Machine and Deep Learning Methods for Predicting 3D Genome Organization.

Three-dimensional (3D) chromatin interactions, such as enhancer-promoter interactions (EPIs), loops, topologically associating domains (TADs), and A/B compartments, play critical roles in a wide range of cellular processes by regulating gene expression. Recent development of chromatin conformation capture technologies has enabled genome-wide profiling of various 3D structures, even with single cells. However, current catalogs of 3D structures remain incomplete and unreliable due to differences in technology, tools, and low data resolution. Machine learning methods have emerged as an alternative to obtain missing 3D interactions and/or improve resolution. Such methods frequently use genome annotation data (ChIP-seq, DNAse-seq, etc.), DNA sequencing information (k-mers and transcription factor binding site (TFBS) motifs), and other genomic properties to learn the associations between genomic features and chromatin interactions. In this review, we discuss computational tools for predicting three types of 3D interactions (EPIs, chromatin interactions, and TAD boundaries) and analyze their pros and cons. We also point out obstacles to the computational prediction of 3D interactions and suggest future research directions.

Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.

Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox. Functional DNA templates composed of multiple parts such as promoter, ribosomal binding site (RBS), coding sequence, and terminator are produced in vitro in a one-pot Golden Gate assembly reaction followed by polymerase chain reaction (PCR) amplification. We demonstrate assembly, cell-free testing of promoter and RBS combinations, as well as characterization of a repressor-promoter pair. By eliminating time-consuming transformation and cloning steps in cells and by taking advantage of modular cloning toolboxes, our cell-free prototyping workflow can produce data for large numbers of new assembled constructs within a single day.

Approaching Pharmacological Space: Events and Components.

The pharmacological space comprises all the dynamic events that determine the bioactivity (and/or the metabolism and toxicity) of a given ligand. The pharmacological space accounts for the structural flexibility and property variability of the two interacting molecules as well as for the mutual adaptability characterizing their molecular recognition process. The dynamic behavior of all these events can be described by a set of possible states (e.g., conformations, binding modes, isomeric forms) that the simulated systems can assume. For each monitored state, a set of state-dependent ligand- and structure-based descriptors can be calculated. Instead of considering only the most probable state (as routinely done), the pharmacological space proposes to consider all the monitored states. For each state-dependent descriptor, the corresponding space can be evaluated by calculating various dynamic parameters such as mean and range values.The reviewed examples emphasize that the pharmacological space can find fruitful applications in structure-based virtual screening as well as in toxicity prediction. In detail, in all reported examples, the inclusion of the pharmacological space parameters enhances the resulting performances. Beneficial effects are obtained by combining both different binding modes to account for ligand mobility and different target structures to account for protein flexibility/adaptability.The proposed computational workflow that combines docking simulations and rescoring analyses to enrich the arsenal of docking-based descriptors revealed a general applicability regardless of the considered target and utilized docking engine. Finally, the EFO approach that generates consensus models by linearly combining various descriptors yielded highly performing models in all discussed virtual screening campaigns.

Targeting a key protein-protein interaction surface on mitogen-activated protein kinases by a precision-guided warhead scaffold.

For mitogen-activated protein kinases (MAPKs) a shallow surface-distinct from the substrate binding pocket-called the D(ocking)-groove governs partner protein binding. Screening of broad range of Michael acceptor compounds identified a double-activated, sterically crowded cyclohexenone moiety as a promising scaffold. We show that compounds bearing this structurally complex chiral warhead are able to target the conserved MAPK D-groove cysteine via reversible covalent modification and interfere with the protein-protein interactions of MAPKs. The electronic and steric properties of the Michael acceptor can be tailored via different substitution patterns. The inversion of the chiral center of the warhead can reroute chemical bond formation with the targeted cysteine towards the neighboring, but less nucleophilic histidine. Compounds bind to the shallow MAPK D-groove with low micromolar affinity in vitro and perturb MAPK signaling networks in the cell. This class of chiral, cyclic and enhanced 3D shaped Michael acceptor scaffolds offers an alternative to conventional ATP-competitive drugs modulating MAPK signaling pathways.

Cryo-EM investigation of ryanodine receptor type 3.

