ACS Bio & Med Chem Au

RNA helicases encoded by positive-strand RNA viruses are essential for genome replication, yet the specific biological functions and mechanochemical basis underlying these functions remain poorly defined. Progress has been limited by the difficulty of resolving individual catalytic steps under single-turnover conditions, which are often experimentally inaccessible for viral enzymes. Alphaviruses replicate within membrane-bound spherules that may alter local metabolite concentrations, raising the possibility that the enzymatic properties of alphaviral proteins differ from those of viruses with greater cytosolic exposure. Here, we present a kinetic and binding analysis of full-length non-structural protein 2 (nsP2) from Chikungunya virus, a multifunctional superfamily 1B NTPase and RNA helicase. Purified nsP2 binds nucleoside triphosphates with high affinity, exhibiting equilibrium dissociation constants in the single digit micromolar range. This property enabled single-turnover, pre-steady-state, and isotope-trapping experiments that are rarely feasible for viral helicases. These analyses identified two sequential conformational-change steps required for nucleotide hydrolysis. Molecular dynamics simulations suggest tightening of the RecA1 and RecA2 domains upon ATP binding followed by compaction of the enzyme mediated by interactions between the 1B subdomain and RecA2 domain. Product inhibition patterns support random release of ADP and inorganic phosphate, with relative binding affinities indicating that ADP dissociates first. The reaction is irreversible. Although nsP2 binds RNA tightly, strand separation under single-turnover conditions is too slow to represent ATP-driven unwinding, instead likely reflecting formation of an unwinding-competent nsP2-RNA complex. Together, these findings establish a quantitative framework for nsP2 function and provide a roadmap for mechanistic studies of alphaviral helicases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/723793v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@13899a1org.highwire.dtl.DTLVardef@ee1aadorg.highwire.dtl.DTLVardef@1991e1org.highwire.dtl.DTLVardef@b877f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Targeted protein degradation using conditional degron tag (CDT) technology is a powerful method for rapidly degrading a protein of interest (POI) upon the addition of a degrader drug. A prerequisite for the temporally controlled degradation of an endogenous POI is the generation of homozygous knock-in cells with the degron tag integrated at either the N- or C-terminus of their gene loci. However, obtaining those homozygous knock-in cells often requires selecting many single-cell clones, as human cells typically exhibit low homology-directed repair (HDR) activities. Additionally, tagging a degron to an endogenous protein may inadvertently reduce protein expression, potentially affecting protein function even before the drug is administered. Here, we develop a method for generating degron-tagged knock-in cells that allows us to skip the laborious single-cell cloning. This method arose from our observation that most knock-in cells carry the degron tag only in one allele (heterozygous), while the other allele typically harbors a frameshift insertion/deletion. This observation allowed us to bypass the need for single-cell cloning. We validated our method by knocking in degron tags at the N-terminus of cytoplasmic dynein1 subunits or Adaptor Protein 2 (AP2) subunit. Our experiments confirmed the rapid degradation of these proteins and their functional inhibition in bulk cell populations. Additionally, to mitigate the reduced expression often associated with the degron tagging, we established a method to control expression levels by inserting a mini-promoter immediately upstream of the knock-in cassette. Our method simplifies the workflow for degron tag knock-ins and enhances the versatility of these valuable technologies.

The nuclear receptor superfamily is comprised of ligand-regulated transcription factors that contain an intrinsically disordered domain at the amino-terminal end, known as the N-terminal domain (NTD). While this poorly conserved domain is known to possess ligand-independent activation function (AF-1), few NTD functions are conserved between nuclear receptors (NRs). Identified roles in other receptors include androgen receptor (AR), estrogen receptor (ER) and mineralocorticoid receptor (MR). Here, we aim to define the function of the NTD of the farnesoid X receptor (FXR), a crucial regulator of lipid and bile acid metabolism. We show that the NTD engages in interdomain contact with other FXR domains. We also observe that the NTD interacts directly with coregulator proteins. Using mutagenesis, mammalian two-hybrid assays and molecular dynamics simulations, we identify and validate a novel SXXLF motif in the NTD which mediates interactions with both coregulators and the ligand binding domain. Mutation of the motif induces large changes in conformational and allosteric coupling in FXR. Our study identifies a new nuclear receptor-interacting motif that modulates the transcriptional activity of FXR. Graphical AbstractFXR-NTD regulates transcriptional activity through interdomain communication with the LBD and is also involved in co-activator recruitment. The SENLF motif is the first defined functional element within the FXR-NTD and mediates both NTD-LBD interaction and selective co-activator engagements to drive NTD-mediated transcriptional activity. O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/724725v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5a37aorg.highwire.dtl.DTLVardef@2fa9e1org.highwire.dtl.DTLVardef@13a19daorg.highwire.dtl.DTLVardef@1775ed2_HPS_FORMAT_FIGEXP M_FIG C_FIG

