Nature Chemical Biology

Life is predicated on chirality, a molecular asymmetry akin to the left and right versions of human hands. Here we show that privileged protein residues are predisposed for chiral regulation. We developed enantiomeric oxaziridine reagents that systematically identify pro-(S) and pro-(R) methionine oxidation sites across proteomes that can be erased by stereospecific methionine sulfoxide reductase enzymes A and B, respectively. These probes reveal that chiral regulation of methionine oxidation-reduction processes can allosterically regulate protein function, as shown in cell and murine models of oxidative stress where selective (R)-methionine sulfoxide formation on M69 of biphenyl hydrolase-like protein leads to hydrolase inhibition and amplification of proteome N-homocysteinylation modifications. This work introduces a platform for characterizing sites of asymmetric methionine oxidation and the functional consequences concomitant with an individual chiral single-atom modification.

Neurons and glial cells are biochemically coupled through the exchange of nutrients, but our knowledge of which metabolites are transferred between them remains limited due to technical challenges. Here, we introduce a strategy to label specific cell types with isotopic tracers so that metabolite transfer can be measured directly in the intact brain. By engineering neurons in mice to metabolize 13C-labeled cellobiose, a glucose dimer that wild-type cells cannot catabolize, we selectively track neuron-derived metabolites by using mass spectrometry-based metabolomics. Applying this approach enabled us to identify myo-inositol as a critical metabolite synthesized by neurons and transferred to oligodendrocyte progenitor cells (OPCs) via the SLC5A3 transporter. The transfer of myo-inositol from neurons to OPCs promotes OPC proliferation and differentiation by enhancing phosphatidylinositol synthesis and upregulating expression of myelin-associated genes. During demyelination, deficient nutrient transfer can be rescued by dietary supplementation of myo-inositol, which accelerates myelin repair. These findings establish a generalizable technology for tracing intercellular metabolite transfer in vivo and identify a previously unrecognized mechanism of myo-inositol transfer from neurons to glial cells in support of CNS regeneration, revealing a potential metabolic target for therapeutic intervention in neurodegenerative disease.

Paclitaxel biosynthesis is limited by the instability of taxadiene-4(5)-epoxide, which readily diverts to the non-productive byproduct 5(12)-oxa-3(11)-cyclotaxane (OCT) instead of rearranging to taxadiene-5-ol. Although FoTO1 suppresses OCT accumulation, its molecular function has been unclear. Here we identify FoTO1 as a dedicated epoxide isomerase that directs productive rearrangement. Biochemical characterization, site-directed mutagenesis, and QM/MM calculations reveal a pre-organized D68-D149 dyad that electrostatically activates epoxide ring opening and stereospecific rearrangement. Modular dissection of the C-terminal extension further reveals a functional partition between catalytic integrity and productive coupling with T5OH, mediated by specific hydrophobic contacts that enforce precise geometric complementarity at the binary complex interface. These results demonstrate how electrostatic activation and enzyme association cooperate to control the fate of a highly reactive intermediate in paclitaxel biosynthesis.

Adenine is a ubiquitous nucleobase found in nucleic acids, cofactors, and signaling molecules and mediates diverse molecular interactions. Here, we identify TvAPT, an adenine prenyltransferase from the cyanobacterium Trichormus variabilis NIES-23. Unlike canonical enzymes limited to C5 dimethylallylation, TvAPT efficiently catalyzes the unprecedented N6-prenylation of adenine-containing substrates using extended prenyl donors (C10 and C15), markedly increasing the hydrophobicity of the adenine moiety. X-ray structural analysis and protein engineering revealed that an enlarged prenyl-binding pocket enables this donor promiscuity, allowing rational tuning of prenyl-donor preference. These findings establish TvAPT as a versatile biocatalytic platform that expands the chemical space of adenine-containing molecules for biomolecular engineering, as demonstrated by the synthesis of membrane-permeable nucleotides and analogues of plant signaling molecules.

