Position Of Binding Site Research Articles

Language models can identify enzymatic binding sites in protein sequences

Recent advances in language modeling have had a tremendous impact on how we handle sequential data in science. Language architectures have emerged as a hotbed of innovation and creativity in natural language processing over the last decade, and have since gained prominence in modeling proteins and chemical processes, elucidating structural relationships from textual/sequential data. Surprisingly, some of these relationships refer to three-dimensional structural features, raising important questions on the dimensionality of the information encoded within sequential data. Here, we demonstrate that the unsupervised use of a language model architecture to a language representation of bio-catalyzed chemical reactions can capture the signal at the base of the substrate-binding site atomic interactions. This allows us to identify the three-dimensional binding site position in unknown protein sequences. The language representation comprises a reaction-simplified molecular-input line-entry system (SMILES) for substrate and products, and amino acid sequence information for the enzyme. This approach can recover, with no supervision, 52.13% of the binding site when considering co-crystallized substrate-enzyme structures as ground truth, vastly outperforming other attention-based models.

Extended Metadynamics Protocol for Binding/Unbinding Free Energies of Peptide Ligands to Class A G-Protein-Coupled Receptors.

A metadynamics protocol is presented to characterize the binding and unbinding of peptide ligands to class A G-protein-coupled receptors (GPCRs). The protocol expands on the one previously presented for binding and unbinding small-molecule ligands to class A GPCRs and accounts for the more demanding nature of the peptide binding-unbinding process. It applies to almost all class A GPCRs. Exemplary simulations are described for subtypes Y1R, Y2R, and Y4R of the neuropeptide Y receptor family, vasopressin binding to the vasopressin V2 receptor (V2R), and oxytocin binding to the oxytocin receptor (OTR). Binding free energies and the positions of alternative binding sites are presented and, where possible, compared with the experiment.

Analysis of Functional Promoter of Camel FGF21 Gene and Identification of Small Compounds Targeting FGF21 Protein.

The fibroblast growth factor 21 (FGF21) gene plays an important role in the mechanism of glucose and lipid metabolism and is a promising therapeutic target for metabolic disease. Camels display a unique regulation characteristic of glucose and lipid metabolism, endowing them with the ability to adapt to survive drought and chronic hunger. However, the knowledge about the camel FGF21 gene regulation and its differences between humans and mice is still limited. In this study, camel FGF21 gene promoter was obtained for ~2000 bp upstream of the transcriptional start site (TSS). Bioinformatics analysis showed that the proximal promoter region sequences near the TSS between humans and camels have high similarity. Two potential core active regions are located in the -445-612 bp region. In addition, camel FGF21 promoter contains three CpG islands (CGIs), located in the -435~-1168 bp regions, significantly more and longer than in humans and mice. The transcription factor binding prediction showed that most transcription factors, including major functional transcription factors, are the same in different species although the binding site positions in the promoter are different. These results indicated that the signaling pathways involved in FGF21 gene transcription regulation are conservative in mammals. Truncated fragments recombinant vectors and luciferase reporter assay determined that camel FGF21 core promoter is located within the 800 bp region upstream of the TSS and an enhancer may exist between the -1000 and -2000 bp region. Combining molecular docking and in silico ADMET druggability prediction, two compounds were screened as the most promising candidate drugs specifically targeting FGF21. This study expanded the functions of these small molecules and provided a foundation for drug development targeting FGF21.

Structural insights of novel mutational frames in Bromodomain Containing-2 gene (BRD2) in juvenile myoclonic epilepsy: bed, bench, and laptop profiles

