Nature Chemical Biology

Deciphering the ubiquitin code requires homogenous, site-specifically ubiquitylated proteins, yet access to such conjugates remains a major challenge. Existing approaches are often constrained by low yields, harsh reaction conditions, engineered recognition motifs or non-native linkage architectures. Here, we present UbyW (Ubiquitylation by UBE2W), a programmable platform for site-specific ubiquitylation that repurposes the E2 enzyme UBE2W to target genetically encoded isopeptidic neo-N-termini. UbyW enables efficient generation of near-native Ub-protein conjugates across diverse protein substrates, including endogenous ubiquitylation sites within folded domains, and can be implemented through a reconstituted intracellular cascade in Escherichia coli for streamlined high-yield production. The platform further enables installation of chemical functionalities adjacent to the isopeptidic linkage, including photocrosslinkers for capturing modification-dependent interactions. Using programmable probes targeting site-specific ubiquitylation of the small GTPase Ran, we identify USP15 as a cognate deubiquitylase and show that Ran K71 monoubiquitylation disrupts key Ran-cycle interactions.

Acetylation of -tubulin K40 by -tubulin acetyltransferase 1 (ATAT1) using acetyl-coenzyme A (Ac-CoA) marks stable microtubule populations, yet chemical tools to directly measure ATAT1 ligand engagement, inhibit its activity, or visualize ATAT1-mediated modification on intact microtubules remain limited. Through the development of a quantitative binding assay, we uncovered that ATAT1 can bind unnatural cofactors but fails to efficiently use them in acyl-transfer reactions. Structure-guided mutation subsequently yielded ATAT1-L163A, which successfully installed clickable handles at the native -tubulin K40 site of synthetic tubulin peptides, -tubulin, and intact microtubules. Cu(I)-catalyzed azide-alkyne cycloaddition enabled visualization of modified microtubules by in-gel fluorescence and microscopy. Moreover, we report a p11-CoA bisubstrate inhibitor that suppressed both native acetylation and engineered acylation. Together, these tools provide chemically controlled access to ATAT1 activity and a site-verified, clickable K40 modification on intact microtubules.

Metabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced anti-leukemic properties via orthogonal chemical screening. Through multi-omics profiling and genome-scale CRISPR/Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate (PI4P) signaling at the endoplasmic reticulum (ER)-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide. Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide.

We describe an engineered "writer/reader" framework for programming post-translational control into synthetic mammalian signaling proteins. In this approach, a bacterially-derived biotin protein ligase (BirA) was used as a "writer" element for the modification of artificial receptors and transcription factors containing a biotin acceptor peptide (AP) fusion tag. To enable modification events to transmit biochemical information, we designed encodable "reader" modules using sequences from a biotinamide-binding antibody. Proteins fused to reader domains were able to interact with AP-tagged polypeptides in a biotinylation-dependent manner, and control over the timing and extent of these interactions could be modulated through both genetic and chemically-based strategies. Genetic and cell-specific control over AP-reader module interactions was achieved via regulated BirA expression, and the interaction states of both intra-and inter-cellular complexes could be modulated with biotinamide-based and bioorthogonally-functionalized compounds. The utility of this approach was demonstrated by installing post-translational and chemogenetic control into synthetic Notch ("SynNotch")-based systems.

Adaptors serve as hubs to regulate diverse protein complexes in cells. This multitude of functions can complicate the study of adaptors, as their genetic disruption may simultaneously impair the activities of several compositionally distinct complexes (or adaptor complexoforms). Here we describe the chemical proteomic discovery of bicyclopyrrolidine acrylamide stereoprobes that react with cysteine-100 (C100) of the methyltransferase (MT) adaptor TRMT112 in human cells. Curiously, the stereoprobes showed negligible reactivity with uncomplexed recombinant TRMT112, and we found that this interaction was restored excluively in the presence of METTL5, but not other MTs. A co-crystal structure revealed stereoprobe binding to a composite pocket proximal to C100 of TRMT112 that is templated by METTL5 and absent in other TRMT112:MT complexes. Structural rearrangements promoted by stereoprobe binding in turn lead to allosteric agonism of METTL5, thus revealing how covalent ligands targeting a pleiotropic adaptor can confer partner-specific functional effects through reactivity with a single complexoform.