Identifying the direct molecular targets of bioactive natural products remains a central challenge in chemical biology. Here we present an integrated experimental-computational framework, that combines matrix-augmented thermal proteomics with HoloGNN, a holistic graph neural network, to systematically prioritize and validate protein-ligand interactions. Benchmarking with PDBbind datasets HoloGNN achieves state-of-the-art performance. Applying this framework to 50 structurally diverse natural products identified Demethylzeylasteral as a direct interactor of ACLY. Orthogonal biochemical assays confirmed micromolar binding and enzymatic inhibition. In an imiquimod-induced psoriasis-like mouse model, Demethylzeylasteral reduced disease severity and inflammatory cytokine expression. Single-cell transcriptomics revealed that Demethylzeylasteral reverses keratinocyte hyperproliferation and suppresses ACLY-dependent lipid metabolic reprogramming. Together, this scalable, closed-loop strategy integrates thermal proteomics and machine learning to uncover direct targets of natural products and provides mechanistic evidence linking ACLY inhibition to therapeutic modulation of inflammatory pathology.

Multicomponent Rieske oxygenases catalyze diverse oxidative transformations but require precisely matched redox partners to sustain efficient electron transfer, severely limiting their modularity and biocatalytic application. Yet, the molecular logic underlying this specificity remains poorly defined. Here we decode the molecular principles governing redox partner specificity in representative three-component Rieske oxygenase systems. Through systematic mutagenesis analysis and cross-component reconstitution assays, we identify a single ferredoxin residue that acts as a class-defining determinant of oxygenase recognition. Guided by this insight, we reprogram electron transfer between non-cognate components by complementary engineering of the oxygenase interface, creating an unnatural redox chain with substantially enhanced catalytic turnover compared to the native system. Spectroscopic, binding and computational analyses reveal that productive electron transfer arises from optimized electrostatic complementarity and redox potential alignment rather than maximal binding affinity. Extending this strategy to another oxygenase system demonstrates its generality. Together, these results establish transferable design rules for rationally engineering electron transfer pathways in multicomponent oxygenases, enabling their predictable adaptation as customizable biocatalysts.

Cell-surface degrader platforms typically require target-specific engineering and have therefore been applied to a relatively small set of protein targets. Here we report Z-TAC, a strategy that enables plug-and-play conversion of existing IgG antibodies into cell-surface protein degraders. Across multiple targets from distinct protein families, Z-TAC induced efficient and sustained degradation of both individual receptors and receptor combinations. For a multi-pass membrane receptor lacking selective antagonists, Z-TAC mediated complete receptor degradation and functional inhibition, demonstrating the ability of this platform to overcome the limitations of conventional pharmacological approaches. This study delineates a generalizable and scalable strategy for functional perturbation of the cell-surface proteome.

Targeted protein degradation (TPD) is a therapeutic strategy to remove disease-causing proteins by routing them to the ubiquitin-proteasome, autophagy, or lysosme machineries. For instance, proteolysis-targeting chimeras (PROTACs) are synthetic hetero-bifunctional small molecules that simultaneously bind the target and an E3 ubiquitin ligase to drive ubiquitination and degradation by the proteasome. Despite considerable success, designing such molecules is challenging and the number of currently addressable ubiquitin E3 ligases is limited. Here we demonstrate hetero-bifunctional de novo designed proteins as alternatives for TPD to access more targets and ligases. First, we develop a stable and highly adaptable helix-turn-helix scaffold for presenting different binding sites. Next, we use computational protein design to incorporate and embellish hot-spot- binding sites to target BCL-xL, plus short linear motifs (SLiMs) for KLHL20 ligase recruitment. The resulting mono- and bi-functionalised proteins bind the targets in vitro, and the latter degrade BCL-xL in cells leading to apoptosis.