PurposeJuvenile myoclonic epilepsy (JME) is an adolescent onset type of idiopathic generalized epilepsies. Bromodomain containing protein-2 gene (BRD2), a transcriptional regulatory protein, has a susceptible role in the expression of JME. Considering the polymorphic variations observed in exon 3 of the BRD2 gene, we evaluated the molecular interactions with anti-seizure medication in individuals diagnosed with JME. MethodsThe genomic DNA was extracted from 5 mL of peripheral venous blood of JME participants (n = 55) and healthy control subjects (n = 55). Detailed anti-seizure medication and outcomes were noted during the study period. Identified novel mutations at nucleotide and protein sequences, compared by multiple sequence alignment. Wild-type (WT) and mutated-type (MT) structures were investigated for molecular docking and interactions with anti-seizure drugs. ResultsA common variant at c.1707G>A was found among 23 participants, while a single variant at c.1663ins C was found in one participant. The deletion positions were observed at c.1890delA, c.1892A>T, c.1895A>T, c.1896G>T, c.1897T>C, c.1898T>C, c.1899C>T, c.1900G>T, c.1901C>T and c.1902A>T exhibiting stop codon after p.111Pro>stop; these variants resulted in a truncated protein. In silico analysis was conducted to validate changes; docking analysis showed that novel variant has a significant role in the interactions with anti-seizure drugs. SignificanceBesides clinical and genetic outcomes, ∼5.45% unique genetical variations were observed in the participants. Significant mimicked at the binding site position (92–111) of human BRD2 ranges ∼8.2%, ∼16.4%, and ∼10.6%. Further, research is needed to identify the importance of polymorphism alterations at the binding site and their molecular interactions with anti-seizure drugs, which might be confirmed in a diverse population with JME.

The severity of COVID-19 in hypertensive patients is associated with mirSNPs in the 3′ UTR of ACE2 that associate with miR-3658: In silico and in vitro studies

The SARS-CoV-2 virus targets the antigen converting enzyme 2 (ACE2) receptor, thus resulting in elevated morbidity and an increased risk of severe and fatal COVID-19 infection in individuals with hypertension and diabetes mellitus. ObjectivesThis study aimed to identify the association between increased susceptibility and severity in order to evaluate their impact in hypertensive COVID-19 patients using in vitro and in silico models. MethodsWe identified 80 miRNA binding sites on ACE2 (for different miRNAs) as well as various 30 SNPs in the miRNA binding sites of the 3′ untranslated region (3′ UTR) in the ACE2 gene using different online software and tools. From August 2020 to August 2021, a total of 200 nasopharyngeal/mouth swabs samples were collected from Multan, Pakistan. In order to quantify the cDNA of ACE2 and miR-3658 genes, we used Rotor Gene qRT-PCR on hypertensive patients with COVID-19 as well as healthy controls. ResultsInterestingly, the binding site of miR-3658 corresponding to the 3′ UTR of ACE2 featured three SNPs (rs1457913029, C>T; rs960535757, A>C, G; rs1423809569, C>T), and its genomic sequence featured a single SNP (rs1024225815, C>T) with the same nucleotide variation (rs1457913029, C>T) which potentially increases the severity of COVID-19. Similarly, three other SNPs (rs1557852115, C>G; rs770335293, A>G; rs1024225815, C>T) were also found on the first binding site positions of miR-3658. Our in vitro study found that ACE2 gene expression had an effect on miR-3658 in COVID-19 patients who also had hypertension. In both cases, our analysis demonstrated that the in silico model captured the same biological mechanisms as the in vitro system. ConclusionThe identified SNPs could represent potential informative signatures owing to their position in the splicing site of the ACE2 gene.

Expression profiling analysis reveals key microRNA-mRNA interactions in patients with transposition of the great arteries and systemic left and right ventricles.