Nitrate functions as a signaling molecule beyond its metabolic intermediate role. Despite progress in plants, the mechanisms underlying mammalian nitrate sensing and signaling remain unclear. The accompanying study identifies Sialin2--a proteolytic fragment of nitrate transporter Sialin--as a mammalian nitrate sensor mediating cellular responses. Here, we demonstrate that nitrate triggers endocytosis, inducing Sialin proteolysis and Sialin2 generation. Nitrate-induced Sialin2 scaffolds Lyn kinase with epidermal growth factor receptor (EGFR) at endosomes, activating phosphatidylinositol 3-kinase (PI3K)-AKT-nitric oxide synthase (NOS) pathway to stimulate localized nitric oxide (NO) production, enhancing angiogenesis and cell survival. In hypertensive rats, nitrate supplementation restores endothelial function and reduces blood pressure through AKT/eNOS-dependent signaling. Unlike the classical nitrate-nitrite-NO pathway, the Sialin2-PI3K-AKT-NOS axis confines NO synthesis to endosomal microdomains, enabling spatiotemporally precise vasodilation. By establishing Sialin2 as a mammalian nitrate sensor, this study unveils a novel paradigm in nitrogen homeostasis and provides targeted therapeutic strategies for vascular disorders.

Efforts to transform polyethylene terephthalate (PET) deconstruction products using live cells have been limited by terephthalic acid (TPA) uptake. Here, we used an intracellular carboxylate reduction assay to show that apparent TPA uptake in E. coli cells that lack a dedicated TPA transporter sharply increases between pH 5-6. Furthermore, we discovered that glycol ester deconstruction products, mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET), surprisingly each result in rapid pH-independent uptake. We exploited glycol ester uptake along with deletion of 22 cellular oxidoreductases to design intracellular hydrolysis routes for synthesis of upcycled reduction products from BHET at >90% yields, and from real PET wastes after tandem catalytic glycolysis and cell-based valorization at >80% combined yields. Our work has important ramifications for PET utilization by cells and adds new perspectives on the evolution of the PETase/MHETase system.

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane embedded signaling proteins. These receptors are involved in a wide array of physiological processes, marking them as attractive targets for drug development. Bitopic ligands, which are comprised of a pharmacophore that targets the receptor orthosteric site and a linked moiety that binds to a separate site, have considerable potential for addressing GPCR function. Here, we report the synthesis and evaluation of novel bitopic conjugates consisting of a small molecule pharmacophore that activates the adenosine A2A receptor (A2AR) linked to antibody fragments (nanobodies, Nbs). This approach leverages the high affinity and specificity binding of Nbs to non-orthosteric sites on engineered A2AR variants to provide bitopic Nb-ligand conjugates that stimulate strong and enduring signaling responses. We further demonstrate that such bitopic conjugates can induce activation by spanning two distinct receptor protomers. This property enables the selective targeting of receptor pairs over either individual receptor, as a form of "logic-gated" activity. We showcase the broad applicability of bitopic conjugates in this context by demonstrating their activity in targeting several pairs of co-expressed receptors, including GPCR monomers from different classes. Furthermore, we demonstrate that this dual-targeting strategy initiates signaling responses that diverge from those induced by monovalent ligands. The ability to target receptor pairs using nanobody-ligand conjugates offers a powerful strategy with potential for cell type-selective signaling and implications for GPCR drug discovery efforts more broadly.

