Structural basis of water-mediated cis Watson-Crick/Hoogsteen base-pair formation in non-CpG methylation.

Non-CpG methylation is associated with several cellular processes, especially neuronal development and cancer, while its effect on DNA structure remains unclear. We have determined the crystal structures of DNA duplexes containing -CGCCG- regions as CCG repeat motifs that comprise a non-CpG site with or without cytosine methylation. Crystal structure analyses have revealed that the mC:G base-pair can simultaneously form two alternative conformations arising from non-CpG methylation, including a unique water-mediated cis Watson-Crick/Hoogsteen, (w)cWH, and Watson-Crick (WC) geometries, with partial occupancies of 0.1 and 0.9, respectively. NMR studies showed that an alternative conformation of methylated mC:G base-pair at non-CpG step exhibits characteristics of cWH with a syn-guanosine conformation in solution. DNA duplexes complexed with the DNA binding drug echinomycin result in increased occupancy of the (w)cWH geometry in the methylated base-pair (from 0.1 to 0.3). Our structural results demonstrated that cytosine methylation at a non-CpG step leads to an anti→syntransition of its complementary guanosine residue toward the (w)cWH geometry as a partial population of WC, in both drug-bound and naked mC:G base pairs. This particular geometry is specific to non-CpG methylated dinucleotide sites in B-form DNA. Overall, the current study provides new insights into DNA conformation during epigenetic regulation.

A survey of recent unusual high-resolution DNA structures provoked by mismatches, repeats and ligand binding.

The structure of the DNA duplex is arguably one of the most important biological structures elucidated in modern history. DNA duplex structure is closely associated with essential biological functions such as DNA replication and RNA transcription. In addition to the classical A-, B- and Z-DNA conformations, DNA duplexes are capable of assuming a variety of alternative conformations depending on the sequence and environmental context. A considerable number of these unusual DNA duplex structures have been identified in the past decade, and some of them have been found to be closely associated with different biological functions and pathological conditions. In this manuscript, we review a selection of unusual DNA duplex structures, particularly those originating from base pair mismatch, repetitive sequence motifs and ligand-induced structures. Although the biological significance of these novel structures has not yet been established in most cases, the illustrated conformational versatility of DNA could have relevance for pharmaceutical or nanotechnology development. A perspective on the future directions of this field is also presented.

Cooperative recognition of T:T mismatch by echinomycin causes structural distortions in DNA duplex.

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.

Parity-dependent hairpin configurations of repetitive DNA sequence promote slippage associated with DNA expansion.

Repetitive DNA sequences are ubiquitous in life, and changes in the number of repeats often have various physiological and pathological implications. DNA repeats are capable of interchanging between different noncanonical and canonical conformations in a dynamic fashion, causing configurational slippage that often leads to repeat expansion associated with neurological diseases. In this report, we used single-molecule spectroscopy together with biophysical analyses to demonstrate the parity-dependent hairpin structural polymorphism of TGGAA repeat DNA. We found that the DNA adopted two configurations depending on the repeat number parity (even or odd). Transitions between these two configurations were also observed for longer repeats. In addition, the ability to modulate this transition was found to be enhanced by divalent ions. Based on the atomic structure, we propose a local seeding model where the kinked GGA motifs in the stem region of TGGAA repeat DNA act as hot spots to facilitate the transition between the two configurations, which may give rise to disease-associated repeat expansion.

CoII(Chromomycin)2 Complex Induces a Conformational Change of CCG Repeats from i-Motif to Base-Extruded DNA Duplex.

We have reported the propensity of a DNA sequence containing CCG repeats to form a stable i-motif tetraplex structure in the absence of ligands. Here we show that an i-motif DNA sequence may transition to a base-extruded duplex structure with a GGCC tetranucleotide tract when bound to the (CoII)-mediated dimer of chromomycin A3, CoII(Chro)2. Biophysical experiments reveal that CCG trinucleotide repeats provide favorable binding sites for CoII(Chro)2. In addition, water hydration and divalent metal ion (CoII) interactions also play a crucial role in the stabilization of CCG trinucleotide repeats (TNRs). Our data furnish useful structural information for the design of novel therapeutic strategies to treat neurological diseases caused by repeat expansions.

Induced-Fit Recognition of CCG Trinucleotide Repeats by a Nickel-Chromomycin Complex Resulting in Large-Scale DNA Deformation.