Patients with transposition of the great arteries (TGA) have different connected systemic chambers and this determines the long-term morbidities and survival. Limited findings have been reported to systematically identify miRNA and mRNA expression levels in such cohorts of patients. In this study, we aimed to characterize miRNAs, mRNAs, and miRNA-mRNA interaction networks in patients with TGA, with a systemic left (LV) and right ventricle (RV). Large panel of human miRNA and mRNA microarrays were conducted to determine the genome-wide expression profiles in the blood of 16 TGA-RV patients, 16 TGA-LV patients, and 16 age and gender-matched controls. Using real-time quantitative PCR (RT-qPCR), the differential expression level of a single miRNA was validated. Enrichment analyses of altered miRNA and mRNA expression levels were identified using bioinformatics tools. Altered miRNA and mRNA expression levels were observed between TGA-RV and TGA-LV patients, together or separated, compared to controls. Among the deregulated miRNAs and mRNAs, 39 and 101 miRNAs were identified as significantly differentially expressed in patients with TGA (both TGA-RV and TGA-LV) and TGA-RV, when compared to matched controls. Furthermore, 51 miRNAs were identified as significantly differentially expressed in patients with TGA-RV when compared to patients with TGA-LV. RT-qPCR relative expression level was highly consistent with microarray analysis results. Similarly, 36 and 164 mRNAs were identified as significantly differentially expressed in patients with TGA (both TGA-RV and TGA-LV) and TGA-RV, when compared to matched controls. Additionally, miR-140-3p showed a higher expression level in patients with overt heart failure (FC = 1.54; P = 0.001) and miR-502-3p showed a higher expression level in patients died due to cardiac death (FC = 1.41; P = 0.011). Integrative analysis resulted in 21 and 23 target genes with higher and lower expression levels, respectively (r ≥ 0.50 and P < 0.05). These target genes (i.e., 21 and 23 target genes) showed an inverse direction of regulation with miRNA and exhibited a miRNA binding site position within the 3'UTR of the target gene. Our findings provide new insights into a potential molecular biomarker(s) for patients with TGA that may guide better risk stratification and the development of novel targeting therapies. Future studies are needed to investigate the potential significance of miRNAs and mRNAs in TGA-related cardiovascular diseases.

OCT4 interprets and enhances nucleosome flexibility.

Pioneer transcription factors are proteins that induce cellular identity transitions by binding to inaccessible regions of DNA in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA elements. The structural features and dynamics modulating their interaction with nucleosomes are still unresolved. From a combination of experiments and molecular simulations, we reveal here how the pioneer factor and master regulator of pluripotency, Oct4, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4’s impact on nucleosome dynamics depends on the binding site position and the mobility of the unstructured tails of nucleosomal histone proteins. Oct4 uses both its DNA binding domains to propagate and stabilize open nucleosome conformations, one for specific sequence recognition and the other for nonspecific interactions with nearby regions of DNA. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics.

Learning probabilistic protein-DNA recognition codes from DNA-binding specificities using structural mappings.

Knowledge of how proteins interact with DNA is essential for understanding gene regulation. Although DNA-binding specificities for thousands of transcription factors (TFs) have been determined, the specific amino acid–base interactions comprising their structural interfaces are largely unknown. This lack of resolution hampers attempts to leverage these data in order to predict specificities for uncharacterized TFs or TFs mutated in disease. Here we introduce recognition code learning via automated mapping of protein–DNA structural interfaces (rCLAMPS), a probabilistic approach that uses DNA-binding specificities for TFs from the same structural family to simultaneously infer both which nucleotide positions are contacted by particular amino acids within the TF as well as a recognition code that relates each base-contacting amino acid to nucleotide preferences at the DNA positions it contacts. We apply rCLAMPS to homeodomains, the second largest family of TFs in metazoans and show that it learns a highly effective recognition code that can predict de novo DNA-binding specificities for TFs. Furthermore, we show that the inferred amino acid–nucleotide contacts reveal whether and how nucleotide preferences at individual binding site positions are altered by mutations within TFs. Our approach is an important step toward automatically uncovering the determinants of protein–DNA specificity from large compendia of DNA-binding specificities and inferring the altered functionalities of TFs mutated in disease.

The relative binding position of Nck and Grb2 adaptors impacts actin-based motility of Vaccinia virus.

Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to assemble complex signalling networks. The concept of phase separation has recently changed our appreciation of multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now investigated this parameter in the operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3 complex-mediated actin polymerisation. Manipulating the position of pTyr motifs in A36 and the unrelated p14 from Orthoreovirus, we find that only specific spatial arrangements of Nck and Grb2 binding sites result in robust N-WASP recruitment, Arp2/3 complex driven actin polymerisation and viral spread. This suggests that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are frequently conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those undergoing phase transitions.