While bioactive compounds are commonly derived both by human design as well as from living organisms, man-made and natural products typically display very different structural characteristics. As such, a longstanding goal in the discovery of new molecular function is to develop approaches to incorporate the advantageous elements of both groups of molecules, thereby expanding the molecular space accessible for this purpose. In this work, we report the engineering a fluorine-selective enzyme that can complement mutated acyltransferase (AT) domains of a modular polyketide synthase, which are the main determinants of the identity and location of substituents on polyketides, to produce different fluorinated regioisomers of the erythromycin precursor in vitro. We further show that by engineering cell uptake of fluorinated building blocks, we can control fluorine selectivity in vivo to produce selectively fluorinated polyketides using engineered E. coli. These results demonstrate that it is possible to introduce fluorine, a key synthetic design element for drug development, selectively into the scaffold of a complex natural product and produce these analogs by microbial fermentation.

Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70s ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

Novel antibodies have been created for targeted degradation of extracellular and membrane proteins in the lysosome. The mechanism of degradation of target proteins for these antibodies has involved either chemical conjugation of synthetic mannose 6-phosphate (M6P) or engineered bispecific antibodies. Currently, recombinant antibodies cannot be produced with naturally phosphorylated N-glycans. Here, we report the development of a novel platform technology for producing bifunctional therapeutic antibodies with high levels of M6P-bearing glycans directly from producing cells. The antibodies designated as phosphorylated N-glycosylated peptide chimeric antibodies (PNCA) maintain their affinity for antigens with concurrent high affinity binding to cell surface cation-independent mannose-6-phosphate receptors that facilitate internalization and delivery of antibody/antigen complexes to lysosomes for efficient degradation of both target extracellular soluble and membrane proteins. This PNCA approach provides a simple, scalable, and viable approach for producing naturally phosphorylated bifunctional antibodies from production cell lines for targeted protein degradation in lysosomes.

Molecular glue degraders (MGDs) reprogram E3 ligases to eliminate neosubstrates, yet their application has largely been confined to CRBN. Here, we identify caspase-2 as a new neosubstrate for von Hippel-Lindau (VHL), expanding the scope of VHL-based MGDs. Guided by a focused VHL ligand library design, we employed TurboID-based proximity labeling to discover stereoisomeric compounds (dCASP2-1 and dCASP2-2) that selectively recruit caspase-2 to VHL and promote its ubiquitin-proteasome system-dependent degradation. Further structure-activity relationship (SAR) studies yielded dCASP2-3 and dCASP2-4, which enhanced degradation potency (by 622-fold relative to dCASP2-1) and abolished enantioselectivity. Mechanistic mapping localized the degrader-induced interface to a two-helix region of the caspase-2 CARD domain, with residues H33, P34, and D100 essential for VHL engagement. Degron-guided computational modeling of the VHL/MGD/caspase-2 ternary complex provided structural insight into neosubstrate recognition. Together, we report the development of VHL molecular glues that selectively and potently degrade caspase-2, offering chemical probes to interrogate its functions in apoptosis and stress responses, while broadening the substrate landscape of VHL-based MGDs.