The repair of photo-induced DNA lesions through nucleotide excision repair machinery is still the source of important questions. It has been observed that the repair rate of the different cyclobutane pyrimidine dimers, i.e. the photoproducts induced by dimerization of two {pi}-stacked pyrimidines (T<>T, T<>C, C<>T, C<>C), depends on the nucleobases involved in the lesion. TT derivatives (T<>T) are removed more slowly than those containing cytosine, especially in 5. Using all-atom molecular dynamics simulations and free-energy calculations, we demonstrate that the variation of the repair rate observed in human skin and in cultured cutaneous cell is associated to the recognition of the four lesions by the DDB2 protein moiety, and more specifically by the differential structural deformation induced on the complementary strand. Indeed, while C<>C and C<>T induce a larger deviation on the groove parameters, T<>T and T<>C, instead, affect DNA structure to a lesser extent. less affected. These effects then hamper differentially the downstream recruitment of the repair complexes. The observed DNA deformation correlates with the experimental repair rate and provides a structural rationale for the different repair rates of CPD by nucleotide excision repair machinery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/724087v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@cf6b6dorg.highwire.dtl.DTLVardef@195e35forg.highwire.dtl.DTLVardef@1829296org.highwire.dtl.DTLVardef@165baba_HPS_FORMAT_FIGEXP M_FIG C_FIG

The self-labeling protein HaloTag is used to install a wide variety of functional small molecules in cells and living organisms with exquisite specificity with respect to cell type and subcellular localization. HaloTag is a core part of many biotechnology-based tools for sensing, tracking, and manipulating biological systems with a high degree of spatial and temporal control. Due to the limitations of fluorescent proteins and other self-labeling proteins, most of these tools have historically been restricted to a single channel. In this work, we used structure-guided rational design and directed evolution to produce an orthogonal HaloTag protein called OrthoTag which reacts selectively with a modified chloroalkane substrate. OrthoTag retains many of HaloTags superior properties, and reaction rate measurements show OrthoTag and its substrate have 60-fold mutual orthogonality to HaloTag. We demonstrate the application of OrthoTag for multiplexed labeling experiments in mammalian cells with minimal optimization. Going forward, OrthoTag can be directly incorporated into any HaloTag-based system to allow simultaneous measurement or manipulation of two biological targets or processes. The availability of multiple high-performance self-labeling proteins will enable the continued development of new multiplexed biotechnology methods.

Protein tyrosine kinases are important regulators of cell signaling, and aberrant kinase activity contributes to many human diseases, including cancers. All protein tyrosine kinases share a highly-conserved ATP binding pocket but diverge in their substrate binding sites in order to mediate distinct signaling events. Many potent and efficacious ATP-competitive tyrosine kinase inhibitors have been developed, however it remains challenging to achieve on-target selectivity across different kinases and target specific disease mutants, given the high degree of conservation in the ATP-binding pocket. By contrast, the variable substrate-binding site offers an opportunity for selective inhibition, provided molecules can be targeted to this site. Here, we present a modular strategy to design selective, peptide-based covalent inhibitors of tyrosine kinases with a distinct binding mode from existing ATP-competitive inhibitors. Using Src kinase as a model system, we demonstrate that Src-selective reactivity can be achieved by first designing an optimized substrate peptide and then strategically positioning an electrophile on the peptide to target a non-conserved cysteine on the kinase. We show that substrate-derived covalent peptides can inhibit kinase activity, bind simultaneously with an ATP-competitive inhibitor, and even inhibit the activity of kinases bearing a common drug resistance mutation. We further explore the application of this approach to develop an inhibitor of the cancer-relevant fibroblast growth factor receptor 1 kinase that shows selectivity for an oncogenic mutant over the wild-type enzyme. Our modular strategy to generate selective covalent peptides targeting protein tyrosine kinases provides a promising framework for future chemical probe and drug development efforts.

