensed than in the dispersed state, which is in line with the theory for random coils in good solvent proposed by de Gennes, Daoud, and Jannink. The estimated concentration in the condensate exceeds the overlap concentration resulting from this theory. The QGSY-rich domain is slightly more extended, slightly more hydrated, and has slightly higher propensity for LLPS than the RGG1 domain. Our results support previous suggestions that LLPS of FUS is driven by multiple transient nonspecific hydrogen bonding and π-sp2 interactions.

Phase Separation of Heterogeneous Nuclear Ribonucleoprotein A1 upon Specific RNA-Binding Observed by Magnetic Resonance.

Interaction of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with specific single-stranded RNA and its relation to liquid-liquid phase separation (LLPS) were studied in vitro by magnetic resonance based on site-directed spin labelling. An ensemble model of dispersed hnRNP A1 in the absence of RNA was derived from distance distributions between spin labelled sites and small angle X-ray scattering. This model revealed a compact state of the low-complexity domain and its interaction with the RNA recognition motifs. Paramagnetic relaxation enhancement NMR spectroscopy confirmed this interaction. Addition of RNA to dispersed hnRNP A1 induced liquid-droplet formation. Such LLPS depended on RNA concentration and sequence, with continuous wave EPR spectroscopy showing an influence of RNA point mutations on local protein dynamics. We propose that an interplay of sequence-specific RNA binding and LLPS contributes to regulation of specific RNA segregation during stress response.

Non-specific adhesive forces between filaments and membraneless organelles.

Many membraneless organelles are liquid-like domains that form inside the active, viscoelastic environment of living cells through phase separation. To investigate the potential coupling of phase separation with the cytoskeleton, we quantify the structural correlations of membraneless organelles (stress granules) and cytoskeletal filaments (microtubules) in a human-derived epithelial cell line. We find that microtubule networks are substantially denser in the vicinity of stress granules. When microtubules are depolymerized, the sub-units localize near the surface of the stress granules. We interpret these data using a thermodynamic model of partitioning of particles to the surface and bulk of the droplets. In this framework, our data are consistent with a weak (≲k B T) affinity of the microtubule sub-units for stress granule interfaces. As microtubules polymerize, their interfacial affinity increases, providing sufficient adhesion to deform droplets and/or the network. Our work suggests that proteins and other objects in the cell have a non-specific affinity for droplet interfaces that increases with the contact area and becomes most apparent when they have no preference for the interior of a droplet over the rest of the cytoplasm. We validate this basic physical phenomenon in vitro through the interaction of a simple protein-RNA condensate with microtubules.

Erratum: Publisher Correction: Non-specific adhesive forces between filaments and membraneless organelles.

[This corrects the article DOI: 10.1038/s41567-022-01537-8.].

Structural biology of RNA-binding proteins in the context of phase separation: What NMR and EPR can bring?

Liquid-liquid phase separation of RNA-binding proteins underlies the formation of membraneless organelles, whose composition is dynamic and whose existence may be transient. These organelles are involved in regulation of RNA processing and translation and, if they behave abnormally, in pathologies. Because disorder phenomena are essential in their formation and dynamics, established methodology is insufficient for characterizing their structure. In this review, we consider the current and potential contribution of NMR and EPR spectroscopy to the understanding of structure and dynamics of phase-separating RNA-binding proteins in, both, their dispersed and condensed state in vitro. We discuss which experiments are applicable under what conditions and which information can be obtained from them. Because for these phenomena, the accessible information depends crucially on metastable phase equilibria, we also consider aspects of sample preparation for NMR and EPR experiments.

Characterization of Weak Protein Domain Structure by Spin-Label Distance Distributions.

Function of intrinsically disordered proteins may depend on deviation of their conformational ensemble from that of a random coil. Such deviation may be hard to characterize and quantify, if it is weak. We explored the potential of distance distributions between spin labels, as they can be measured by electron paramagnetic resonance techniques, for aiding such characterization. On the example of the intrinsically disordered N-terminal domain 1-267 of fused in sarcoma (FUS) we examined what such distance distributions can and cannot reveal on the random-coil reference state. On the example of the glycine-rich domain 188-320 of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) we studied whether deviation from a random-coil ensemble can be robustly detected with 19 distance distribution restraints. We discuss limitations imposed by ill-posedness of the conversion of primary data to distance distributions and propose overlap of distance distributions as a fit criterion that can tackle this problem. For testing consistency and size sufficiency of the restraint set, we propose jack-knife resampling. At current desktop computers, our approach is expected to be viable for domains up to 150 residues and for between 10 and 50 distance distribution restraints.

NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation.

Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.

Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method.

Phase separated macromolecules play essential roles in many biological and synthetic systems. Physical characterization of these systems can be challenging because of limited sample volumes, particularly for phase-separated proteins. Here, we demonstrate that a classic method for measuring the surface tension of liquid droplets, based on the analysis of the shape of a sessile droplet, can be effectively scaled down to measure the interfacial tension between a macromolecule-rich droplet phase and its co-existing macromolecule-poor continuous phase. The connection between droplet shape and surface tension relies on the density difference between the droplet and its surroundings. This can be determined with small sample volumes in the same setup by measuring the droplet sedimentation velocity. An interactive MATLAB script for extracting the capillary length from a droplet image is included in the ESI.