Programmable molecular biology increasingly requires strategies for converting engineered recognition or proximity modules into measurable outputs, particularly within transcriptional regulation, RNA imaging, and CRISPR-associated systems. Synthetic chemically induced dimerization (CID) systems provide a class of programmable recognition modules for such applications, yet generalized strategies for coupling structurally diverse CIDs to functional readouts remain limited. Here, we introduce a CID-to-output conversion strategy based on engineering of the linker-mediated coupling interface. Using single-fluorescent-protein sensors as an experimentally tractable optical model readout, we systematically varied paired N- and C-terminal linkers flanking circularly permuted green fluorescent protein (cpGFP) to map coupling landscapes across synthetic CID systems derived from combinatorial selection and computational protein design. The results revealed strong non-additive interactions across paired linkers and suggest that linker length is a first-order determinant of CID-to-output coupling. Across nanobody-, monobody-, and de novo-designed CID architectures, this framework yielded functional sensors with dynamic ranges up to 1270% and robust responses in mammalian cells. Together, this work demonstrates that effective CID-to-output conversion can be achieved by empirically mapping the linker-mediated coupling interface, providing a practical route for adapting synthetic CID to diverse programmable molecular readouts and nucleic-acid-associated synthetic biology systems O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/735888v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1111094org.highwire.dtl.DTLVardef@1579e8aorg.highwire.dtl.DTLVardef@16981feorg.highwire.dtl.DTLVardef@1d588f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fluorescence lifetime imaging microscopy (FLIM) can visualize multiple targets in a single spectral window, making it a powerful tool to overcome multiplexing limitations during live cell fluorescence microscopy. Here we show that small molecule probes-which are well-suited for imaging applications due to high specificity, low toxicity, and the elimination of transfection requirements-can be fine-tuned via bioorthogonal chemistry to exhibit predictably different fluorescent lifetimes suitable for FLIM multiplexing.

Blockade of signaling through the angiotensin II type 1 receptor (AT1R), a prototypical G protein-coupled receptor (GPCR), by angiotensin receptor blockers (ARBs) is a major therapeutic approach to treating a wide variety of cardiovascular and renal diseases1. Like most GPCRs, the AT1R signals through two transducers, G proteins and {beta}-arrestins2,3. Previous reports have described {beta}-arrestin-biased peptide orthosteric agonists for the AT1R with potential therapeutic advantages over currently available unbiased ARBs4-6. Here we report the DNA- encoded library screening-guided isolation and pharmacological characterization of the first small molecule AT1R allosteric ligands. We use cryo-electron microscopy, double electron- electron resonance spectroscopy, molecular dynamics simulations, and targeted mutagenesis to determine their binding sites, binding modes and conformational mechanisms driving their unique and divergent modulatory effects on G protein and {beta}-arrestin pathways. Our findings uncover new mechanisms for precisely controlling the dynamic behavior of the AT1R with implications for drug development targeting this pathophysiologically important receptor family.

C-Terminal cyclic imides are posttranslational modifications on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by Western blot and enrichment from whole cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.

Substance use and related mental health epidemics are causing increasing suffering and death in diverse communities.1,2 Despite extensive efforts focused on developing pharmacotherapies for treating substance use disorders, there is an urgent need for radically different therapeutic approaches.3,4 Ibogaine provides an important drug prototype in this direction, as a psychoactive iboga alkaloid suggested to have the ability to interrupt opioid use in drug-dependent humans.5 However, ibogaine and its major metabolite noribogaine present considerable safety risk associated with cardiac arrhythmias.6 We introduce a new class of iboga alkaloids - "oxa-iboga" - defined as benzofuran-containing iboga analogs and created via structural editing of the iboga skeleton. The oxa-iboga compounds act as potent kappa opioid receptor agonists in vitro and in vivo, but exhibit atypical behavioral features compared to standard kappa psychedelics. We show that oxa-noribogaine has greater therapeutic efficacy in rat models of opioid use, and no cardiac pro-arrhythmic potential, in contrast to noribogaine. Oxa-noribogaine induces long-lasting suppression of morphine and fentanyl intake after a single dose, persistent reduction of morphine intake and reinforcing efficacy after a short treatment regimen, and suppression of morphine and fentanyl drug seeking in relapse models. Oxa-noribogaine also induces a lasting elevation of neurotrophin proteins in the ventral tegmental area and medial prefrontal cortex, consistent with targeted neuroplasticity induction and alteration of addiction-like states. As such, oxa-iboga compounds represent candidates for a novel type of pharmacotherapy for treatment of opioid use disorder.

