Structure of an RNA G-quadruplex from the West Nile virus genome.

Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes.

Dissection of integrated readout reveals the structural thermodynamics of DNA selection by transcription factors.

Nucleobases such as inosine have been extensively utilized to map direct contacts by proteins in the DNA groove. Their deployment as targeted probes of dynamics and hydration, which are dominant thermodynamic drivers of affinity and specificity, has been limited by a paucity of suitable experimental models. We report a joint crystallographic, thermodynamic, and computational study of the bidentate complex of the arginine side chain with a Watson-Crick guanine (Arg×GC), a highly specific configuration adopted by major transcription factors throughout the eukaryotic branches in the Tree of Life. Using the ETS-family factor PU.1 as a high-resolution structural framework, inosine substitution for guanine resulted in a sharp dissection of conformational dynamics and hydration and elucidated their role in the DNA specificity of PU.1. Our work suggests an under-exploited utility of modified nucleobases in untangling the structural thermodynamics of interactions, such as the Arg×GC motif, where direct and indirect readout are tightly integrated.

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy.

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.

Self-Consistent Parameterization of DNA Residues for the Non-Polarizable AMBER Force Fields.

Fixed-charge (non-polarizable) forcefields are accurate and computationally efficient tools for modeling the molecular dynamics of nucleic acid polymers, particularly DNA, well into the µs timescale. The continued utility of these forcefields depends in part on expanding the residue set in step with advancing nucleic acid chemistry and biology. A key step in parameterizing new residues is charge derivation which is self-consistent with the existing residues. As atomic charges are derived by fitting against molecular electrostatic potentials, appropriate structural models are critical. Benchmarking against the existing charge set used in current AMBER nucleic acid forcefields, we report that quantum mechanical models of deoxynucleosides, even at a high level of theory, are not optimal structures for charge derivation. Instead, structures from molecular mechanics minimization yield charges with up to 6-fold lower RMS deviation from the published values, due to the choice of such an approach in the derivation of the original charge set. We present a contemporary protocol for rendering self-consistent charges as well as optimized charges for a panel of nine non-canonical residues that will permit comparison with literature as well as studying the dynamics of novel DNA polymers.

A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

Intrinsic disorder controls two functionally distinct dimers of the master transcription factor PU.1.

Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.

Neutrophils suppress tumor-infiltrating T cells in colon cancer via matrix metalloproteinase-mediated activation of TGFß.

High T-cell infiltration in colorectal cancer (CRC) correlates with a favorable disease outcome and immunotherapy response. This, however, is only observed in a small subset of CRC patients. A better understanding of the factors influencing tumor T-cell responses in CRC could inspire novel therapeutic approaches to achieve broader immunotherapy responsiveness. Here, we investigated T cell-suppressive properties of different myeloid cell types in an inducible colon tumor mouse model. The most potent inhibitors of T-cell activity were tumor-infiltrating neutrophils. Gene expression analysis and combined in vitro and in vivo tests indicated that T-cell suppression is mediated by neutrophil-secreted metalloproteinase activation of latent TGFß. CRC patient neutrophils similarly suppressed T cells via TGFß in vitro, and public gene expression datasets suggested that T-cell activity is lowest in CRCs with combined neutrophil infiltration and TGFß activation. Thus, the interaction of neutrophils with a TGFß-rich tumor microenvironment may represent a conserved immunosuppressive mechanism in CRC.

Second Generation G-Quadruplex Stabilizing Trimethine Cyanines.

G-Quadruplex DNA has been recognized as a highly appealing target for the development of new selective chemotherapeutics, which could result in markedly reduced toxicity toward normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex structures without targeting duplex DNA have attracted attention due to their high amenability to structural modifications that allows fine-tuning of their biomolecular interactions. We have previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind G-quadruplexes through end stacking interactions. Herein, we are reporting a second generation of drug candidates, the asymmetric trimethine cyanines. These have been synthesized and evaluated for their quadruplex binding properties. Incorporating a benz[c,d]indolenine heterocyclic unit increased overall quadruplex binding, and elongating the alkyl length increases the quadruplex-to-duplex binding specificity.

NMR Structure Determination for Oligonucleotides.

NMR spectroscopy is a versatile tool for determining the structure and dynamics of nucleic acids under solution conditions. In this unit, we provide an overview and detail of the experiments and methods used in our laboratory to determine the structure of oligonucleotides at natural abundance, thus limiting our approach to 1 H, 13 C, and 31 P NMR techniques. Isotopic labeling is heavily used in RNA NMR studies, however, labeling of DNA is still less common and, if modified nucleotides are investigated, is exceptionally expensive or not feasible. Each method described here is extensively documented and annotated with tips and observations to facilitate their application. Sections are devoted to sample preparation, NMR experiments and setup, resonance assignment, structure generation protocols, evaluation, tips that may be useful, and software sources. © 2018 by John Wiley & Sons, Inc.

First Structure of a Designed Minor Groove Binding Heterocyclic Cation that Specifically Recognizes Mixed DNA Base Pair Sequences.