Small-molecule compounds targeting trinucleotide repeats in DNA have considerable potential as therapeutic or diagnostic agents against many neurological diseases. NiII (Chro)2 (Chro=chromomycin A3) binds specifically to the minor groove of (CCG)n repeats in duplex DNA, with unique fluorescence features that may serve as a probe for disease detection. Crystallographic studies revealed that the specificity originates from the large-scale spatial rearrangement of the DNA structure, including extrusion of consecutive bases and backbone distortions, with a sharp bending of the duplex accompanied by conformational changes in the NiII chelate itself. The DNA deformation of CCG repeats upon binding forms a GGCC tetranucleotide tract, which is recognized by NiII (Chro)2 . The extruded cytosine and last guanine nucleotides form water-mediated hydrogen bonds, which aid in ligand recognition. The recognition can be accounted for by the classic induced-fit paradigm.

Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element.

The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.

Investigation of C-terminal domain of SARS nucleocapsid protein-Duplex DNA interaction using transistors and binding-site models.

AlGaN/GaN high electron mobility transistors (HEMTs) were used to sense the binding between double stranded DNA (dsDNA) and the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (N protein). The sensing signals were the drain current change of the HEMTs induced by the protein-dsDNA binding. Binding-site models using surface coverage ratios were utilized to analyze the signals from the HEMT-based sensors to extract the dissociation constants and predict the number of binding sites. Two dissociation constants, K D1 = 0.0955 nM, K D2 = 51.23 nM, were obtained by fitting the experimental results into the two-binding-site model. The result shows that this technique is more competitive than isotope-labeling electrophoretic mobility shift assay (EMSA). We demonstrated that AlGaN/GaN HEMTs were highly potential in constructing a semiconductor-based-sensor binding assay to extract the dissociation constants of nucleotide-protein interaction.

The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity.

TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

Genomic and Metabolomic Landscape of Right-Sided and Left-Sided Colorectal Cancer: Potential Preventive Biomarkers.

Colorectal cancer (CRC) is the third most common cancer worldwide. The incidence and mortality rates of CRC are significantly higher in Taiwan than in other developed countries. Genes involved in CRC tumorigenesis differ depending on whether the tumor occurs on the left or right side of the colon, and genomic analysis is a keystone in the study and treatment of CRC subtypes. However, few studies have focused on the genetic landscape of Taiwanese patients with CRC. This study comprehensively analyzed the genomes of 141 Taiwanese patients with CRC through whole-exome sequencing. Significant genomic differences related to the site of CRC development were observed. Blood metabolomic profiling and polygenic risk score analysis were performed to identify potential biomarkers for the early identification and prevention of CRC in the Taiwanese population. Our findings provide vital clues for establishing population-specific treatments and health policies for CRC prevention in Taiwan.

Targeting protein-protein interaction interfaces in COVID-19 drug discovery.

To date, the COVID-19 pandemic has claimed over 1 million human lives, infected another 50 million individuals and wreaked havoc on the global economy. The crisis has spurred the ongoing development of drugs targeting its etiological agent, the SARS-CoV-2. Targeting relevant protein-protein interaction interfaces (PPIIs) is a viable paradigm for the design of antiviral drugs and enriches the targetable chemical space by providing alternative targets for drug discovery. In this review, we will provide a comprehensive overview of the theory, methods and applications of PPII-targeted drug development towards COVID-19 based on recent literature. We will also highlight novel developments, such as the successful use of non-native protein-protein interactions as targets for antiviral drug screening. We hope that this review may serve as an entry point for those interested in applying PPIIs towards COVID-19 drug discovery and speed up drug development against the pandemic.

Multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging.

The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains--the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)--flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the "coupled-allostery" model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.

SLC38A2 Overexpression Induces a Cancer-like Metabolic Profile and Cooperates with SLC1A5 in Pan-cancer Prognosis.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pan-cancer cohort and found that the SLC1A5hi /SLC38A2lo group did not relate to a poor survival rate, whereas the SLC1A5lo /SLC38A2hi group significantly aggravated the lethality. Intriguingly, the SLC1A5hi /SLC38A2hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.

Blood multiomics reveal insights into population clusters with low prevalence of diabetes, dyslipidemia and hypertension.

Diabetes, dyslipidemia and hypertension are important metabolic diseases that impose a great burden on many populations worldwide. However, certain population strata have reduced prevalence for all three diseases, but the underlying mechanisms are poorly understood. We sought to identify the phenotypic, genomic and metabolomic characteristics of the low-prevalence population to gain insights into possible innate non-susceptibility against metabolic diseases. We performed k-means cluster analysis of 16,792 subjects using anthropometric and clinical biochemistry data collected by the Taiwan Biobank. Nuclear magnetic resonance spectra-based metabolome analysis was carried out for 217 subjects with normal body mass index, good exercise habits and healthy lifestyles. We found that the gene APOA5 was significantly associated with reduced prevalence of disease, and lesser associations included the genes HIF1A, LIMA1, LPL, MLXIPL, and TRPC4. Blood plasma of subjects belonging to the low disease prevalence cluster exhibited lowered levels of the GlycA inflammation marker, very low-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, valine and leucine compared to controls. Literature mining revealed that these genes and metabolites are biochemically linked, with the linkage between lipoprotein metabolism and inflammation being particularly prominent. The combination of phenomic, genomic and metabolomic analysis may also be applied towards the study of metabolic disease prevalence in other populations.