Ryanodine Receptor isoform 3 (RyR3) is a large ion channel found in the endoplasmic reticulum membrane of many different cell types. Within the hippocampal region of the brain, it is found in dendritic spines and regulates synaptic plasticity. It controls myogenic tone in arteries and is upregulated in skeletal muscle in early development. RyR3 has a unique functional profile with a very high sensitivity to activating ligands, enabling high gain in Ca2+-induced Ca2+ release. Here we solve high-resolution cryo-EM structures of RyR3 in non-activating and activating conditions, revealing structural transitions that occur during channel opening. Addition of activating ligands yields only open channels, indicating an intrinsically high open probability under these conditions. RyR3 has reduced binding affinity to the auxiliary protein FKBP12.6 due to several sequence variations in the binding interface. We map disease-associated sequence variants and binding sites for known pharmacological agents. The N-terminal region contains ligand binding sites for a putative chloride anion and ATP, both of which are targeted by sequence variants linked to epileptic encephalopathy.

Rational design based on multi-monomer simultaneous docking for epitope imprinting of SARS-CoV-2 spike protein.

Among biomimetic strategies shaping engineering designs, molecularly imprinted polymer (MIP) technology stands out, involving chemically synthesised receptors emulating natural antigen-antibody interactions. These versatile 'designer polymers' with remarkable stability and low cost, are pivotal for in vitro diagnostics. Amid the recent global health crisis, we probed MIPs' potential to capture SARS-CoV-2 virions. Large biotemplates complicate MIP design, influencing generated binding site specificity. To precisely structure recognition sites within polymers, we innovated an epitope imprinting method supplemented by in silico polymerization component screening. A viral surface Spike protein informed epitope selection was targeted for MIP development. A novel multi-monomer docking approach (MMSD) was employed to simulate classical receptor-ligand interactions, mimicking binding reinforcement across multiple amino acids. Around 40 monomer combinations were docked to the epitope sequence and top performers experimentally validated via rapid fluorescence binding assays. Notably, high imprinting factor polymers correlated with MMSD predictions, promising rational MIP design applicable to diverse viral pathologies.

Identifying the "stripe" transcription factors and cooperative binding related to DNA methylation.

DNA methylation plays a critical role in gene regulation by modulating the DNA binding of transcription factors (TFs). This study integrates TFs' ChIP-seq profiles with WGBS profiles to investigate how DNA methylation affects protein interactions. Statistical methods and a 5-letter DNA motif calling model have been developed to characterize DNA sequences bound by proteins, while considering the effects of DNA modifications. By employing these methods, 79 significant universal "stripe" TFs and cofactors (USFs), 2360 co-binding protein pairs, and distinct protein modules associated with various DNA methylation states have been identified. The USFs hint a regulatory hierarchy within these protein interactions. Proteins preferentially bind to non-CpG sites in methylated regions, indicating binding affinity is not solely CpG-dependent. Proteins involved in methylation-specific USFs and cobinding pairs play essential roles in promoting and sustaining DNA methylation through interacting with DNMTs or inhibiting TET binding. These findings underscore the interplay between protein binding and methylation, offering insights into epigenetic regulation in cellular biology.

Cryo-EM structures of the human P2X1 receptor reveal subtype-specific architecture and antagonism by supramolecular ligand-binding.

P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design.

The molecular dissection of TRIM25's RNA-binding mechanism provides key insights into its antiviral activity.

TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity.

Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket.

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca2+) binding in the low-affinity Ca2+-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca2+-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca2+ binding in the low-affinity calcium-binding pocket.

PAIP1 binds to pre-mRNA and regulates alternative splicing of cancer pathway genes including VEGFA.

BACKGROUND: Poly (A) binding protein interacting protein 1 (PAIP1) has been shown to causally contribute to the development and progression of cancer. However, the mechanisms of the PAIP1 regulation in tumor cells remain poorly understood. RESULTS: Here, we used a recently developed UV cross-linking and RNA immunoprecipitation method (iRIP-seq) to map the direct and indirect interaction sites between PAIP1 and RNA on a transcriptome-wide level in HeLa cells. We found that PAIP1 not only binds to 3'UTRs, but also to pre-mRNAs/mRNAs with a strong bias towards the coding region and intron. PAIP1 binding sites are enriched in splicing enhancer consensus GA-rich motifs. RNA-seq analysis revealed that PAIP1 selectively modulates the alternative splicing of genes in some cancer hallmarks including cell migration, the mTOR signaling pathway and the HIF-1 signaling pathway. PAIP1-regulated alternative splicing events were strongly associated with PAIP1 binding, demonstrating that the binding may promote selection of the nearby splice sites. Deletion of a PAIP1 binding site containing seven repeats of GA motif reduced the PAIP1-mediated suppression of the exon 6 inclusion in a VEGFA mRNA isoform. Proteomic analysis of the PAIP1-interacted proteins revealed the enrichment of the spliceosome components and splicing factors. CONCLUSIONS: These findings suggest that PAIP1 is both a polyadenylation and alternative splicing regulator, that may play a large role in RNA processing via its role in alternative splicing regulation.

