ACS Chemical Biology最新文献

Developing novel nonribosomal peptides (NRPs) requires a comprehensive understanding of the enzymes involved in their biosynthesis, particularly the substrate amino acid recognition mechanisms in the adenylation (A) domain. This study focused on the A domain responsible for adenylating l-2,4-diaminobutyric acid (l-Dab) within the synthetase of polymyxin, an NRP produced by Paenibacillus polymyxa NBRC3020. To date, investigations into recombinant proteins that selectively adenylate l-Dab─exploring substrate specificity and enzymatic activity parameters─have been limited to reports on A domains found in enzymes synthesizing l-Dab homopolymers (pldA from S. celluloflavus USE31 and pddA from S. hindustanus NBRC15115), which remain exceedingly rare. The polymyxin synthetase in NBRC3020 contains five A domains specific to l-Dab, distributed across five distinct modules (modules 1, 3, 4, 5, 8, and 9). In this study, we successfully obtained soluble A domain proteins from modules 1, 5, 8, and 9 by preparing module-specific recombinant proteins. These proteins were expressed in E. coli BAP-1, purified via Ni-affinity chromatography, and demonstrated high specificity for l-Dab. Through sequence homology analysis, three-dimensional structural modeling, docking simulations to estimate substrate-binding sites, and functional validation using alanine mutants, we identified Glu281 and Asp344 as critical residues for recognizing the side chain amino group of l-Dab, and Asp238 as essential for recognizing its main chain amino group in the A domain. Notably, these key residues were conserved not only across the A domains in modules 1, 5, 8, and 9 of P. polymyxa NBRC3020 but also in those of the P. polymyxa PKB1 strain, as confirmed by sequence homology analysis. Interestingly, in pldA and pddA, the key residues involved in recognizing the side-chain amino group of l-Dab, which are conserved among polymyxin synthetases of NBRC3020 and PKB1 strain, were not observed. This suggests a potentially different mechanism for l-Dab recognition.

OaPAC, the photoactivated adenylyl cyclase from Oscillatoria acuminata, is composed of a blue light using FAD (BLUF) domain fused to an adenylate cyclase (AC) domain. Since both the BLUF and AC domains are part of the same protein, OaPAC is a model for understanding how the ultrafast modulation of the chromophore binding pocket caused by photoexcitation results in the activation of the output domain on the μs-s time scale. In the present work, we use unnatural amino acid mutagenesis to identify specific sites in the protein that are involved in transducing the signal from the FAD binding site to the ATP binding site. To provide insight into site-specific structural dynamics, we replaced W90 which is close to the chromophore pocket, F103 which interacts with W90 across the dimer interface, and F180 in the central core of the AC domain, with the infrared probe azido-Phe (AzPhe). Using ultrafast IR, we show that AzPhe at position 90 responds on multiple time scales following photoexcitation. In contrast, the light minus dark IR spectrum of AzPhe103 shows only a minor perturbation in environment between the dark and light states, while replacement of F180 with AzPhe resulted in a protein with no catalytic activity. We also replaced Y125, which hydrogen bonds with N256 across the dimer interface, with fluoro-Tyr residues. All the fluoro-Tyr substituted proteins retained the light-induced red shift in the flavin absorption spectrum; however, only the 3-FY125 OaPAC retained photoinduced catalytic activity. The loss of activity in 3,5-F₂Y125 and 2,3,5-F₃Y125 OaPAC, which potentially increase the acidity of the Y125 phenol by more than 1000-fold, suggests that deprotonation of Y125 disrupts the signal transduction pathway from the BLUF to the AC domain.

The National Institutes of Health molecular probe ML283 was synthesized as a potent, selective inhibitor of the helicase encoded by the hepatitis C virus. Because modeling with AutoDock Vina predicted that ML283 might bind the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 13 (nsp13) helicase, the effects of a collection of ML283 analogs and other hepatitis C virus (HCV) helicase inhibitors on the SARS-CoV-2 helicase were analyzed. Only modest impacts on nsp13-catalyzed ATP hydrolyses were observed with some compounds, most of which were analogs of the drug ebselen, not ML283. In contrast, a new molecular-beacon-based helicase assay revealed that ML283 and many ML283 analogs are potent SARS-CoV-2 helicase inhibitors. Analog potencies correlate with the binding energies predicted by modeling, which suggests that a pocket surrounded by the carboxy-terminal nsp13 RecA-like helicase motor domain might be exploitable for antiviral drug development.

