Exploration of microRNAs as transcriptional regulator in mumps virus infection through computational studies.

Mumps is a common childhood infection caused by the mumps virus (MuV). Aseptic meningitis and encephalitis are usual symptoms of mumps together with orchitis and oophoritis that can arise in males and females, respectively. We have used computational tools: RNA22, miRanda and psRNATarget to predict the microRNA-mRNA binding sites to find the putative microRNAs playing role in the host response to mumps virus infection. Our computational studies indicate that hsa-mir-3155a is most likely involved in mumps infection. This was further investigated by the prediction of binding sites of hsa-mir-3155a to the MuV genome. Additionally, structure prediction using MC-Fold and MC-Sym, respectively has been applied to predict the 3D structures of miRNA and mRNA. The miRNA-mRNA interaction profile between has been confirmed through molecular docking simulation studies. Taken together, the putative miRNA (hsa_miR_6794_5p) has been found to be most likely involved in the regulation of transcriptional activity in the MuV infection.

Molecular basis of facilitated target search and sequence discrimination of TALE homeodomain transcription factor Meis1.

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD-DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease.

FDA-approved small molecule kinase inhibitors for cancer treatment (2001-2015): Medical indication, structural optimization, and binding mode Part I.

The dysregulation of kinases has emerged as a major class of targets for anticancer drug discovery given its node roles in the etiology of tumorigenesis, progression, invasion, and metastasis of malignancies, which is validated by the FDA approval of 28 small molecule kinase inhibitor (SMKI) drugs for cancer treatment at the end of 2015. While the preclinical and clinical data of these drugs are widely presented, it is highly essential to give an updated review on the medical indications, design principles and binding modes of these anti-tumor SMKIs approved by the FDA to offer insights for the future development of SMKIs with specific efficacy and safety.

Channelrhodopsins with distinct chromophores and binding patterns.

Channelrhodopsins are popular optogenetic tools in neuroscience, but remain poorly understood mechanistically. Here we report the cryo-EM structures of channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii and H. catenoides kalium channelrhodopsin (KCR1). We show that ChR2 recruits an endogenous N-retinylidene-PE-like molecule to a previously unidentified lateral retinal binding pocket, exhibiting a reduced light response in HEK293 cells. In contrast, H. catenoides kalium channelrhodopsin (KCR1) binds an endogenous retinal in its canonical retinal binding pocket under identical condition. However, exogenous ATR reduces the photocurrent magnitude of wild type KCR1 and also inhibits its leaky mutant C110T. Our results uncover diverse retinal chromophores with distinct binding patterns for channelrhodopsins in mammalian cells, which may further inspire next generation optogenetics for complex tasks such as cell fate control.

MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing.

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.

The Chlamydia pneumoniae effector SemD exploits its host's endocytic machinery by structural and functional mimicry.

To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization.

Structural characterization of DNA-binding domain of essential mammalian protein TTF 1.

Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues). Despite its critical role in various cellular processes, the physical structure of TTF1 remains unsolved. Attempts to purify the functional TTF1 protein have been unsuccessful till date. Therefore, we focused on characterizing the Myb domain of this essential protein. We started with predicting a 3-D model of the Myb domain using homology modelling, and ab-initio method. We then determined its stability through MD simulation in an explicit solvent. The model predicted is highly stable, which stabilizes at 200ns. To experimentally validate the computational model, we cloned and expressed the codon optimized Myb domain into a bacterial expression vector and purified the protein to homogeneity. Further, characterization of the protein shows that, Myb domain is predominantly helical (65%) and is alone sufficient to bind the Sal Box DNA. This is the first-ever study to report a complete in silico model of the Myb domain, which is physically characterized. The above study will pave the way towards solving the atomic structure of this essential mammalian protein.

Distinct granzyme k expression in immune cells: a single-cell rna-seq meta-analysis.

BACKGROUND: Granzymes are essential serine proteases in cytotoxic T cells and natural killer (NK) cells, with GZMK's expression being less understood. This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. OBJECTIVE: This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. METHODS: We conducted a meta-analysis using cellxgene, an interactive data exploration platform developed by the Chan Zuckerberg Initiative. We focused on mature T cells, NK cells, B cells, and NKT cells. We also checked transcription factor binding sites at the granzyme gene promoter regions using JASPAR. Comparative analysis was also done using mouse single-cell RNA sequencing data. RESULTS: GZMK was the most lowly expressed in NK cells and mature NKT cells in most tissues except for colon and lymph nodes. In mature T cells, GZMK is similarly or more highly expressed than other granzymes. HBCA data revealed weak expression of GZMK in NK cells but strong expression in effector memory CD8-positive, alpha-beta T cells. Combined data shows no significant difference in GZMK expression between cell types. Subtype analysis shows that GZMK expression was higher in CD16-negative, CD56-bright NK cells when compared to CD16-positive, CD56-dim NK cells. We also identified unique transcription factor binding sites for GZMK. While this pattern in mouse data with low Gzmk expression in NK cells and higher T cells was repeated. CONCLUSION: GZMK expression is distinctively regulated among immune cells and tissues, with unique promoter regions and transcription factor binding sites contributing to this differential expression. These insights into GZMK's role in immune function and regulation offer potential therapeutic targets.