A conserved peptide-binding pocket in HyNaC/ASIC ion channels.

The only known peptide-gated ion channels-FaNaCs/WaNaCs and HyNaCs-belong to different clades of the DEG/ENaC family. FaNaCs are activated by the short neuropeptide FMRFamide, and HyNaCs by Hydra RFamides, which are not evolutionarily related to FMRFamide. The FMRFamide-binding site in FaNaCs was recently identified in a cleft atop the large extracellular domain. However, this cleft is not conserved in HyNaCs. Here, we combined molecular modeling and site-directed mutagenesis and identified a putative binding pocket for Hydra-RFamides in the extracellular domain of the heterotrimeric HyNaC2/3/5. This pocket localizes to only one of the three subunit interfaces, indicating that this trimeric ion channel binds a single peptide ligand. We engineered an unnatural amino acid at the putative binding pocket entrance, which allowed covalent tethering of Hydra RFamide to the channel, thereby trapping the channel in an open conformation. The identified pocket localizes to the same region as the acidic pocket of acid-sensing ion channels (ASICs), which binds peptide ligands. The pocket in HyNaCs is less acidic, and both electrostatic and hydrophobic interactions contribute to peptide binding. Collectively, our results reveal a conserved ligand-binding pocket in HyNaCs and ASICs and indicate independent evolution of peptide-binding cavities in the two subgroups of peptide-gated ion channels.

MLSNet: a deep learning model for predicting transcription factor binding sites.

Accurate prediction of transcription factor binding sites (TFBSs) is essential for understanding gene regulation mechanisms and the etiology of diseases. Despite numerous advances in deep learning for predicting TFBSs, their performance can still be enhanced. In this study, we propose MLSNet, a novel deep learning architecture designed specifically to predict TFBSs. MLSNet innovatively integrates multisize convolutional fusion with long short-term memory (LSTM) networks to effectively capture DNA-sparse higher-order sequence features. Further, MLSNet incorporates super token attention and Bi-LSTM to systematically extract and integrate higher-order DNA shape features. Experimental results on 165 ChIP-seq (chromatin immunoprecipitation followed by sequencing) datasets indicate that MLSNet consistently outperforms several state-of-the-art algorithms in the prediction of TFBSs. Specifically, MLSNet reports average metrics: 0.8306 for ACC, 0.8992 for AUROC, and 0.9035 for AUPRC, surpassing the second-best methods by 1.82%, 1.68%, and 1.54%, respectively. This research delineates the effectiveness of combining multi-size convolutional layers with LSTM and DNA shape-based features in enhancing predictive accuracy. Moreover, this study comprehensively assesses the variability in model performance across different cell lines and transcription factors. The source code of MLSNet is available at https://github.com/minghaidea/MLSNet.

Memory effects of transcription regulator-DNA interactions in bacteria.

Memory effect refers to the phenomenon where past events influence a system's current and future states or behaviors. In biology, memory effects often arise from intra- or intermolecular interactions, leading to temporally correlated behaviors. Single-molecule studies have shown that enzymes and DNA-binding proteins can exhibit time-correlated behaviors of their activity. While memory effects are well documented and studied in vitro, no such examples exist in cells to our knowledge. Combining single-molecule tracking (SMT) and single-cell protein quantitation, we find in living Escherichia coli cells distinct temporal correlations in the binding/unbinding events on DNA by MerR- and Fur-family metalloregulators, manifesting as memory effects with timescales of ~1 s. These memory effects persist irrespective of the type of the metalloregulators or their metallation states. Moreover, these temporal correlations of metalloregulator-DNA interactions are associated with spatial confinements of the metalloregulators near their DNA binding sites, suggesting microdomains of ~100 nm in size that possibly result from the spatial organizations of the bacterial chromosome without the involvement of membranes. These microdomains likely facilitate repeated binding events, enhancing regulator-DNA contact frequency and potentially gene regulation efficiency. These findings provide unique insights into the spatiotemporal dynamics of protein-DNA interactions in bacterial cells, introducing the concept of microdomains as a crucial player in memory effect-driven gene regulation.

Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3.

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

Experimental localization of metal-binding sites reveals the role of metal ions in type II DNA topoisomerases.