The high-resolution NMR structure of the first heterocyclic, non-amide, organic cation that strongly and selectively recognizes mixed AT/GC bp (bp=base pair) sequences of DNA in a 1:1 complex is described. Compound designs of this type provide essential methods for control of functional, non-genomic DNA sequences and have broad cell uptake capability, based on studies from animals to humans. The high-resolution structural studies described in this report are essential for understanding the molecular basis for the sequence-specific binding as well as for new ideas for additional compound designs for sequence-specific recognition. The molecular features, in this report, explain the mechanism of recognition of both A⋅T and G⋅C bps and are an interesting molecular recognition story. Examination of the experimental structure and the NMR restrained molecular dynamics model suggests that recognition of the G⋅C base pair involves two specific H-bonds. The structure illustrates a wealth of information on different DNA interactions and illustrates an interfacial water molecule that is a key component of the complex.

Multiple DNA-binding modes for the ETS family transcription factor PU.1.

The eponymous DNA-binding domain of ETS (E26 transformation-specific) transcription factors binds a single sequence-specific site as a monomer over a single helical turn. Following our previous observation by titration calorimetry that the ETS member PU.1 dimerizes sequentially at a single sequence-specific DNA-binding site to form a 2:1 complex, we have carried out an extensive spectroscopic and biochemical characterization of site-specific PU.1 ETS complexes. Whereas 10 bp of DNA was sufficient to support PU.1 binding as a monomer, additional flanking bases were required to invoke sequential dimerization of the bound protein. NMR spectroscopy revealed a marked loss of signal intensity in the 2:1 complex, and mutational analysis implicated the distal surface away from the bound DNA as the dimerization interface. Hydroxyl radical DNA footprinting indicated that the site-specifically bound PU.1 dimers occupied an extended DNA interface downstream from the 5'-GGAA-3' core consensus relative to its 1:1 counterpart, thus explaining the apparent site size requirement for sequential dimerization. The site-specifically bound PU.1 dimer resisted competition from nonspecific DNA and showed affinities similar to other functionally significant PU.1 interactions. As sequential dimerization did not occur with the ETS domain of Ets-1, a close structural homolog of PU.1, 2:1 complex formation may represent an alternative autoinhibitory mechanism in the ETS family at the protein-DNA level.

Spectral and Hydrodynamic Analysis of West Nile Virus RNA-Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge.

Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein-RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable.

Structural Impact of Single Ribonucleotide Residues in DNA.

Single ribonucleotide intrusions represent the most common nonstandard nucleotide type found incorporated in genomic DNA, yet little is known of their structural impact. This lesion incurs genomic instability in addition to affecting the physical properties of the DNA. To probe for structural and dynamic effects of single ribonucleotides in various sequence contexts-AxC, CxG, and GxC, where x=rG or dG-we report the structures of three single-ribonucleotide-containing DNA duplexes and the corresponding DNA controls. The lesion subtly and locally perturbs the structure asymmetrically on the 3' side of the lesion in both the riboguanosine-containing and the complementary strand of the duplex. The perturbations are mainly restricted to the sugar and phosphodiester backbone. The ribose and 3'-downstream deoxyribose units are predominately in N-type conformation; backbone torsion angles ϵ and/or ζ of the ribonucleotide or upstream deoxyribonucleotide are affected. Depending on the flanking sequences, the C2'-OH group forms hydrogen bonds with the backbone, 3'-neighboring base, and/or sugar. Interestingly, even in similar purine-rG-pyrimidine environments (A-rG-C and G-rG-C), a riboguanosine unit affects DNA in a distinct manner and manifests different hydrogen bonds, which makes generalizations difficult.

Using NMR and molecular dynamics to link structure and dynamics effects of the universal base 8-aza, 7-deaza, N8 linked adenosine analog.

A truly universal nucleobase enables a host of novel applications such as simplified templates for PCR primers, randomized sequencing and DNA based devices. A universal base must pair indiscriminately to each of the canonical bases with little or preferably no destabilization of the overall duplex. In reality, many candidates either destabilize the duplex or do not base pair indiscriminatingly. The novel base 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin- 4-amine) N8-(2'deoxyribonucleoside), a deoxyadenosine analog (UB), pairs with each of the natural DNA bases with little sequence preference. We have utilized NMR complemented with molecular dynamic calculations to characterize the structure and dynamics of a UB incorporated into a DNA duplex. The UB participates in base stacking with little to no perturbation of the local structure yet forms an unusual base pair that samples multiple conformations. These local dynamics result in the complete disappearance of a single UB proton resonance under native conditions. Accommodation of the UB is additionally stabilized via heightened backbone conformational sampling. NMR combined with various computational techniques has allowed for a comprehensive characterization of both structural and dynamic effects of the UB in a DNA duplex and underlines that the UB as a strong candidate for universal base applications.

Imino proton NMR guides the reprogramming of A•T specific minor groove binders for mixed base pair recognition.