Structure-Activity Relationship in Pyrazolo[4,3-c]pyridines, First Inhibitors of PEX14-PEX5 Protein-Protein Interaction with Trypanocidal Activity.

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against Trypanosoma, including the human pathogen Trypanosoma brucei gambiense and Trypanosoma cruzi parasites.

Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands.

The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.

Inhibitors of glycosomal protein import provide new leads against trypanosomiasis.

Vector-borne trypanosomatid parasite infections in tropical and sub-tropical countries constitute a major threat to humans and livestock. Trypanosoma brucei parasites are transmitted by tsetse fly and lead to African sleeping sickness in humans and Nagana in cattle. In Latin American countries, Trypanosoma cruzi infections spread by triatomine kissing bugs lead to Chagas disease. Various species of Leishmania transmitted to humans by phlebotomine sandflies manifest in a spectrum of diseases termed Leishmaniasis. 20 million people are currently infected with trypanosomatid parasites, leading to over 30,000 deaths annually and half billion people at risk of the infection. It is estimated that 300,000 Chagas infected people reside in the United States and 100,000 in Europe. Glycosomes are peroxisome-like organelles found only in trypanosomatids. Glycolysis occurs in the cytosol in all other organisms, but glycolytic enzymes and other metabolic pathways are compartmentalized inside glycosomes in trypanosomatids. Glycosomes are essential for the parasite survival and hence thought to be an attractive drug target. Our recent study [Dawidowski et al. Science (2017)] is the first to report small molecule inhibitors of glycosomal protein import. Using structure-based drug design, we developed small molecule inhibitors of the Trypanosoma PEX5-PEX14 protein-protein interaction that disrupt glycosomal protein import and kill the parasites. Oral treatment of T. brucei infected mice with PEX14 inhibitor significantly reduced the parasite levels with no adverse effect on mice. The study provides the grounds for further development of the glycosome inhibitors into clinical candidates and validates the parasite protein-protein interactions as drug targets.

Allosteric modulation of peroxisomal membrane protein recognition by farnesylation of the peroxisomal import receptor PEX19.

The transport of peroxisomal membrane proteins (PMPs) requires the soluble PEX19 protein as chaperone and import receptor. Recognition of cargo PMPs by the C-terminal domain (CTD) of PEX19 is required for peroxisome biogenesis in vivo. Farnesylation at a C-terminal CaaX motif in PEX19 enhances the PMP interaction, but the underlying molecular mechanisms are unknown. Here, we report the NMR-derived structure of the farnesylated human PEX19 CTD, which reveals that the farnesyl moiety is buried in an internal hydrophobic cavity. This induces substantial conformational changes that allosterically reshape the PEX19 surface to form two hydrophobic pockets for the recognition of conserved aromatic/aliphatic side chains in PMPs. Mutations of PEX19 residues that either mediate farnesyl contacts or are directly involved in PMP recognition abolish cargo binding and cannot complement a ΔPEX19 phenotype in human Zellweger patient fibroblasts. Our results demonstrate an allosteric mechanism for the modulation of protein function by farnesylation.

Structural biology of the import pathways of peroxisomal matrix proteins.

The peroxisomal proteins (peroxins) that mediate the import of peroxisomal matrix proteins have been identified. Recently, the purification of a functional peroxisomal translocon has been reported. However, the molecular details of the import pathways and the mechanisms by which the cargo is translocated into the lumen of the organelle are still poorly understood. Structural studies have begun to provide insight into molecular mechanisms of peroxisomal import pathways for cargo proteins that harbor peroxisomal targeting signals, PTS1 and PTS2, at their C- and N-termini, respectively. So far structures have been reported for binary or tertiary protein-protein interfaces, and highlight the role of intrinsically disordered regions for these interactions. Here, we provide an overview of the currently available structural biology of peroxisomal import pathways. Current challenges and future perspectives of the structural biology of peroxisomal protein translocation are discussed.

Towards the molecular mechanism of the integration of peroxisomal membrane proteins.

The correct topogenesis of peroxisomal membrane proteins is a crucial step for the formation of functioning peroxisomes. Although this process has been widely studied, the exact mechanism with which it occurs has not yet been fully characterized. Nevertheless, it is generally accepted that peroxisomes employ three proteins - Pex3, Pex19 and Pex16 in mammals - for the insertion of peroxisomal membrane proteins into the peroxisomal membrane. Structural biology approaches have been utilized for the elucidation of the mechanistic questions of peroxisome biogenesis, mainly by providing information on the architecture of the proteins significant for this process. This review aims to summarize, compare and put into perspective the structural knowledge that has been generated mainly for Pex3 and Pex19 and their interaction partners in recent years.