Pathogenic R163W Variant of the Copper Chaperone for Sod1 (Ccs) Functions as an Anti-chaperone.

The copper chaperone for Sod1 (Ccs) is a metallochaperone that plays a multifaceted role in the maturation of Cu,Zn superoxide dismutase (Sod1). The Ccs mutation R163W was identified in an infant with fatal neurological abnormalities. Based on a comprehensive structural and functional analysis, we developed the first data-driven model for R163W-related pathogenic phenotypes. The work here confirms previous findings that the substitution of arginine with tryptophan at this site, which is located adjacent to a conserved Zn binding site, creates an unstable Zn-deficient protein that loses its ability to efficiently activate Sod1. Intriguingly, R163W Ccs can reduce copper (i.e., Cu(II) → Cu(I)) bound in its Sod1-like domain (D2), and this novel redox event is accompanied by disulfide bond formation. The loss of Zn binding, along with the unusual ability to bind copper in D2, diverts R163W Ccs toward aggregation. The remarkably high affinity of D2 Cu(I) binding converts R163W from a Cu chaperone to a Cu scavenger that accelerates Sod1 deactivation (i.e., an Anti-chaperone). Overall, these findings present a first-of-its-kind molecular mechanism for Ccs dysfunction that leads to pathogenesis in humans.

Dopamine reuptake and inhibitory mechanisms in human dopamine transporter.

The dopamine transporter has a crucial role in regulation of dopaminergic neurotransmission by uptake of dopamine into neurons and contributes to the abuse potential of psychomotor stimulants1-3. Despite decades of study, the structure, substrate binding, conformational transitions and drug-binding poses of human dopamine transporter remain unknown. Here we report structures of the human dopamine transporter in its apo state, and in complex with the substrate dopamine, the attention deficit hyperactivity disorder drug methylphenidate, and the dopamine-uptake inhibitors GBR12909 and benztropine. The dopamine-bound structure in the occluded state precisely illustrates the binding position of dopamine and associated ions. The structures bound to drugs are captured in outward-facing or inward-facing states, illuminating distinct binding modes and conformational transitions during substrate transport. Unlike the outward-facing state, which is stabilized by cocaine, GBR12909 and benztropine stabilize the dopamine transporter in the inward-facing state, revealing previously unseen drug-binding poses and providing insights into how they counteract the effects of cocaine. This study establishes a framework for understanding the functioning of the human dopamine transporter and developing therapeutic interventions for dopamine transporter-related disorders and cocaine addiction.

Mispacking of the F87 sidechain drives aggregation-promoting conformational fluctuations in the subunit interfaces of the transthyretin tetramer.

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The x-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4% and 7% (ΔG ~ 1.5-1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer (ΔG ~ 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

Glucocorticoid Receptor-Dependent Binding Analysis Using Chromatin Immunoprecipitation and Quantitative Polymerase Chain Reaction.

ChIP-qPCR offers the opportunity to identify interactions of DNA-binding proteins such as transcription factors and their respective DNA binding sites. Thereby, transcription factors can interfere with gene expression, resulting in up- or downregulation of their target genes. Utilizing ChIP, it is possible to identify specific DNA binding sites that are bound by the DNA-binding proteins in dependence on treatment or prevailing conditions. During ChIP, DNA-binding proteins are reversibly cross-linked to their DNA binding sites and the DNA itself is fragmented. Using bead-captured antibodies, the target proteins are isolated while still binding their respective DNA response element. Using quantitative PCR, these DNA fragments are amplified and quantified. In this protocol, DNA binding sites of the glucocorticoid receptor are identified by treatment with the synthetic glucocorticoid Dexamethasone in murine bone marrow-derived macrophages.

Differential Analysis of Protein-DNA Binding Using ChIP-Seq Data.

Chromatin immunoprecipitation in combination with next-generation sequencing (ChIP-Seq) allows probing of protein-DNA binding in a rapid and genome-wide fashion. Herein we describe the required steps to preprocess ChIP-Seq data and to analyze the differential binding of proteins to DNA for perturbation experiments. In these experiments, different conditions are compared to find the underlying biological mechanisms caused by the stimulus or treatment. In addition, we provide a sample analysis using the steps outlined in the chapter.