Along the Bos taurus genome, uncover candidate imprinting control regions

BackgroundIn mammals, Imprinting Control Regions (ICRs) regulate a subset of genes in a parent-of-origin-specific manner. In both human and mouse, previous studies identified a set of CpG-rich motifs occurring as clusters in ICRs and germline Differentially Methylated Regions (gDMRs). These motifs consist of the ZFP57 binding site (ZFBS) overlapping a subset of MLL binding units known as MLL morphemes. MLL or MLL1 (Mixed Lineage Leukemia 1) is a relatively large multidomain protein that plays a central role in the regulation of transcription. The structures of both MLL1 and MLL2 include a domain (MT) that binds CpG-rich DNA and a conserved domain (SET) that methylates lysine 4 in histone H3 producing H3K4me3 marks in chromatin.ResultsSince genomic imprinting impacts many developmental and key physiological processes, we followed a previous bioinformatics strategy to pinpoint ICR positions in the Bos taurus genome. Initial genome-wide analyses involved finding the positions of ZFP57 binding sites, and the CpG-rich motifs (ZFBS-morph overlaps) along cattle chromosomal DNA. By creating plots displaying the density of ZFBS-morph overlaps, we removed background noise and thus improved signal detection. With the density-plots, we could view the positions of peaks locating known and candidate ICRs in cattle DNA. Our evaluations revealed the correspondence of peaks in plots to reported known and inferred ICRs/DMRs in cattle. Beside peaks pinpointing such ICRs, the density-plots also revealed additional peaks. Since evaluations validated the robustness of our approach, we inferred that the additional peaks may correspond to candidate ICRs for imprinted gene expression.ConclusionOur bioinformatics strategy offers the first genome-wide approach for systematically localizing candidate ICRs. Furthermore, we have tailored our datasets for upload onto the UCSC genome browser so that researchers could find known and candidate ICRs with respect to a wide variety of annotations at all scales: from the positions of Single Nucleotide Polymorphisms (SNPs), to positions of genes, transcripts, and repeated DNA elements. Furthermore, the UCSC genome browser offers tools to produce enlarged views: to uncover the genes in the vicinity of candidate ICRs and thus discover potential imprinted genes for experimental validations.

Characterization of Hemagglutinin Gene Fragment of H9N2 Avian Influenza Virus Isolated from Environmental Live Bird Market in the Greater Jakarta Area

Virus H9N2 has become the most common subtype of low pathogenic avian influenza (LPAI) in poultry and shows the ability to infect humans. One of the important factors triggering the virus ​​pandemic is the live bird market (LBM). The virus acts as an internal gene donor in the other subtypes, such as H5N1, H5N2, H7N9, and H10N8, in poultry and humans. This study was conducted to detect the presence of the H9N2 virus and the molecular characteristics of the hemagglutinin gene fragment at the cleavage site, receptor binding site, antigenic site, and glycosylation site positions of the H9N2 subtype AI virus isolated from the LBM environment. This study used Disease Investigation Center Subang isolates from environmental samples of LBM in the Greater Jakarta area (DKI Jakarta, Bogor, Depok, Tangerang, and Bekasi) in 2019. Based on molecular detection using RT-PCR and RT-qPCR, it was found that avian influenza subtype H9N2 was detected. This indicates that LBM has the potential to be the source of the spread of the ​​H9N2 virus. The pattern of the amino acid cleavage site PSRSSR↓GLF indicates that the research sample belongs to the low pathogenic AI. The substitution of amino acids in the receptor binding site and antigenic site increases the specificity of non-human host recognition. The potential glycosylation site, with the NCS motif found in amino acid position 295-297, close to the receptor-binding site. Based on genetic analysis and phylogenetic topography, the virus is included in the CVI (China, Vietnam, Indonesia) lineage and subclade H9.4.2.5.

