Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding.

Phase separation can concentrate biomolecules and accelerate reactions. However, the mechanisms and principles connecting this mesoscale organization to signaling dynamics are difficult to dissect because of the pleiotropic effects associated with disrupting endogenous condensates. To address this limitation, we engineered new phosphorylation reactions within synthetic condensates. We generally found increased activity and broadened kinase specificity. Phosphorylation dynamics within condensates were rapid and could drive cell-cycle-dependent localization changes. High client concentration within condensates was important but not the main factor for efficient phosphorylation. Rather, the availability of many excess client-binding sites together with a flexible scaffold was crucial. Phosphorylation within condensates was also modulated by changes in macromolecular crowding. Finally, the phosphorylation of the Alzheimer's-disease-associated protein Tau by cyclin-dependent kinase 2 was accelerated within condensates. Thus, condensates enable new signaling connections and can create sensors that respond to the biophysical properties of the cytoplasm.

Multimodal interactions drive chromatin phase separation and compaction.

Gene silencing is intimately connected to DNA condensation and the formation of transcriptionally inactive heterochromatin by Heterochromatin Protein 1α (HP1α). Because heterochromatin foci are dynamic and HP1α can promote liquid-liquid phase separation, HP1α-mediated phase separation has been proposed as a mechanism of chromatin compaction. The molecular basis of HP1α-driven phase separation and chromatin compaction and the associated regulation by trimethylation of lysine 9 in histone 3 (H3K9me3), which is the hallmark of constitutive heterochromatin, is however largely unknown. Using a combination of chromatin compaction and phase separation assays, site-directed mutagenesis, and NMR-based interaction analysis, we show that human HP1α can compact chromatin in the absence of liquid-liquid phase separation. We further demonstrate that H3K9-trimethylation promotes compaction of chromatin arrays through multimodal interactions. The results provide molecular insights into HP1α-mediated chromatin compaction and thus into the role of human HP1α in the regulation of gene silencing.

Small-molecule targeting of GPCR-independent noncanonical G-protein signaling in cancer.

Activation of heterotrimeric G-proteins (Gαßγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class small-molecule inhibitor of noncanonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking noncanonical G-protein signaling in tumor cells and inhibiting proinvasive traits of metastatic cancer cells. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable noncanonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.

Determining the Physico-Chemical Composition of Biomolecular Condensates from Spatially-Resolved NMR.

Liquid-Liquid phase separation has emerged as fundamental process underlying the formation of biomolecular condensates. Insights into the composition and structure of biomolecular condensates is, however, complicated by their molecular complexity and dynamics. Here, we introduce an improved spatially-resolved NMR experiment that enables quantitative analysis of the physico-chemical composition of multi-component biomolecular condensates in equilibrium and label-free. Application of spatially-resolved NMR to condensates formed by the Alzheimer's disease-associated protein Tau demonstrates decreased water content, exclusion of the molecular crowding agent dextran, presence of a specific chemical environment of the small molecule DSS, and ≈150-fold increased concentration of Tau inside the condensate. The results suggest that spatially-resolved NMR can have a major impact in understanding the composition and physical chemistry of biomolecular condensates.

Molecular Basis of Small-Molecule Binding to α-Synuclein.

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.

A Novel SNCA A30G Mutation Causes Familial Parkinson's Disease.

BACKGROUND: The SNCA gene encoding α-synuclein (αSyn) is the first gene identified to cause autosomal-dominant Parkinson's disease (PD). OBJECTIVE: We report the identification of a novel heterozygous A30G mutation of the SNCA gene in familial PD and describe clinical features of affected patients, genetic findings, and functional consequences. METHODS: Whole exome sequencing was performed in the discovery family proband. Restriction digestion with Bbvl was used to screen SNCA A30G in two validation cohorts. The Greek cohort included 177 familial PD probands, 109 sporadic PD cases, and 377 neurologically healthy controls. The German cohort included 136 familial PD probands, 380 sporadic PD cases, and 116 neurologically healthy controls. We also conducted haplotype analysis using 13 common single nucleotide variants around A30G to determine the possibility of a founder effect for A30G. We then used biophysical methods to characterize A30G αSyn. RESULTS: We identified a novel SNCA A30G (GRCh37, Chr4:90756730, c.89 C>G) mutation that co-segregated with the disease in five affected individuals of three Greek families and was absent from controls. A founder effect was strongly suggested by haplotype analysis. The A30G mutation had a local effect on the intrinsically disordered structure of αSyn, slightly perturbed membrane binding, and promoted fibril formation. CONCLUSION: Based on the identification of A30G co-segregating with the disease in three families, the absence of the mutation in controls and population databases, and the observed functional effects, we propose SNCA A30G as a novel causative mutation for familial PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The p12 subunit of human polymerase δ uses an atypical PIP box for molecular recognition of proliferating cell nuclear antigen (PCNA).