An Optimized High-Resolution Mapping Method for Glucocorticoid Receptor-DNA Binding in Mouse Primary Macrophages.

ChIP-exo is a powerful tool for achieving enhanced sensitivity and single-base-pair resolution of transcription factor (TF) binding, which utilizes a combination of chromatin immunoprecipitation (ChIP) and lambda exonuclease digestion (exo) followed by high-throughput sequencing. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) is an updated and simplified version of the original ChIP-exo method, which has reported an efficient adapter ligation through the DNA circularization step. Building upon an established method, we present a protocol for generating NGS (next-generation sequencing) ready and high-quality ChIP-nexus library for glucocorticoid receptor (GR). This method is specifically optimized for bone marrow-derived macrophage (BMDM) cells. The protocol is initiated by the formation of DNA-protein cross-links in intact cells. This is followed by chromatin shearing, chromatin immunoprecipitation, ligation of sequencing adapters, digestion of adapter-ligated DNA using lambda exonuclease, and purification of single-stranded DNA for circularization and library amplification.

Sequential ChIP-Seq.

ChIP-Seq has been used extensively to profile genome-wide transcription factor binding and post-translational histone modifications. A sequential ChIP assay determines the in vivo co-localization of two proteins to the same genomic locus. In this chapter, we combine the two protocols in Sequential ChIP-Seq, a method for identifying genome-wide sites of in vivo protein co-occupancy.

Estrogen Receptor Chromatin Profiling by CUT&RUN.

Gonadal steroid hormones, namely, testosterone, progesterone, and estrogens, influence the physiological state of an organism through the regulation of gene transcription. Steroid hormones activate nuclear hormone receptor (HR), transcription factors (TFs), which bind DNA in a tissue- and cell type-specific manner to influence cellular function. Identifying the genomic binding sites of HRs is essential to understanding mechanisms of hormone signaling across tissues and disease contexts. Traditionally, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been used to map the genomic binding of HRs in cancer cell lines and large tissues. However, ChIP-seq lacks the sensitivity to detect TF binding in small numbers of cells, such as genetically defined neuronal subtypes in the brain. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) resolves most of the technical limitations of ChIP-seq, enabling the detection of protein-DNA interactions with as few as 100-1000 cells. In this chapter, we provide a stepwise CUT&RUN protocol for detecting and analyzing the genome-wide binding of estrogen receptor α (ERα) in mouse brain tissue. The steps described here can be used to identify the genomic binding sites of most TFs in the brain.

ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.

A versatile residue numbering scheme for Nav and Cav channels.

Voltage-gated sodium (Nav) and calcium (Cav) channels are responsible for the initiation of electrical signals. They have long been targeted for the treatment of various diseases. The mounting number of cryoelectron microscopy (cryo-EM) structures for diverse subtypes of Nav and Cav channels from multiple organisms necessitates a generic residue numbering system to establish the structure-function relationship and to aid rational drug design or optimization. Here we suggest a structure-based residue numbering scheme, centering around the most conserved residues on each of the functional segments. We elaborate the generic numbers through illustrative examples, focusing on representative drug-binding sites of eukaryotic Nav and Cav channels. We also extend the numbering scheme to compare common disease mutations among different Nav subtypes. Application of the generic residue numbering scheme affords immediate insights into hotspots for pathogenic mutations and critical loci for drug binding and will facilitate drug discovery targeting Nav and Cav channels.

The plasma membrane inner leaflet PI(4,5)P2 is essential for the activation of proton-activated chloride channels.

Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl- levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P2 depletion by activating phosphatidylinositol 5-phosphatases or Gq protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P2-binding site is localized within the cytosol in resting state but shifts to the cell membrane's inner surface in an activated state and interacts with inner leaflet PI(4,5)P2. Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P2.

FitScore: a fast machine learning-based score for 3D virtual screening enrichment.

Enhancing virtual screening enrichment has become an urgent problem in computational chemistry, driven by increasingly large databases of commercially available compounds, without a commensurate drop in in vitro screening costs. Docking these large databases is possible with cloud-scale computing. However, rapid docking necessitates compromises in scoring, often leading to poor enrichment and an abundance of false positives in docking results. This work describes a new scoring function composed of two parts - a knowledge-based component that predicts the probability of a particular atom type being in a particular receptor environment, and a tunable weight matrix that converts the probability predictions into a dimensionless score suitable for virtual screening enrichment. This score, the FitScore, represents the compatibility between the ligand and the binding site and is capable of a high degree of enrichment across standardized docking test sets.