Precise control over protein secretion is essential for programming intercellular communication and coordinating complex physiological responses. However, conventional methods relying on transcriptional regulation or chemical induction often lack the spatiotemporal precision and reversibility required to mimic endogenous signaling dynamics. Here, we present the Blue Light-Assisted Secretion Toolkit (BLAST), a genetically encoded system that orchestrates protein release from the endoplasmic reticulum via light-tunable protein-protein interactions. BLAST comprises two complementary modules utilizing both light-induced iLID/SspB association (a-BLAST) and LOV2/Zdk1 dissociation (d-BLAST). Both modules harness the highly conserved RXR motif to enforce strict ER confinement in the dark state. Most importantly, by utilizing non-destructive steric masking rather than enzymatic cleavage, BLAST achieves unprecedented temporal resolution with strict reversibility. We demonstrate that both systems can be repeatedly toggled ON and OFF, instantaneously arresting cargo release upon light withdrawal to generate highly controlled, pulsatile secretion profiles. Leveraging this dynamic control, we successfully achieved the rapid, robust, and light-triggered secretion of complex therapeutic proteins, including insulin and interleukin-12. By bypassing transcriptional delays and irreversible activation steps, BLAST provides a generalized, plug-and-play platform for the on-demand delivery of therapeutic proteins, significantly expanding the optogenetic toolbox for synthetic biology and cell-based therapies.

Cardiac Calsequestrin (CASQ2) polymerizes within the junctional sarcoplasmic reticulum to buffer Ca{superscript 2} and regulate ryanodine receptor 2 (RyR2) gating, yet the molecular mechanism governing this process remains poorly understood. Using an integrated set of complementary approaches spanning single-particle biophysics, bulk solution measurements, and polymer chemistry, we demonstrate that CASQ2 is an intrinsic dimer at nanomolar concentrations and under physiological ionic conditions, independently of Ca{superscript 2}. In addition, Ca{superscript 2}-dependent polymerization operates as a highly cooperative switch between a stable oligomeric phase and a high-order polymeric state. Physiological amounts of K ions modulate this switch through a biphasic electrostatic mechanism, supporting polymerization at low concentrations and inhibiting it beyond charge neutralization ([~]194 mM). These findings redefine CASQ2 as an intrinsic dimer with polymerization-switch properties, and provide a mechanistic framework for understanding how catecholaminergic polymorphic ventricular tachycardia type 2 mutations, distributed evenly across the CASQ2 surface, cause disease through two distinct pathological trajectories.

Steroid-based fluorescent-quencher probes now enable real-time, residue-level mapping of previously inaccessible cholesterol-binding sites on G-protein-coupled receptors. We designed Tide Quencher 1 (TQ1) conjugated steroids that target two distinct peripheral sites on the M1 muscarinic receptor. One near the extracellular N-terminus and another adjacent to the intracellular C-terminus. Using pregnanolone glutamate as a versatile scaffold, we synthesised a library of probes varying in C-3 linker length ({gamma}-aminobutyric acid vs. L-glutamic acid) and C-3/C-5 stereochemistry (3/3{beta}/5/5{beta}). Fluorescence-quenching assays with CFP-tagged receptors revealed that TQ1 probes consistently outperformed Dabcyl, delivering up to 40 % quenching within minutes and sub-micromolar EC50 values. The most potent N-terminal probe (35-PRG-Glu-TQ1 (5)) achieved 300 nM potency, while the best C-terminal probe (35{beta}-PRG-Glu-TQ1 (3)) reached 1 {micro}M potency with rapid association. Molecular docking and MD simulations identified key residues (K20, Q24, W405 at the N-site; K57, Y62, W150 at the C-site) mediating binding, a prediction confirmed by alanine-scan mutagenesis that markedly reduced quenching at the N-terminus and only modestly affected the C-terminus. Competition experiments with non-quenching analogues further validated probe specificity. Crucially, the pregnane core proved essential; alternative steroid backbones failed to generate robust quenching. This fluorescence-quenching platform overcomes the limitations of traditional radioligand assays, providing kinetic insight, high-throughput compatibility, and the ability to dissect lipid-GPCR interactions in native membranes. The approach is readily extensible to other GPCR families, opening new avenues for structure-guided drug discovery targeting allosteric cholesterol sites.