RNA interference (RNAi) has rapidly matured as a novel therapeutic approach. In this field, chemical modifications have been critical to the clinical success of short interfering RNAs (siRNAs). Notwithstanding the significant advances, achieving robust durability and gene silencing in extrahepatic tissues, as well as reducing off-target effects of siRNA, are areas where chemical modifications can still improve siRNA performance. The present study developed the challenging synthesis of amide-linked guanosine dimers (G_AM1G and G_AM1A) and completed an "amide walk" one by one, systematically replacing every internucleoside phosphate with an amide linkage in a guide strand targeting the PIK3CB gene. Dual-luciferase and RT-qPCR assays in HeLa cells showed that, in a model system of unmodified siRNAs, the amide linkage at position 3 (between nucleosides 3 and 4) suppressed the cleavage of off-target YY1 and FADD mRNAs similarly to the industry gold standard modification glycol nucleic acid (GNA). These results suggest that amide linkages in the seed region have strong potential to improve the specificity of siRNAs by suppressing the microRNA-like off-target activity.

Since the middle of the 20th century, long-distance avian migration has been known to rely partly on geomagnetic field. However, the underlying sensory mechanism is still not fully understood. Cryptochrome-4a (ErCry4a), found in European robin (Erithacus rubecula), a night-migratory songbird, has been suggested to be a magnetic sensory molecule. It is sensitive to external magnetic fields via the so-called radical-pair mechanism. ErCry4a is primarily located in the outer segments of the double-cone photoreceptor cells in the eye, which contain stacked and highly ordered membranes that could facilitate the anisotropic attachment of ErCry4a needed for magnetic compass sensing. Here, we investigate possible interactions of ErCry4a with a model membrane that mimics the lipid composition of outer segments of vertebrate photoreceptor cells using experimental and computational approaches. Experimental results show that the attachment of ErCry4a to the membrane could be controlled by the physical state of lipid molecules (average area per lipid) in the outer leaflet of the lipid bilayer. Furthermore, polarization modulation infrared reflection absorption spectroscopy allowed us to determine the conformation, motional freedom, and average orientation of the α-helices in ErCry4a in a membrane-associated state. Atomistic molecular dynamics studies supported the experimental results. A ∼ 1000 kcal mol^-1 decrease in the interaction energy as a result of ErCry4a membrane binding was determined compared to cases where no protein binding to the membrane occurred. At the molecular level, the binding seems to involve negatively charged carboxylate groups of the phosphoserine lipids and the C-terminal residues of ErCry4a. Our study reveals a potential direct interaction of ErCry4a with the lipid membrane and discusses how this binding could be an essential step for ErCry4a to propagate a magnetic signal further and thus fulfill a role as a magnetoreceptor.

Dimethyl fumarate (DMF) is an established oral therapy for multiple sclerosis worldwide. Although the clinical efficacy of these fumarate esters has been extensively investigated, the mode of action and pharmacokinetics of fumarates have not been fully elucidated due to their broad-spectrum reactivity and complex metabolism in vivo. To better understand the mechanism of action of DMF and its active metabolite, monomethyl fumarate (MMF), we designed and utilized clickable probes to visualize and enrich probe-modified proteins. We further perform quantitative chemoproteomics analysis for proteome-wide target identification and validate several unique and shared targets of DMF and MMF, which provide insight into the reactivity, selectivity, and target engagement of fumarates.

Protein interactions play a crucial role in regulating cellular mechanisms, highlighting the need for effective methods to control these processes. In this regard, chemical inducers of proximity (CIPs) offer a promising approach to precisely manipulate protein-protein interactions in live cells and in vivo. In this study, we introduce pMandi, a photocaged version of the plant hormone-based CIP mandipropamid (Mandi), which allows the use of light as an external trigger to induce protein proximity in live mammalian cells. Furthermore, we present opabactin (OP) as a new plant hormone-based CIP that is effective in live mammalian cells at low nanomolar concentration and in live medaka embryos at submicromolar concentration. Its photocaged derivative, pOP, enables the induction of protein proximity upon light exposure in individual cells, enhancing spatiotemporal control to the level of single-cell resolution. Additionally, we explored the use of both photocaged CIPs to promote protein proximity in live medaka embryos.