Metal ions have important roles in supporting the catalytic activity of DNA-regulating enzymes such as topoisomerases (topos). Bacterial type II topos, gyrases and topo IV, are primary drug targets for fluoroquinolones, a class of clinically relevant antibacterials requiring metal ions for efficient drug binding. While the presence of metal ions in topos has been elucidated in biochemical studies, accurate location and assignment of metal ions in structural studies have historically posed significant challenges. Recent advances in X-ray crystallography address these limitations by extending the experimental capabilities into the long-wavelength range, exploiting the anomalous contrast from light elements of biological relevance. This breakthrough enables us to confirm experimentally the locations of Mg2+ in the fluoroquinolone-stabilized Streptococcus pneumoniae topo IV complex. Moreover, we can unambiguously identify the presence of K+ and Cl- ions in the complex with one pair of K+ ions functioning as an additional intersubunit bridge. Overall, our data extend current knowledge on the functional and structural roles of metal ions in type II topos.

Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist.

The melanocortin receptor 4 (MC4R) belongs to the melanocortin receptor family of G-protein coupled receptors and is a key switch in the leptin-melanocortin molecular axis that controls hunger and satiety. Brain-produced hormones such as α-melanocyte-stimulating hormone (agonist) and agouti-related peptide (inverse agonist) regulate the molecular communication of the MC4R axis but are promiscuous for melanocortin receptor subtypes and induce a wide array of biological effects. Here, we use a chimeric construct of conformation-selective, nanobody-based binding domain (a ConfoBody Cb80) and active state-stabilized MC4R-ß2AR hybrid for efficient de novo discovery of a sequence diverse panel of MC4R-specific, potent and full agonistic nanobodies. We solve the active state MC4R structure in complex with the full agonistic nanobody pN162 at 3.4 Å resolution. The structure shows a distinct interaction with pN162 binding deeply in the orthosteric pocket. MC4R peptide agonists, such as the marketed setmelanotide, lack receptor selectivity and show off-target effects. In contrast, the agonistic nanobody is highly specific and hence can be a more suitable agent for anti-obesity therapeutic intervention via MC4R.

The cAMP receptor protein from Gardnerella vaginalis is not regulated by ligands.

Overgrowth of Gardnerella vaginalis causes an imbalance in vaginal microecology. The pathogenicity of G. vaginalis is directly regulated by the cAMP receptor protein (CRP). In this study, we resolve the crystal structure of CRPGv at a resolution of 2.22 Å and find some significant differences from homologous proteins. The first 23 amino acids of CRPGv are inserted into the ligand binding pocket, creating a strong steric barrier to ligand entry that has not been seen previously in its homologues. In the absence of ligands, the two α helices used by CRPGv to bind oligonucleotide chains are exposed and can specifically bind TGTGA-N6-TCACA sequences. cAMP and other ligands of CRP homologs are not cofactors of CRPGv. There is no coding gene of the adenylate cyclase, and cAMP could not be identified in G. vaginalis by liquid chromatography tandem mass spectrometry. We speculate that CRPGv may achieve fine regulation through a conformational transformation different from that of its homologous proteins, and this conformational transformation is no longer dependent on small molecules, but may be aided by accessory proteins. CRPGv is the first discovered CRP that is not ligand-regulated, and its active conformation provides a structural basis for drug screening.

[The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages].

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

New dynamic scoring method for deep evaluation of naloxegol as ß-tubulin binding inhibitor.

We report a new scoring method for rating the performance of ligands on same protein, using their extensive dynamic flexibility properties, binding with protein and impact on receptor protein. Based on molecular dynamics (MD), this method is more accurate than single-point energy calculations. This method identified an ideal FDA-approved drug as ß-tubulin microtubule inhibitor with improved attributes compared to commercial microtubule disassembly inhibitor, Paclitaxel (PTX). We started with virtual screening (VS) of FDA-approved drugs inside PTX's binding pocket (A) of human ß-tubulin protein. Screened ligands (>80% score) were evaluated for non-permeation through blood-brain barrier (BBB) as targets were body cancers, gastrointestinal absorption, Lipinski, non-efflux from central nervous system (CNS) by p-glycoprotein (Pgp), and ADMET analysis. This identified FDA-approved Naloxegol drug with superior attributes compared to PTX. Pocket (A) specific docking of chain length variable derivatives of Naloxegol gave docked poses that underwent MD run to give a range of properties and their descriptors (RMSD, RMSF, RoG, H-bonds, hydrophobic interaction and SASA). QSPR validated that MD properties dependent upon [-CH2-CH2-O-]n=0-7 chain length of Naloxegol. MD data underwent normalization, PCA analysis and scoring against PTX. One Naloxegol derivative scored higher than PTX as a potential microtubule disassembly inhibitor.