The influenza virus poses a significant global health threat due to its continuous evolution, immune evasion, and zoonotic spillover. The rise of drug resistance, reduced susceptibility to existing antiviral medications, and the limited effectiveness of annual vaccines underscore the need for new antiviral strategies. To infect, the influenza virus binds to sialic acid (SA)-containing molecules on host cell membranes through hemagglutinin (HA). Blocking this interaction represents a promising antiviral approach. Herein, we report that SA containing plasma membrane-derived vesicles (PMV) efficiently inhibits in vitro Influenza A virus (IAV) infection. Using orthogonal methods, we demonstrate that PMV derived from A549, MDCK, and HEK cells competitively bind to H1N1 (WSN) and H3N2 (X-31) IAV strains, block entry and infection in human respiratory epithelial cells in a dose-dependent manner, without causing significant toxicity. When the size of the vesicles was reduced through extrusion, the antiviral activity was enhanced, and this was found to be correlated with a size-dependent increase in hemagglutination inhibition and reduced IAV internalisation. Plasma membrane-derived vesicles may serve as a novel antiviral strategy against influenza virus infections due to their simple production method and conserved SA binding site on HA.

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

In an infection, Hepatitis B Virus (HBV) core protein (HBc) normally assembles into icosahedral capsids. Capsid Assembly Modulators (CAMs) are direct acting antivirals that induce HBc mis-assembly and are the subject of active research and development. Two versions of HBc are used in structural studies of CAM-HBc complexes: Cp150 and Cp149-Y132A. Cp150 forms empty icosahedral capsids that are structurally indistinguishable from those found in virions. The Y132A mutation of Cp149 leads to an assembly defective soluble protein that crystalizes as flat hexagonal sheets, where the hexagons resemble icosahedral quasi-sixfold vertices. In this study, we compare structures of CAM-bound Cp150 to CAM-bound Cp149-Y132A. In capsids, the residues forming the CAM site shift to match the structure of bound CAMs, an induced fit. In Cp149-Y132A crystals, CAM sites show little structural adjustment in response to different CAMs binding. In turn, the array of residues that interact with CAMs varies from CAM to CAM in capsid structures but remains nearly constant in Cp149-Y132A crystals. These results illustrate important differences between CAM binding in Cp149-Y132A and Cp150 structures that will contribute to future CAM design.

Ligand optimization is central to drug discovery as hundreds of analogs might be designed and synthesized between an initial hit and a therapeutic candidate. The efficiency of this process is unclear, at least partly because there is no random background for optimization against which to compare. Such a random background might emerge from synthetically accessible but otherwise systematic random small substitutions across starting ligands, measuring likelihood of achieving a substantial improvement in affinity/potency or other property by any single perturbation. Recent literature and ligand-affinity/potency databases suggest that perhaps 10% of analogs with minor modifications improve upon a parents potency substantially (by [&ge;]10-fold), but this number is clouded by reporting bias, intentional improvement, and inter-group reproducibility. To begin to establish a background expectation for ligand optimization, we comprehensively and systematically modified 18 lead molecules across six targets with single atom changes; 257 compounds were synthesized. Unexpectedly, 11.2% of these random small perturbation analogs improved potency by [&ge;]10-fold over their parents. Conversely, these more potent analogs typically had worse in vitro pharmacokinetics (e.g. reduced metabolic stability, lower plasma free fraction). While it was possible to find analogs where the potency increase compensated for inferior exposure and half-life, resulting in more potent compounds in vivo, overall a frustrated landscape for ligand optimization is revealed. This study begins to establish a background expectation for ligand potency optimization and offers a simple strategy to do so. It also begins to quantify the challenges confronting the field in moving beyond in vitro potency.