Structure-Based Stabilization of Non-native Protein-Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design.

Structure-based stabilization of protein-protein interactions (PPIs) is a promising strategy for drug discovery. However, this approach has mainly focused on the stabilization of native PPIs, and non-native PPIs have received little consideration. Here, we identified a non-native interaction interface on the three-dimensional dimeric structure of the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV N-NTD). The interface formed a conserved hydrophobic cavity suitable for targeted drug screening. By considering the hydrophobic complementarity during the virtual screening step, we identified 5-benzyloxygramine as a new N protein PPI orthosteric stabilizer that exhibits both antiviral and N-NTD protein-stabilizing activities. X-ray crystallography and small-angle X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD dimers through simultaneous hydrophobic interactions with both partners, resulting in abnormal N protein oligomerization that was further confirmed in the cell. This unique approach based on the identification and stabilization of non-native PPIs of N protein could be applied toward drug discovery against CoV diseases.

Thermostability of the N-terminal RNA-binding domain of the SARS-CoV nucleocapsid protein: experiments and numerical simulations.

Differential scanning calorimetry, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and numerical simulations were used to study the thermostability of the N-terminal RNA-binding domain (RBD) of the SARS-CoV nucleocapsid protein. The transition temperature of the RBD in a mixing buffer, composed of glycine, sodium acetate, and sodium phosphate with 100 mM sodium chloride, at pH 6.8, determined by differential scanning calorimetry and circular dichroism, is 48.74 degrees C. Experimental results showed that the thermal-induced unfolding-folding transition of the RBD follows a two-state model with a reversibility >90%. Using a simple Go-like model and Langevin dynamics we have shown that, in agreement with our experiments, the folding of the RBD is two-state. Theoretical estimates of thermodynamic quantities are in reasonable agreement with the experiments. Folding and thermal unfolding pathways of the RBD also were experimentally and numerically studied in detail. It was shown that the strand beta(1) from the N-terminal folds last and unfolds first, while the remaining beta-strands fold/unfold cooperatively.

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA.

Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248-365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248-280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248-280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.

Recent insights into the development of therapeutics against coronavirus diseases by targeting N protein.

The advent of severe acute respiratory syndrome (SARS) in the 21st century and the recent outbreak of Middle-East respiratory syndrome (MERS) highlight the importance of coronaviruses (CoVs) as human pathogens, emphasizing the need for development of novel antiviral strategies to combat acute respiratory infections caused by CoVs. Recent studies suggest that nucleocapsid (N) proteins from coronaviruses and other viruses can be useful antiviral drug targets against viral infections. This review aims to provide readers with a concise survey of the structural features of coronavirus N proteins and how these features provide insights into structure-based development of therapeutics against coronaviruses. We will also present our latest results on MERS-CoV N protein and its potential as an antiviral drug target.

Structure-based virtual screening and experimental validation of the discovery of inhibitors targeted towards the human coronavirus nucleocapsid protein.

Nucleocapsid protein (NP), an essential RNA-binding viral protein in human coronavirus (CoV)-infected cells, is required for the replication and transcription of viral RNA. Recent studies suggested that human CoV NP is a valid target for antiviral drug development. Based on this aspect, structure-based virtual screening targeting nucleocapsid protein (NP) was performed to identify good chemical starting points for medicinal chemistry. The present study utilized structure-based virtual screening against human CoV-OC43 using the Zinc database, which is performed through docking with varying precisions and computational intensities to identify eight potential compounds. The chosen potential leads were further validated experimentally using biophysical means. Surface plasmon resonance (SPR) analysis indicated that one among the potential leads, 6-chloro-7-(2-morpholin-4-yl-ethylamino) quinoxaline-5,8-dione (small-compound H3), exhibited a significant decrease of RNA-binding capacity of NP by more than 20%. The loss of binding activity was manifested as a 20% decrease in the minimum on-rate accompanied with a 70% increase in the maximum off-rate. Fluorescence titration and X-ray crystallography studies indicated that H3 antagonizes the binding between HCoV-OC43 NP and RNA by interacting with the N-terminal domain of the NP. Our findings provide insight into the development of new therapeutics that disrupt the interaction between RNA and viral NP in the HCoV. The discovery of the new compound would be an impetus to design novel NP inhibitors against human CoV.

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure.

We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five alpha helices and a beta-hairpin. The dimer interface consists of a continuous four-stranded beta-sheet superposed by two long alpha helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the beta-sheet and the alpha helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.