Abstract P3-06-04: Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer

Abstract Introduction: The Notch and Wnt signaling pathways play critical role in mammary development and tumourigenesis. The epithelia-mesenchymal transition (EMT) is an important process of breast cancer development. Growing evidence shows that Glycogen Synthase Kinase 3 Beta (GSK 3β) is considered as an EMT suppressor of its downregulation of protein snail, a zinc-finger transcriptional repressor that regulates epithelia marker E-cadherin. Previous studies by our group demonstrated that Notch3 may inhibit the emergence and progression of breast cancer. In mammalian cell, GSK3β has been show to physically bind and phosphorylate the intracellular domain of two Notch paralogues. But the relationship between Notch3,GSK3β and EMT in breast cancer still unclear. Intriguingly, we found that knockdown of Notch3 in luminal subtype of breast cancer cells MCF-7 downregulated GSK3β and promoted EMT. In contrast, overexpression of Notch3 in triple-negative cell lines MDA-MB-231 upregulated GSK3βand inhibited EMT. Mechanistically, our results revealed that Notch3 specifically binds to the CSL binding element of the GSK3β promoter and trans-activates its expression. Furthermore, Notch3 and GSK3β expression were positively associated in human breast cancer. Moreover, the mRNA expression of both Notch3 and GSK3βpredicted a better Overall-Survival for breast cancer patients. Our findings demonstrate that Notch3 inhibits breast cancer cells EMT via trans-activating GSK3β expression. Methods: 1. Western blot and/or qRT-PCR were performed to study the expressions of Notch3,GSK3β, β-catenin and EMT related-molecules in breast cancer cell line. 2. Chromatin immunoprecipitation (ChIP) and dual-luciferase assays were carried out to investigate whether Notch3 bound to GSK3β promoter and mediatesGSK3β expression on transcriptional level. 3. Wound healing assay, migration and invasion assays were performed to analyze the effect of Notch3 and GSK3β on breast cancer cell lines migration abilities. 4. The association of Notch3 and GSK3β expression in human breast cancer samples and correlation between Notch3 or GSK3β expression and patient survival in a cohort of human breast cancer patients in Kaplan Meier Plotter database were analyzed. Results: 1. Notch3 up-regulates GSK3β expression in mRNA and protein levels. 2. Notch3 regulates GSK3β expression by binding to the CSL antisense binding site (TTCCCA) positions -308bp to -303bp in the GSK3β promoter. 3. Notch3 intracellular domain (N3ICD) down-regulates the expression of Vimentin, β-catenin, but up-regulates E-cadherin expression. 4. Notch3 inhibits the ability of migration and invasion in breast cancer cell lines. 5. Notch3 and GSK3β expression were positively associated in human breast cancer. The mRNA expression of both Notch3 and GSK3β predicted a better Overall-Survival for breast cancer patients. Summary: We have identified a positive correlation between Notch3 and GSK3β in breast cancer and confirmed a role for the Notch3- GSK3β axis in inhibiting EMT. In addition, we explored in detail and first clarified the possible molecular mechanism of regulating this axis. Our results provide new insights into the complex regulation of breast cancer proliferation and tumorigenesis. We believe that the Notch3- GSK3β axis may be a prospective prognostic predictor for breast cancer. Citation Format: Chen Wei-Ling, Yong-Qu Zhang, Wen-He Huang, Min Chen, Yao-Chen Li, Guo-Jun Zhang. Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-04.

Examining the influence of bilayer structure on energy transfer and molecular photon upconversion in metal ion linked multilayers.

Metal ion linked multilayers is a unique motif to spatially control and geometrically restrict molecules on a metal oxide surface and is of interest in a number of promising applications. Here we use a bilayer composed of a metal oxide surface, an anthracene annihilator molecule, Zn(II) linking ion, and porphyrin sensitizers to probe the influence of the position of the metal ion binding site on energy transfer, photon upconversion, and photocurrent generation. Despite being energetically similar, varying the position of the carboxy metal ion binding group (i.e. ortho, meta, para) of the Pt(II) tetraphenyl porphyrin sensitizer had a large impact on energy transfer rates and upconverted photocurrent that can be attributed to differences in their geometries. From polarized attenuated total reflectance measurements of the bilayers on ITO, we found that the orientation of the first layer (anthracene) was largely unperturbed by subsequent layers. However, the tilt angle of the porphyrin plane varies dramatically from 41° to 64° to 57° for the para-, meta-, and ortho-COOH substituted porphyrin molecules, which is likely responsible for the variation in energy transfer rates. We go on to show using molecular dynamics simulations that there is considerable flexibility in porphyrin orientation, indicating that an average structure is insufficient to predict the ensemble behavior. Instead, even a small subset of the population with highly favorable energy transfer rates can be the primary driver in increasing the likelihood of energy transfer. Gaining control of the orientation and its distribution will be a critical step in maximizing the potential of the metal ion linked structures.

Site-specific recombinase genome engineering toolkit in maize.