Double-stranded RNA (dsRNA) is recognized by cellular receptors as a sign of viral infection, triggering the innate immune response. Increasing evidence shows that cellular dysregulation, for example in immune disorders and neurodegenerative diseases, can also lead to accumulation of endogenously produced dsRNA that stimulates a viral-like immune response. Additionally, dsRNA contamination in RNA therapeutics can lead to harmful side effects via a similar pathway. Despite the clinical relevance of dsRNA, reliable tools for its detection remain limited. At present, dsRNA detection relies almost exclusively on the monoclonal antibodies J2 and K1, which suffer from sequence bias and low sensitivity, limiting their reliability. To address this challenge, we aimed to repurpose naturally occurring dsRNA-binding domains (dsRBDs) to produce reliable, pan-specific affinity reagents for dsRNA. We first systematically screened the dsRBDs of the three human adenosine deaminases acting on RNA (ADARs). This analysis identified ADAR3 dsRBDs as promising candidates due to their reduced sequence dependence compared to the dsRBDs of ADAR1 and ADAR2. We then engineered ADAR3-derived dsRBD constructs having varying linker lengths and domain combinations, allowing us to specifically vary the length cutoff of dsRNA detected, thus creating dsRNA accumulation detected by ADAR3 RBDs (dsRADAR) affinity reagents. Finally, we demonstrate the superior performance of dsRADAR over currently available dsRNA antibodies in a cell model of viral infection and a tissue model of gastric inflammation. Together, dsRADAR provides a sensitive and reliable approach for imaging and quantifying diverse dsRNA structures in a variety of biological contexts. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/724404v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d89c30org.highwire.dtl.DTLVardef@1f64fc1org.highwire.dtl.DTLVardef@1ee391forg.highwire.dtl.DTLVardef@e834a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Different small-molecule drugs targeting the same protein can produce divergent clinical outcomes through poorly characterized interactome changes. Existing proximity labeling approaches for target identification suffer from background biotinylation independent of small-molecule recruitment, obscuring true drug targets and their binding partners. Here, we incorporate a destabilizing domain (DD) into the biotin targeting chimera (BioTAC) framework to create ddBioTAC, wherein the proximity labeling enzyme TurboID is selectively stabilized only upon binding of a bifunctional targeting molecule. Using the bromodomain-targeting molecule NICE-01 in HeLa cells, we demonstrate that, in the absence of the bifunctional targeting molecule the destabilized TurboID enzyme (TurboID-DD) exhibits reduced protein levels and biotinylation activity compared to the control TurboID-FKBP (FK506-binding protein), while recovering comparable activity upon NICE-01 treatment. This results in an eightfold improvement in specific enrichment of the known target bromodomain containing protein 4 (BRD4) and its interactors, including MED1 and EF1D. Proteome-wide mass spectrometry confirms that ddBioTAC more accurately discriminates drug targets and proximal interactors from non-specific background, advancing unbiased drug-induced interactome profiling.

Rhodoquinone (RQ) is a recently discovered component of the mammalian electron transport chain (ETC) with a high degree of tissue-specificity. Currently, a lack of pure analytical standards limits efforts to precisely quantify its levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and interrogate its biochemical functions within mammalian ETC complexes. Here, rhodoquinone-9 (RQ-9) and rhodoquinone-10 (RQ-10), and their isomeric by-products isorhodoquinone-9 (isoRQ-9) and isorhodoquinone-10 (isoRQ-10), were synthesized from ubiquinone-9 and ubiquinone-10 starting materials. Isomers were separated and purified by flash chromatography and structurally confirmed with nuclear magnetic resonance (NMR) spectroscopy. The chromatographic and fragmentation patterns of both the oxidized and reduced forms of these electron carriers were further characterized by LC-MS/MS, establishing signatures for their confident identification in lipidomics studies. LC-MS/MS analysis of murine kidney tissue with RQ-9 analytical standard spike-in corroborate the identity of the endogenous murine RQ-9 and enable absolute quantification of its levels. Thus, we synthesized and purified RQ-9 and RQ-10 analytical standards that will enable absolute quantification in mammalian tissues and in vitro reconstitution studies on RQ-9 and RQ-10 in the mammalian ETC.

Caulobacter crescentus is a gram-negative bacterium that produces the anionic sphingolipid ceramide diphosphoglycerate that can substitute for the lipopolysaccharide component of the outer membrane. ccna_01210 is a gene in the operon for ceramide diphosphoglycerate synthesis and encodes for the enzyme, CpgD. CpgD is a magnesium-dependent CTP:phosphoglycerate cytidylyltransferase that catalyzes the synthesis of CDP-glycerate, and a pyrophosphate byproduct. CpgD displays substrate specificity for the nucleotide CTP and a preference for 2-phospho-D-glycerate over other phosphoglycerate enantiomers and isomers. Here, we present five high resolution structures for CpgD in various catalytic states that rationalize the specificity and preference of CpgD for its two substrates. This includes structures of apo CpgD, a CpgD-CTP-Mg2+ ternary complex, and three CpgD-CDP-glycerate-pyrophosphate-Mg2+ product bound complexes. The structures reveal CpgD nucleotide specificity is mediated by favorable hydrogen bonding interactions with the cytosine nucleobase of CTP, while the preference for 2-phospho-D-glycerate occurs due to favorable van der Waals interactions with the 2D enantiomer and unfavorable steric clashes with the 2L enantiomer. A catalytic mechanism involving a pentacoordinate transition state is proposed based on the observed stereochemical inversion of the -phosphate in the substrate CTP in comparison to the -phosphate of the product CDP-glycerate. Overall, this provides insights into the catalytic mechanism, nucleotide specificity, and enantiomeric substrate preference of the cytidylyltransferase CpgD that participates in a unique pathway of bacterial sphingolipid synthesis.