Bile salt hydrolases (BSHs) are gut microbial enzymes that catalyze the deconjugation of glycine-or taurine-conjugated bile acids (BAs), a key step in shaping the BA pool in the human gastrointestinal tract and modulating host-gut microbiome interactions.1-3 All known BSHs are members of the N-terminal nucleophile (Ntn) hydrolase superfamily and share a conserved architecture and mechanism involving a nucleophilic active site cysteine.4,5 This knowledge has guided predictions and study of BSH activity in the gut microbiome6,7 as well as the development of BSH inhibitors8. Here, we report the discovery and characterization of a previously unknown BSH from the human gut bacterium Bilophila wadsworthia that belongs to the metal-dependent amidohydrolase superfamily and exhibits robust and specific activity toward taurine-conjugated bile salts. We show this secreted enzyme, metalloBSH, utilizes a metallocofactor for BA deconjugation, a mechanism distinct from that of canonical Ntn-type BSHs. MetalloBSHs are conserved in B. wadsworthia and present in many other Desulfovibrionaceae found in vertebrate gut microbiomes. Analysis of multi-omic datasets indicates metalloBSHs are expressed in vivo and correlate with BA metabolism. Overall, our findings reshape our understanding of BSH activity in the gut microbiome and highlight the promise of activity guided discovery in revealing previously overlooked gut microbial enzymes.

Gene regulation through translation is critical for spatiotemporal protein expression. Internal ribosomal entry sites (IRESes) mediate mRNA-specific translation by recruiting ribosomes to 5 untranslated regions. Circular RNAs (circRNAs), naturally occurring and stable RNA species, are increasingly used as synthetic tools for sustained therapeutic protein translation by IRES-driven initiation. However, the functionality of different IRESes in synthetic circRNAs remains sparsely characterized. We systematically examine circRNA reporter translation by viral and cellular IRESes in human cells and in diverse in vitro translation systems. Improved circRNA purification by urea-PAGE and RNase R-treatment removes contaminants that induce RNA sensing. Viral CVB3 and HCV, as well as cellular Hoxa9, Chrdl1, Cofilin and c-Myc IRESes, effectively drive circRNA translation. We also establish circRNA translation in an improved human cell-free extract that recapitulates IRES-dependent regulation, and allows for precise engineering of HCV IRES-mediated translation. These findings inform IRES selection for synthetic circRNA translation relevant for circRNA-based medicines.

Proteolysis-targeting chimeras (PROTACs) are emerging as potent tools for targeted protein degradation that overcome many of the limitations of traditional small molecule inhibitors. Yet how these hetero-bifunctional therapeutics enter cells remains a mystery. While passive diffusion is conventionally assumed, the bulky structure of PROTACs suggests that active transport may be required. Recently, the fatty acid transporter CD36 was identified as a key receptor for PROTACs. However, because the uptake mechanism of CD36 is itself unknown, how PROTACs enter cells remains a mystery. Here we show that PROTAC uptake and function require clathrin-mediated endocytosis. We uncover previously unrecognized clathrin adaptor-binding motifs in the CD36 C-terminus and use live-cell imaging to visualize the recruitment of both CD36 and PROTACs to sites of clathrin-mediated endocytosis on the cellular plasma membrane. Strikingly, disruption of clathrin assembly through either genetic or pharmacological means abolishes all detectable PROTAC-induced protein degradation, demonstrating that the clathrin pathway is required for the function of PROTACs that utilize diverse E3 enzymes against multiple targets. These results elucidate the molecular mechanism of PROTAC entry into cells, providing critical information for optimizing cellular uptake and response to targeted degraders.