Site‐specific recombinase enzymes function in heterologous cellular environments to initiate strand‐switching reactions between unique DNA sequences termed recombinase binding sites. Depending on binding site position and orientation, reactions result in integrations, excisions, or inversions of targeted DNA sequences in a precise and predictable manner. Here, we established five different stable recombinase expression lines in maize through Agrobacterium‐mediated transformation of T‐DNA molecules that contain coding sequences for Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase. Through the bombardment of recombinase activated DsRed transient expression constructs, we have determined that all five recombinases are functional in maize plants. These recombinase expression lines could be utilized for a variety of genetic engineering applications, including selectable marker removal, targeted transgene integration into predetermined locations, and gene stacking.

High-Resolution Scanning of Optimal Biosensor Reporter Promoters in Yeast.

Small-molecule binding allosteric transcription factors (aTFs) derived from bacteria enable real-time monitoring of metabolite abundances, high-throughput screening of genetic designs, and dynamic control of metabolism. Yet, engineering of reporter promoter designs of prokaryotic aTF biosensors in eukaryotic cells is complex. Here we investigate the impact of aTF binding site positions at single-nucleotide resolution in >300 reporter promoter designs in Saccharomyces cerevisiae. From this we identify biosensor output landscapes with transient and distinct aTF binding site position effects for aTF repressors and activators, respectively. Next, we present positions for tunable reporter promoter outputs enabling metabolite-responsive designs for a total of four repressor-type and three activator-type aTF biosensors with dynamic output ranges up to 8- and 26-fold, respectively. This study highlights aTF binding site positions in reporter promoters as key for successful biosensor engineering and that repressor-type aTF biosensors allows for more flexibility in terms of choice of binding site positioning compared to activator-type aTF biosensors.

Expanding the Dynamic Range of a Transcription Factor-Based Biosensor in Saccharomyces cerevisiae.

Metabolite biosensors are useful tools for high-throughput screening approaches and pathway regulation approaches. An important feature of biosensors is the dynamic range. To expand the maximum dynamic range of a transcription factor-based biosensor in Saccharomyces cerevisiae, using the fapO/FapR system from Bacillus subtilis as an example case, five native promoters, including constitutive and glucose-regulated ones, were modified. By evaluating different binding site (BS) positions in the core promoters, we identified locations that resulted in a high maximum dynamic range with low expression under repressed conditions. We further identified BS positions in the upstream element region of the TEF1 promoter that did not influence the native promoter strength but resulted in repression in the presence of a chimeric repressor consisting of FapR and the yeast repressor Mig1. These modified promoters with broad dynamic ranges will provide useful information for the engineering of future biosensors and their use in complex genetic circuits.

Predicting Diffusion Coefficients of DNA-Protein Complexes by Convex Hull Modelling

The recent development of single-molecule diffusometry establishes the diffusion coefficient as a new observable in single-molecule experiments to visualize biomolecular interactions. To decipher the rich information available from these experiments, accurate modeling of the hydrodynamic properties of biomolecular complexes is needed. Currently available tools for predicting diffusion coefficients of biomolecules, such as Hydropro and HullRad, rely on the existence of atomic coordinates for the molecule of interest. Composite molecular complexes that lack detailed Protein Data Bank entries, but can be modeled structurally from their constituent parts (i.e. a complex with multiple proteins bound to a single DNA) are frequently encountered in experiments but cannot yet be properly modeled for hydrodynamic properties. We present a simple program to predict the diffusion coefficients of composite DNA-protein complexes. Our algorithm extends the computational framework introduced by Fleming et al. in the HullRad software to construct a convex hull of the molecular complex and then uses an ellipsoidal approximation to compute hydrodynamic parameters. In order to construct the convex hull of the whole complex, the algorithm incorporates a priori knowledge of the complex such as the crystal structures of the constituent biomolecules, binding site positions, geometry and mechanics of DNA duplexes. The program is computationally efficient, integrates naturally with PyMOL, and outputs the 3D convex hull for visualization. We validate the program by accurately predicting hydrodynamic parameters of known crystal structures of DNA-protein complexes. We then predict diffusion coefficients of the restriction enzyme BamHI bound to double strand DNA of different lengths and compare these predictions with single-molecule diffusometry measurements. We envision that this program will aid researchers in using diffusion coefficient to identify and characterize relevant biological structures at the nanometer scale.