Small molecules play indispensable roles in living systems as hormones, membrane bilayer constituents, enzyme cofactors, metal chelators, and defensive chemicals. The exceptional structural diversity of such molecules underpins their wide-ranging biological functions. Precise functional group insertion into various molecular scaffold classes is a hallmark of small molecule biosynthesis and is frequently important for biological activity. Functional group deletion is also important, but mechanisms are less well understood. Here, we report deletion of a cysteine-derived nitrogen atom during assembly of the conserved pharmacophore in the anticancer drug romidepsin, and related depsipeptide HDAC inhibitors. A shunt metabolite hydroxylated at the cysteine--carbon-derived position is a thousand-fold less active, indicating nitrogen deletion is important for potent HDAC inhibition. In vitro reconstitution and dissection of the complete nonribosomal peptide synthetase-polyketide synthase-mediated pathway for pharmacophore assembly reveal that cryptic S-octanoylation is catalysed by an atypical heterocyclisation domain, while multifunctional dehydratase and ketoreductase domains and trans-acting phosphotransferase and flavin-dependent oxidoreductase enzymes catalyse successive transformations in nitrogen deletion. Our findings significantly advance the understanding of heteroatom deletion mechanisms in small molecule biosynthesis and highlight the key role this can play in enhancing bioactivity. One-pot biocatalytic synthesis of the pharmacophore provides foundations for chemoenzymatic approaches to next-generation HDAC inhibitors.

Transcription factor-based biosensors monitor metabolites and control genetic programs, but their wider use is constrained by the limited repertoire of characterized, mutually compatible sensor parts. Here we combine a curated screen of natural LysR-type transcriptional regulators (LTTRs), the largest family of bacterial transcription factors, with systematic domain swapping. Using a standardized construction platform, we convert 17 LTTRs into whole-cell reporters in Escherichia coli. Of 16 viable circuits, nine show regulatory activity, including six ligand-inducible biosensors for acetate, benzoate, -ketoglutarate, chlorohydroquinone, L-homocysteine and salicylate. Mapping interactions across 11 LTTR systems identifies seven mutually orthogonal regulator pairs, providing, to our knowledge, the first orthogonality map for this family. We next construct 108 chimeras across three domain-swap architectures; 69 retain measurable activity, with functional outcomes enriched when the native hinge-ligand-binding-domain association is preserved. As proof of principle, we redesign a cross-reactive regulator: replacing its DNA-binding domain with one from an orthogonal regulator abolishes unwanted promoter crosstalk while preserving ligand-inducible activation of its own target, transferring orthogonality to a previously incompatible pair. Together, natural-diversity screening and domain swapping emerge as complementary routes to expand LTTR biosensor repertoires, revealing a strong link between connector architecture and chimera function.

Recent evidence indicates that mono - and poly-ADP ribosylation (MARylation and PARylation) are not limited to proteins but extend to DNA. Notably, in vitro base PARylation by PARP1 in single stranded DNA (ssDNA) was demonstrated at N1-deoxyadenosine (N1-dA). Here, we report that PARP1 catalyzes N3-specific ADP-ribosylation of deoxycytidine (N3-dC) in single-stranded DNA. Analogous to N1-dA PARylation, which is prone to spontaneous adenine-to-inosine deamination, N3-dC PARylation promotes cytosine deamination, yielding N3-PARylated-deoxyuridine. These deamination products yield diagnostic PARylation signatures in LC-MS/MS, namely N1-ribosyl-deoxyinosine (N1-r-dI) and N3-ribosyl-deoxyuridine (N3-r-dU). We synthesized both N1-r-dI and N3-r-dU as diagnostic standards and established absolute quantification of base ADP-ribosylations by LC-MS/MS. Quantitative analysis of PARylated dA and dC in ssDNA reveals pronounced sequence preferences of PARP1. Removal of these base modifications differs markedly, since ADP-ribose glycohydrolase TARG1 removes PAR from both dA and dC, whereas PARG acts exclusively on dA. Our results establish cytidine ADP-ribosylation as a novel DNA modification, with potential roles in DNA metabolism, epigenetic regulation, or genome stability.