T cell receptor mimic (TCRm) antibodies and nanobodies that specifically bind peptide-HLA complexes have great therapeutic potential, as they can target polymorphic HLA on tumour cells furnishing peptides derived from tumour-associated antigens. MR1 is an MHC class-I-like molecule that exhibits limited polymorphism that binds and presents conserved metabolites, such as 5-OP-RU, derived from microbial riboflavin biosynthesis. Whether antibodies targeting such MR1-5-OP-RU complexes can be generated remains unclear. Using yeast display technology and in vitro affinity maturation, a nanobody with high affinity and fine specificity toward MR1-5-OP-RU complex was generated. These nanobodies bind both mouse and human MR1-5-OP-RU and inhibited MAIT cell responses to 5-OP-RU in vitro and in vivo demonstrating therapeutic potential. Moreover, we provide a molecular basis underpinning the fine specificity of these nanobodies, solving the crystal structures of MR1 in complex with either 5-OP-RU or Ac-6-FP. Here, the nanobody co-bound MR1 and 5-OP-RU, akin to a TCRm antibody. Moreover, we engineer bispecific antibodies targeting both MR1-5-OP-RU and CD3, that drive broad T cell killing of bacterially-infected cells as well as tumour cells treated with 5-OP-RU, thereby providing proof-of-principle for targeting the MR1 molecule with with TCRm-based nanobodies. One Sentence SummaryWe report the development of a nanobody targeting MR1-5-OP-RU complex and demonstrate its utility to modulate MAIT cells responses, and as a bispecific engager.

Tubulin glycylation, a cilia-specific posttranslational modification is emerging as a potentially key regulator of ciliary axonemal microtubules. However, insights into the functional consequences of glycylation have remained limited. Here, using in vitro reconstitution assays with unmodified or custom-glycylated tubulin, we provide a systematic mechanistic analysis of glycylation-dependent regulation of motors and microtubule-associated proteins. Our studies highlight that glycylation selectively enhances ciliary kinesin-2 motility while reducing kinesin-1 activity, suggesting a role in promoting efficient intraflagellar transport along axonemal microtubules. Moreover, glycylation protects microtubules from decay by suppressing the activities of the depolymerase MCAK and severing enzyme spastin, thereby enhancing stability. Notably, this regulation is dependent on the proportion of glycylation on the microtubule surface, coupled with concomitant reduction of glutamylation. Thus, by generating microtubule surfaces with distinct biochemical states, we establish that combinatorial modification patterns define functional microtubule properties especially in cilia. Together, our findings provide the first comprehensive mechanistic framework for tubulin glycylation in regulating molecular motors and MAPs in cilia, establishing glycylation as a key determinant of motor selectivity and microtubule stability within the axoneme.

Engineering bacterial promoters to integrate multiple regulatory signals remains a formidable challenge. Juxtaposing operator sites frequently increases basal leakiness, compresses the fully induced state, and introduces severe sequence-context dependencies. Here, we systematically engineered two-input combinatorial promoters in Escherichia coli that integrate signals from multiple transcription factors. To achieve precise operational control over these regulators, we drove the promoters using highly optimised, small-molecule-responsive sensors from the Marionette transcription-factor cassette, allowing us to assemble 19 reporter-specific, four-state truth tables across 12 distinct promoter architectures. We evaluated each design against a stringent statistical criterion for inducer-conditioned coincidence responses. Nine architectures satisfied this criterion, yielding a robust set of operational AND switches. By comparing successful and unsuccessful designs, we reveal that performance hinges primarily on suppressing partially induced states, ensuring structural compatibility between the promoter scaffold and the inserted operator, and precisely managing the orientation of long operators to avoid recreating unintended promoter-like motifs. Furthermore, reciprocal architectures and alternate downstream reporters frequently display divergent behaviours, underscoring profound asymmetries and local genetic-context dependencies. Ultimately, these findings deliver versatile combinatorial switches alongside practical, sequence-aware design rules for engineering multi-input bacterial promoters.