We present versatile tools for intersectional optical and chemical tagging of live cells. Photocaged tetrazines serve as "photo-click" adapters between recognition groups on the cell surface and diverse chemical payloads. We describe two new functionalized photocaged tetrazine structures which add a light-gating step to three common cell-targeting chemical methods: HaloTag/chloroalkane labeling, nonspecific primary amine labeling, and antibody labeling. We demonstrate light-gated versions of these three techniques in live cultured cells. We then explore two applications: monitoring tissue flows on the surface of developing zebrafish embryos, and combinatorial multicolor labeling and sorting of optically defined groups of cells. Photoclick adapters add optical control to cell tagging schemes, with modularity in both tag and cell attachment chemistry.

Carbohydrate sulfation plays a pivotal role in modulating the strength of Siglec-glycan interactions. Recently, new aspects of Siglec binding to sulfated cell surface carbohydrates have been discovered, but the class of glycan presenting these sulfated Siglec ligands has not been fully elucidated. In this study, the contribution of different classes of glycans to cis and trans Siglec ligands was investigated within cells expressing the carbohydrate sulfotransferase 1 (CHST1) or CHST2. For some Siglecs, the glycan class mediating binding was clear, such as O-glycans for Siglec-7 and N-glycans for Siglec-2 and Siglec-9. Both N-glycans and mucin-type O-glycans contributed to ligands for Siglec-3, -5, -8, and -15. However, significant levels of Siglec-3 and -8 ligands remained in CHST1-expressing cells lacking complex N-glycans and mucin-type O-glycans. A combination of genetic, pharmacological, and enzymatic treatment strategies ruled out heparan sulfates and glycoRNA as contributors, although Siglec-8 did exhibit some binding to glycolipids. Genetic disruption of O-mannose glycans within CHST1-expressing cells had a small but significant impact on Siglec-3 and -8 binding, demonstrating that this class of glycans can present sulfated Siglec ligands. We also investigated the ability of sulfated cis ligands to mask Siglec-3 and Siglec-7. For Siglec-7, cis ligands were again found to be mucin-type O-glycans. While N-glycans were the major sulfated trans ligands for Siglec-3, disruption of complex mucin-type O-glycans had the largest impact on Siglec-3 masking. Overall, this study enhances our knowledge of the types of sulfated glycans that can serve as Siglec ligands.

Rhynchosporium commune, the causal agent of barley scald disease, poses a major threat to global barley production. Despite its significant impact, the molecular mechanisms underlying R. commune's infection process remain largely unexplored. To address this, we analyzed the differential gene expression data of R. commune WAI453 cultivated under both in planta and in vitro conditions, aiming to identify secondary metabolite biosynthetic gene clusters that are potentially involved in the pathogenicity of R. commune. Our analysis revealed increased expression of a polyketide-terpene gene cluster (the rhy cluster), containing a specific myeloblastosis (MYB)-type transcription factor gene rhyM, during in planta growth. Overexpression of rhyM in an axenic culture activated the expression of the rhy cluster, resulting in the production of a series of new meroterpenoid metabolites, which we named rhynchospenes A-E. Their structures were elucidated through a combination of spectroscopic methods and single crystal X-ray diffraction analysis. Infiltration of rhynchospenes into barley leaves resulted in strong necrosis, with rhynchospene B demonstrating the highest phytotoxicity and causing necrosis at a minimum concentration of 50 ppm. Silencing rhyM in R. commune WAI453 confirmed the role of rhynchospenes as virulence factors in barley disease. The resulting mutant showed significantly reduced expression of the rhy cluster in planta compared to the wild-type strain and decreased virulence in seedling pathogenicity assays on barley. The characterization of the rhy cluster and rhynchospenes provided insights into the role of secondary metabolites in R. commune virulence and barley scald disease development. The study also highlights the potential use of MYB-type transcription factor overexpression in uncovering cryptic SMs involved in pathogenicity and host adaptations.