Human DNA polymerase δ is essential for DNA replication and acts in conjunction with the processivity factor proliferating cell nuclear antigen (PCNA). In addition to its catalytic subunit (p125), pol δ comprises three regulatory subunits (p50, p68, and p12). PCNA interacts with all of these subunits, but only the interaction with p68 has been structurally characterized. Here, we report solution NMR-, isothermal calorimetry-, and X-ray crystallography-based analyses of the p12-PCNA interaction, which takes part in the modulation of the rate and fidelity of DNA synthesis by pol δ. We show that p12 binds with micromolar affinity to the classical PIP-binding pocket of PCNA via a highly atypical PIP box located at the p12 N terminus. Unlike the canonical PIP box of p68, the PIP box of p12 lacks the conserved glutamine; binds through a 2-fork plug made of an isoleucine and a tyrosine residue at +3 and +8 positions, respectively; and is stabilized by an aspartate at +6 position, which creates a network of intramolecular hydrogen bonds. These findings add to growing evidence that PCNA can bind a diverse range of protein sequences that may be broadly grouped as PIP-like motifs as has been previously suggested.

Determination of Complex Small-Molecule Structures Using Molecular Alignment Simulation.

Correct structural assignment of small molecules and natural products is critical for drug discovery and organic chemistry. Anisotropy-based NMR spectroscopy is a powerful tool for the structural assignment of organic molecules, but it relies on the utilization of a medium that disrupts the isotropic motion of molecules in organic solvents. Here, we establish a quantitative correlation between the atomic structure of the alignment medium, the molecular structure of the small molecule, and molecule-specific anisotropic NMR parameters. The quantitative correlation uses an accurate three-dimensional molecular alignment model that predicts residual dipolar couplings of small molecules aligned by poly(γ-benzyl-l-glutamate). The technique facilitates reliable determination of the correct stereoisomer and enables unequivocal, rapid determination of complex molecular structures from extremely sparse NMR data.

p15PAF is an intrinsically disordered protein with nonrandom structural preferences at sites of interaction with other proteins.

We present to our knowledge the first structural characterization of the proliferating-cell-nuclear-antigen-associated factor p15(PAF), showing that it is monomeric and intrinsically disordered in solution but has nonrandom conformational preferences at sites of protein-protein interactions. p15(PAF) is a 12 kDa nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15(PAF) gene is overexpressed in several types of human cancer. The nearly complete NMR backbone assignment of p15(PAF) allowed us to measure 86 N-H(N) residual dipolar couplings. Our residual dipolar coupling analysis reveals nonrandom conformational preferences in distinct regions, including the proliferating-cell-nuclear-antigen-interacting protein motif (PIP-box) and the KEN-box (recognized by the ubiquitin ligase that targets p15(PAF) for degradation). In accordance with these findings, analysis of the (15)N R2 relaxation rates shows a relatively reduced mobility for the residues in these regions. The agreement between the experimental small angle x-ray scattering curve of p15(PAF) and that computed from a statistical coil ensemble corrected for the presence of local secondary structural elements further validates our structural model for p15(PAF). The coincidence of these transiently structured regions with protein-protein interaction and posttranslational modification sites suggests a possible role for these structures as molecular recognition elements for p15(PAF).

Impact of distinct FG nucleoporin repeats on Nup98 self-association.