Characteristics of a PHD Finger Subtype.

Although the plant homeodomain (PHD) finger superfamily is known for its site-specific readouts of histone tails, the origins of the mechanistic differences in histone H3 readout by different PHD subtypes remain less clear. We show that sequences containing the xCDxCDx motif in the PHD treble clef (xCDxCDx-PHD) constitute a distinct subtype, based on the following observations: (i) the amino acid composition of the binding site is strikingly different from other subtypes due to position-specific enrichment of negatively charged and bulky nonpolar residues, (ii) the binding site positions are mutually and positively correlated, and this correlation is absent in other subtypes, and (iii) there are only small structural deviations, despite low sequence similarity. The xCDxCDx-PHD constitutes ∼20% of the PHD family, and the double PHD fingers (DPFs) are 10% of the total number of xCDxCDx-PHDs. This subtype originated early in the evolution of eukaryotes but has diversified within the metazoan lineage. Despite sequence diversification, the positions of the enriched nonpolar residues, in particular, show very small structural deviations, suggesting critical contributions of nonpolar residues in the binding mechanism of this subtype. Using mutagenesis, we probed the contributions of the binding-site positions enriched in nonpolar residues in four xCDxCDx-PHD proteins and found that they contribute to the tight packing of the H3 residues. This effect may potentially be exploited, as we observed affinity enhancement upon substituting a bulky nonpolar residue at the same binding site in another histone reader. Overall, we present a detailed characterization of PHD subtypes.

Interaction of Au(iii) and Pt(ii) complexes with Na/K-ATPase: experimental and theoretical study of reaction stoichiometry and binding sites.

The present paper deals with investigation of the interaction between selected simple structure Au(iii) ([AuCl4]-, [AuCl2(dmso)2]+, [AuCl2(bipy)]+) and Pt(ii) ([PtCl2(dmso)2]) complexes with Na/K-ATPase as the target enzyme, using an experimental and theoretical approach. Reaction stoichiometries and binding constants for these enzyme/complex systems were determined, while kinetic measurements were used in order to reveal the type of inhibition. Based on the results obtained by quantum mechanical calculations (electrostatic surface potential (ESP), volume and surface of the complexes) the nature of the investigated complexes was characterized. By using the solvent accessible surface area (SASA) applied on specific inhibitory sites (ion channel and intracellular domains) the nature of these sites was described. Docking studies were used to determine the theoretical probability of the non-covalent metal binding site positions. Inhibition studies implied that all the investigated complexes decreased the activity of the enzyme while the kinetic analysis indicated an uncompetitive mode of inhibition for the selected complexes. Docking results suggested that the main inhibitory site of all these complexes is located in the ion translocation pathway on the extracellular side in the E2P enzyme conformation, similar to the case of cardiac glycosides, specific Na/K-ATPase inhibitors. Also, based on our knowledge, the hydrolyzed forms of [AuCl4]- and [PtCl2(dmso)2] complexes were investigated for the first time by theoretical calculations in this paper. Thereby, a new inhibitory site situated between the M2 and M4 helices was revealed. Binding in this site induces conformational changes in the enzyme domains and perturbs the E1-E2P conformational equilibrium, causing enzyme inhibition.

Experimental assessment and analysis of super-resolution in fluorescence microscopy based on multiple-point spread function fitting of spectrally demultiplexed images

This paper presents an experimental assessment and analysis of super-resolution microscopy based on multiple-point spread function fitting of spectrally demultiplexed images using a designed DNA structure as a test target. For the purpose, a DNA structure was designed to have binding sites at a certain interval that is smaller than the diffraction limit. The structure was labeled with several types of quantum dots (QDs) to acquire their spatial information as spectrally encoded images. The obtained images are analyzed with a point spread function multifitting algorithm to determine the QD locations that indicate the binding site positions. The experimental results show that the labeled locations can be observed beyond the diffraction-limited resolution using three-colored fluorescence images that were obtained with a confocal fluorescence microscope. Numerical simulations show that labeling with eight types of QDs enables the positions aligned at 27.2-nm pitches on the DNA structure to be resolved with high accuracy.