Conformationally responsive DNA nanoswitches have previously been developed and validated for a variety of biosensing applications including detection of DNA, microRNA, and viral RNA/DNA. Here we develop new methodology for enhancing the sensitivity of DNA-based sensing by recycling a fixed number of targets for repeated reuse. We achieved target-dependent enzymatic ligation of looped nanoswitches and showed that subsequent removal of target does not affect the ligated loop. Through cyclic annealing, ligation, and target removal, we can linearly control signal amplification up to hundreds of cycles. This method adds an important new capability for low abundance targets without the need for target amplification.

Based on the success in pre-clinical models, methods that reduce sialylation in tumors have progressed to clinical trials, as this improves anti-tumor cellular responses. Immune responses against cancer can also be mediated by soluble, non-cellular mechanisms, such as the complement system. Dysregulation of the complement cascade and hypersialylation are hallmarks found across tumor types. Sialic acids are known to interact with complement proteins. However, the downstream pathways involved in the regulation of the complement cascade when reducing sialylation in tumors remain unclear. Here, using human melanoma cell lines and patient samples, we show that metabolic or enzymatic targeting of sialylation directly increases the activation of the complement, enhancing C3 opsonization of tumor cells and the formation of the membrane attack complex. This is mediated by the classical pathway of the complement system, in line with increased binding of immunoglobulins to tumor cells when sialylation is impaired. Our work positions the complement cascade as a relevant anti-tumor response playing a role when sialylation is targeted for cancer treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/723302v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@8ef68forg.highwire.dtl.DTLVardef@1dd30d4org.highwire.dtl.DTLVardef@b0c9ecorg.highwire.dtl.DTLVardef@98cc49_HPS_FORMAT_FIGEXP M_FIG C_FIG

Thermal proteome profiling (TPP) and its higher-throughput derivative, the proteome integral solubility alteration (PISA) assay, measure changes in protein thermal stability upon ligand binding or other perturbations and have been widely adopted in drug discovery and biomedical research. Though the PISA workflow is straightforward, key parameters, including detergent concentration, methods for removing denatured aggregates, and temperature range selection, vary across studies and can markedly influence assay outcomes. Yet these factors have not been systematically evaluated, limiting rational experimental design and data interpretation. Here, through a combined use of TPP, PISA, tandem mass tag (TMT)-based multiplexing, and computational simulation, we systematically characterize these parameters based on the melting behavior of [~]9,000 proteins. We find that reducing detergent concentration elevates apparent Tm by 1.5-2{degrees}C proteome-wide, and aggregate removal by filtration versus centrifugation further alters measurements. We leverage these observations to optimize PISA then apply the optimized conditions to identify the aminopeptidase NPEPPS as a previously uncharacterized binding partner of angiotensin II, a key vasoactive peptide hormone in blood pressure regulation. Together, this work provides a general framework for assay design and data interpretation, and extends the utility of PISA beyond small molecules to dissecting peptide-protein interactions, an increasingly important modality in drug discovery.

DNA topology is a key regulator of chromatin structure and transcription, yet its direct role in transcription factor recognition remains unclear. Here, we investigate how distinct DNA topological states modulate binding of the Saccharomyces cerevisiae bZIP transcription factor GCN4 using topologically defined plasmids. By combining, complementary biochemical approaches, including Bio-Layer Interferometry applied here for the first time to topology-dependent protein-DNA interactions, we show that DNA supercoiling directly reshapes GCN4-DNA recognition. Positively supercoiled DNA forms more stable and persistent complexes, whereas negatively supercoiled DNA retains greater conformational heterogeneity. To interpret these effects, we performed multiscale molecular simulations. Coarse-grained simulations of plasmids recapitulate the global topology-dependent trends observed experimentally, while matched minicircle models reproduce the same behaviour at the local scale. In strong agreement with experimental data, simulations reveal that DNA topology modulates the conformational ensemble of the GCN4 basic region. Overall, positively supercoiled DNA promotes a more ordered binding mode and localized protein distribution, whereas negatively supercoiled DNA supports increased structural plasticity. These findings identify DNA topology as an active determinant of transcription factor recognition and provide a multiscale framework linking global DNA mechanics to local protein-DNA interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/722604v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@18f8ba9org.highwire.dtl.DTLVardef@11a395dorg.highwire.dtl.DTLVardef@ac093borg.highwire.dtl.DTLVardef@923212_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1