Nucleoporins rich in phenylalanine/glycine (FG) residues form the permeability barrier within the nuclear pore complex and are implicated in several pathological cellular processes, including oncogenic fusion condensates. The self-association of FG-repeat proteins and interactions between FG-repeats play a critical role in these activities by forming hydrogel-like structures. Here we show that mutation of specific FG repeats of Nup98 can strongly decrease the protein's self-association capabilities. We further present a cryo-electron microscopy structure of a Nup98 peptide fibril with higher stability per residue compared with previous Nup98 fibril structures. The high-resolution structure reveals zipper-like hydrophobic patches which contain a GLFG motif and are less compatible for binding to nuclear transport receptors. The identified distinct molecular properties of different regions of the nucleoporin may contribute to spatial variations in the self-association of FG-repeats, potentially influencing transport processes through the nuclear pore.

Membrane partitioning of the pore-forming domain of colicin A. Role of the hydrophobic helical hairpin.

The colicins are bacteriocins that target Escherichia coli and kill bacterial cells through different mechanisms. Colicin A forms ion channels in the inner membranes of nonimmune bacteria. This activity resides exclusively in its C-terminal fragment (residues 387-592). The soluble free form of this domain is a 10 α-helix bundle. The hydrophobic helical hairpin, H8-H9, is buried inside the structure and shielded by eight amphipathic surface helices. The interaction of the C-terminal colicin A domain and several chimeric variants with lipidic vesicles was examined here by isothermal titration calorimetry. In the mutant constructions, natural sequences of the hydrophobic helices H8 and H9 were either removed or substituted by polyalanine or polyleucine. All the constructions fully associated with DOPG liposomes including the mutant that lacked helices H8 and H9, indicating that amphipathic rather than hydrophobic helices were the major determinants of the exothermic binding reactions. Alanine is not specially favored in the lipid-bound form; the chimeric construct with polyalanine produced lower enthalpy gain. On the other hand, the large negative heat capacities associated with partitioning, a characteristic feature of the hydrophobic effect, were found to be dependent on the sequence hydrophobicity of helices H8 and H9.

Genipin prevents alpha-synuclein aggregation and toxicity by affecting endocytosis, metabolism and lipid storage.

Parkinson's Disease (PD) is a common neurodegenerative disorder affecting millions of people worldwide for which there are only symptomatic therapies. Small molecules able to target key pathological processes in PD have emerged as interesting options for modifying disease progression. We have previously shown that a (poly)phenol-enriched fraction (PEF) of Corema album L. leaf extract modulates central events in PD pathogenesis, namely α-synuclein (αSyn) toxicity, aggregation and clearance. PEF was now subjected to a bio-guided fractionation with the aim of identifying the critical bioactive compound. We identified genipin, an iridoid, which relieves αSyn toxicity and aggregation. Furthermore, genipin promotes metabolic alterations and modulates lipid storage and endocytosis. Importantly, genipin was able to prevent the motor deficits caused by the overexpression of αSyn in a Drosophila melanogaster model of PD. These findings widens the possibility for the exploitation of genipin for PD therapeutics.

Contributions of the N-terminal intrinsically disordered region of the severe acute respiratory syndrome coronavirus 2 nucleocapsid protein to RNA-induced phase separation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein's ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by mutations found in human COVID-19 infections, including in the Omicron variant of concern. Here, we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2 . In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTE's structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein. STATEMENT: The nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the protein's RNA-mediated liquid-liquid phase separation.

A NAC domain mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein and recapitulates its pathological diversity.

The alpha-synuclein mutation E83Q, the first in the NAC domain of the protein, was recently identified in a patient with dementia with Lewy bodies. We investigated the effects of this mutation on the aggregation of aSyn monomers and the structure, morphology, dynamic, and seeding activity of the aSyn fibrils in neurons. We found that it markedly accelerates aSyn fibrillization and results in the formation of fibrils with distinct structural and dynamic properties. In cells, this mutation is associated with higher levels of aSyn, accumulation of pS129, and increased toxicity. In a neuronal seeding model of Lewy body (LB) formation, the E83Q mutation significantly enhances the internalization of fibrils into neurons, induces higher seeding activity, and results in the formation of diverse aSyn pathologies, including the formation of LB-like inclusions that recapitulate the immunohistochemical and morphological features of brainstem LBs observed in brains of patients with Parkinson's disease.