The eukaryotic chaperonin TRiC/CCT is essential for folding complex proteins, yet how it folds substrates that exceed its closed chamber capacity remains a longstanding paradox. Here, we define the folding pathway of IFT172, the largest subunit ([~]200 kDa) of the intraflagellar transport (IFT) machinery, and uncover a "divide-and-conquer" mechanism. TRiC and HSP70 engage IFT172 concurrently but on distinct domains: TRiC captures the N-terminal WD40 {beta}-propellers within its chamber, whereas HSP70 independently stabilizes the C-terminal TPR domain in the cytosol. To accommodate this oversized client, specific TRiC subunits (CCT4, CCT2, and CCT7) undergo pronounced Z-shaped outward bending, thereby expanding the chamber. Unexpectedly, the first WD40 domain reaches a near-folded state within the open, ATP-bound chamber, and subsequent TRiC ring closure triggers substrate ejection rather than encapsulation. This non-canonical "fold-and-eject" mechanism challenges the classical view that the closed chamber is an obligate folding cage. We further demonstrate that this pathway is essential for ciliary functions in vivo, and reveal a conserved mode of chaperonin recognition among IFT components bearing tandem WD40-TPR architectures. Together, our findings establish a new paradigm for the folding of oversized, multi-domain proteins and identify TRiC as a central proteostasis hub in ciliary biogenesis, with direct implications for ciliopathy pathogenesis.

Despite decades of biochemical study, a comprehensive map of the mammalian metabolome remains elusive. Mass spectrometry-based metabolomics detects thousands of small molecule-associated signals in mammalian tissues, but it is currently unclear how many of these reflect products of endogenous metabolism. Here, we leverage systematic in vivo isotope tracing to infer the biosynthetic origins of unidentified metabolites. We administered 26 different isotopically labelled nutrients to mice, measured circulating and tissue metabolite labelling by mass spectrometry, and developed a statistical framework to infer the number of carbon atoms incorporated from each of these precursors into more than 4,000 putative metabolites. We show this information can be harnessed for biosynthesis-aware structure elucidation using a multimodal AI model that co-embeds isotopic labelling patterns with chemical structures. This approach revealed several previously unrecognized families of mammalian metabolites, including cysteine-derived alkylthiazolidines, dithioacetal mercapturic acid derivatives, short-chain N-acyltaurines, acylglycyltaurines, and N-oxidized taurines. It further uncovered a family of mevalonate-derived isoprenoid metabolites that includes 2,3-dihydrofarnesoic acid, which is markedly depleted in both mouse and human aging. Age-related depletion of these isoprenoids is driven by impaired coenzyme A synthesis. Our work establishes the biosynthetic precursors for thousands of unidentified metabolites and reveals multiple previously unrecognized branches of mammalian metabolism.

Chromatin accessibility is essential for maintaining the fidelity of gene regulation and is dynamically regulated by epigenetic enzymes that are often dysregulated in cancer. The most commonly mutated regulator is the modular, multi-subunit Brahma-associated factor (BAF) chromatin remodeling complex. Previous attempts to exploit BAF complex mutations as potential tumor vulnerabilities using loss-of-function approaches have shown limited clinical success. Here, we instead propose a gain-of-function (GOF) strategy and establish Transcriptional/Remodeling chemical Inducers of Proximity (TRIPs), a class of neomorphic molecules that recruit active BAF complexes to rewire an oncogenic repressor, B-cell lymphoma 6 (BCL6). TRIPs potently induce transcriptional de-repression and apoptosis in Diffuse Large B-cell Lymphoma (DLBCL), enabled by ternary complex formation between BCL6 and BAF. CRISPR knockout screening identifies the PBAF complex as an essential contributor to cellular TRIP efficacy. Finally, we demonstrate that TRIP induces chromatin enrichment of BAF at BCL6-bound sites, resulting in ATPase-dependent eviction of BCL6, and de-repression of pro-apoptotic BCL6 target genes. We establish BAF recruitment for targeted chromatin remodeling as a viable GOF pharmacological strategy for tackling diseases driven by aberrant gene repression.