Sequence-specific binding to DNA is crucial for targeting transcription factor-DNA complexes to modulate gene expression. The heterocyclic diamidine, DB2277, specifically recognizes a single G•C base pair in the minor groove of mixed base pair sequences of the type AAAGTTT. NMR spectroscopy reveals the presence of major and minor species of the bound compound. To understand the principles that determine the binding affinity and orientation in mixed sequences of DNA, over thirty DNA hairpin substrates were examined by NMR and thermal melting. The NMR exchange dynamics between major and minor species shows that the exchange is much faster than compound dissociation determined from biosensor-surface plasmon resonance. Extensive modifications of DNA sequences resulted in a unique DNA sequence with binding site AAGATA that binds DB2277 in a single orientation. A molecular docking result agrees with the model representing rapid flipping of DB2277 between major and minor species. Imino spectral analysis of a (15)N-labeled central G clearly shows the crucial role of the exocyclic amino group of G in sequence-specific recognition. Our results suggest that this approach can be expanded to additional modules for recognition of more sequence-specific DNA complexes. This approach provides substantial information about the sequence-specific, highly efficient, dynamic nature of minor groove binding agents.

Effect of methylation on the side-chain pKa value of arginine.

Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pKa values of methylated arginine variants were determined using NMR data. The pKa values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pKa value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition.

Defective Myb Function Ablates Cyclin E1 Expression and Perturbs Intestinal Carcinogenesis.

UNLABELLED: Cyclin E1 is essential for the reentry of quiescent cells into the cell cycle. When hypomorphic mutant Myb mice (Myb(Plt4)) were examined, it was noted that Cyclin E1 (Ccne1) expression was reduced. Furthermore, the induction of Ccne1 in recovering intestinal epithelia following radiation-induced damage was ablated in Myb-mutant mice. These data prompted us to investigate whether Myb directly regulated Ccne1 and to examine whether elevated Myb in colorectal cancer is responsible for Cyclin E1-driven tumor growth. Here, it was found that Myb/MYB and Ccne1/CCNE1 expressions were coupled in both mouse and human adenomas. In addition, the low molecular weight Cyclin E1 was the predominant form in intestinal crypts and adenomatous polyposis coli (Apc)-mutant adenomas. Chromatin immunoprecipitation (ChIP) analysis confirmed that Myb bound directly to the Ccne1 promoter and regulated its endogenous expression. In contrast, Myb(Plt4) served as a dominant-negative factor that inhibited wild-type Myb and this was not apparently compensated for by the transcription factor E2F1 in intestinal epithelial cells. Myb(Plt4/Plt4) mice died prematurely on an Apc(Min/) (+) background associated with hematopoietic defects, including a myelodysplasia; nevertheless, Apc(Min/) (+) mice were protected from intestinal tumorigenesis when crossed to Myb(Plt4/) (+) mice. Knockdown of CCNE1 transcript in murine colorectal cancer cells stabilized chromosome ploidy and decreased tumor formation. These data suggest that Cyclin E1 expression is Myb dependent in normal and transformed intestinal epithelial cells, consistent with a cell-cycle progression and chromosome instability role in cancer. IMPLICATIONS: This study demonstrates that Myb regulates Cyclin E1 expression in normal gastrointestinal tract epithelial cells and is required during intestinal tumorigenesis.

Peritoneal Tumorigenesis and Inflammation are Ameliorated by Humidified-Warm Carbon Dioxide Insufflation in the Mouse.

BACKGROUND: Conventional laparoscopic surgery uses CO2 that is dry and cold, which can damage peritoneal surfaces. It is speculated that disseminated cancer cells may adhere to such damaged peritoneum and metastasize. We hypothesized that insufflation using humidified-warm CO2, which has been shown to reduce mesothelial damage, will also ameliorate peritoneal inflammation and tumor cell implantation compared to conventional dry-cold CO2. METHODS: Laparoscopic insufflation was modeled in mice along with anesthesia and ventilation. Entry and exit ports were introduced to maintain insufflation using dry-cold or humidified-warm CO2 with a constant flow and pressure for 1 h; then 1000 or 1 million fluorescent-tagged murine colorectal cancer cells (CT26) were delivered into the peritoneal cavity. The peritoneum was collected at intervals up to 10 days after the procedure to measure inflammation, mesothelial damage, and tumor burden using fluorescent detection, immunohistochemistry, and scanning electron microscopy. RESULTS: Rapid temperature control was achieved only in the humidified-warm group. Port-site tumors were present in all mice. At 10 days, significantly fewer tumors on the peritoneum were counted in mice insufflated with humidified-warm compared to dry-cold CO2 (p < 0.03). The inflammatory marker COX-2 was significantly increased in the dry-cold compared to the humidified-warm cohort (p < 0.01), while VEGFA expression was suppressed only in the humidified-warm cohort. Significantly less mesothelial damage and tumor cell implantation was evident from 2 h after the procedure in the humidified-warm cohort. CONCLUSIONS: Mesothelial cell damage and inflammation are reduced by using humidified-warm CO2 for laparoscopic oncologic surgery and may translate to reduce patients' risk of developing peritoneal metastasis.