Molecular interactions of FG nucleoporin repeats at high resolution.

Proteins that contain repeat phenylalanine-glycine (FG) residues phase separate into oncogenic transcription factor condensates in malignant leukaemias, form the permeability barrier of the nuclear pore complex and mislocalize in neurodegenerative diseases. Insights into the molecular interactions of FG-repeat nucleoporins have, however, remained largely elusive. Using a combination of NMR spectroscopy and cryoelectron microscopy, we have identified uniformly spaced segments of transient ß-structure and a stable preformed α-helix recognized by messenger RNA export factors in the FG-repeat domain of human nucleoporin 98 (Nup98). In addition, we have determined at high resolution the molecular organization of reversible FG-FG interactions in amyloid fibrils formed by a highly aggregation-prone segment in Nup98. We have further demonstrated that amyloid-like aggregates of the FG-repeat domain of Nup98 have low stability and are reversible. Our results provide critical insights into the molecular interactions underlying the self-association and phase separation of FG-repeat nucleoporins in physiological and pathological cell activities.

Extending the applicability of P3D for structure determination of small molecules.

The application of anisotropic nuclear magnetic resonance (NMR) parameters for the correct structural assignment of small molecules requires the use of partially ordered media. Previously we demonstrated that the use of P3D simulations using poly(γ-benzyl-L-glutamate) (PBLG) as an alignment medium allows for the determination of the correct diastereomer from extremely sparse NMR data. Through the analysis of the structural characteristics of small molecules in different alignment media, here we show that when steric or electrostatic factors dominate the alignment, P3D-PBLG retains its diastereomer discrimination power. We also demonstrate that P3D simulations can define the different conformations of a flexible small molecule from sparse NMR data.

Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates.

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

Effects of pharmacological modulators of α-synuclein and tau aggregation and internalization.

Parkinson's disease (PD) and Alzheimer's disease (AD) are common neurodegenerative disorders of the elderly and, therefore, affect a growing number of patients worldwide. Both diseases share, as a common hallmark, the accumulation of characteristic protein aggregates, known as Lewy bodies (LB) in PD, and neurofibrillary tangles in AD. LBs are primarily composed of misfolded α-synuclein (aSyn), and neurofibrillary tangles are primarily composed of tau protein. Importantly, upon pathological evaluation, most AD and PD/Lewy body dementia cases exhibit mixed pathology, with the co-occurrence of both LB and neurofibrillary tangles, among other protein inclusions. Recent studies suggest that both aSyn and tau pathology can spread and propagate through neuronal connections. Therefore, it is important to investigate the mechanisms underlying aggregation and propagation of these proteins for the development of novel therapeutic strategies. Here, we assessed the effects of different pharmacological interventions on the aggregation and internalization of tau and aSyn. We found that anle138b and fulvic acid decrease aSyn and tau aggregation, that epigallocatechin gallate decreases aSyn aggregation, and that dynasore reduces tau internalization. Establishing the effects of small molecules with different chemical properties on the aggregation and spreading of aSyn and tau will be important for the development of future therapeutic interventions.

The Tumor Suppressor ING5 Is a Dimeric, Bivalent Recognition Molecule of the Histone H3K4me3 Mark.

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex. We show that ING5 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING5, which contains the nuclear localization sequence, is flexible and disordered, but it binds dsDNA with micromolar affinity. NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 in the same way as the isolated PHD. We have observed that ING5 can form heterodimers with the highly homologous ING4, and that two of three primary tumor-associated mutants in the N-terminal domain strongly destabilize the coiled-coil structure. They also affect cell proliferation and cell cycle phase distribution, suggesting a driver role in cancer progression.

A generalized approach for NMR studies of lipid-protein interactions based on sparse fluorination of acyl chains.

Sparse lipid fluorination enhances the lipids' 1H signal dispersion, enables clean molecular distinction by 19F NMR, and evinces micelle insertion of proteins via fluorine-induced signal shifts. We present a minimal fluorination scheme, and illustrate the concept on di-(4-fluoro)-heptanoylphosphatidylcholine micelles and solubilised seven-helix transmembrane pSRII protein.