We discover that biomolecular condensates, formed by intrinsically disordered proteins without inherent chemical activity, can spontaneously drive nonenzymatic reductive amination. These condensates facilitate reactions...We discover that biomolecular condensates, formed by intrinsically disordered proteins without inherent chemical activity, can spontaneously drive nonenzymatic reductive amination. These condensates facilitate reactions between amines and aldehydes or ketones, yielding imines, which are subsequently hydrogenated to form alkylated amines leading to C-N bond formation. Our experiments show that condensates modulate the reductive amination of diverse types of metabolite containing carbonyl groups. Using combinatorial metabolomics, we found that condensates generate previously unknown metabolites through the dimerization of natural amines with ketones and aldehydes. Metabolomics in living cells confirms that the ability of condensates in mediating C-N bond formation enables the synthesis of new metabolites and regulates cellular pathways. These findings uncover a previously unrecognized inherent function of biomolecular condensates, redefining their roles in metabolism. This further highlights the broader influence of condensates on chemical homeostasis and biochemical regulation in biological and prebiotic chemistry.
Akaike T, Alvarez B, Banerjee R
… +10 more, Chung CY, Dick TP, Furdui C, Hampton M, Hidalgo E, Hoxhaj G, Neumann C, Rivera-Fuentes P, Weerapana E, Xian M
Schneider FF, Martin Del Campo JS, Zhang L
… +2 more, Dean DR, Einsle O
Nat Chem Biol
· 2026 May · PMID 41872497
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The maturation of the unique FeMo-cofactor of molybdenum nitrogenase is a multistep process requiring the sequential action of a series of maturase complexes. As a final step, the NifEN complex forms FeMo-cofactor from t...The maturation of the unique FeMo-cofactor of molybdenum nitrogenase is a multistep process requiring the sequential action of a series of maturase complexes. As a final step, the NifEN complex forms FeMo-cofactor from the precursor NifB-co, also called L-cluster, through replacement of an apical iron ion by molybdenum and the attachment of an organic homocitrate ligand. NifB-co is delivered by a small cofactor chaperone, NifX, and initially bound near the surface of the maturase NifEN. Here, we report high-resolution cryo-electron microscopy structures of NifEN in complex with NifX, showing NifB-co binding to NifEN in full detail, capturing both interacting partners in the act of cluster transfer. In a dynamic transfer complex, the large metal cluster is coordinated by single residues from both NifEN and NifX. In silico studies concur with these structures but suggest a third, internal conversion site where cluster maturation likely takes place.
Diep P, Madsen CS, Choi W
… +8 more, Dong Z, Kang-Yun CS, Uychoco PFV, Seidel JA, Eaton SA, Jiao Y, Cotruvo JA, Park DM
Nat Chem Biol
· 2026 May · PMID 41851437
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Proteins offer a molecular design space to create bespoke ligands for the separation of critical metals like rare earth elements (REs). However, data-intensive approaches to tune metalloprotein selectivity are constraine...Proteins offer a molecular design space to create bespoke ligands for the separation of critical metals like rare earth elements (REs). However, data-intensive approaches to tune metalloprotein selectivity are constrained by the low-throughput nature of existing characterization methods. Here we invented an assay called 'SpyTag-Catcher Immobilization of Lanmodulin for Assaying Metal-Binding Selectivity' (SpyCI-LAMBS) to measure metalloprotein selectivity en masse. This 96-format workflow was used to study the selectivity of 621 lanmodulin (LanM) orthologs for 15 REs, revealing eight distinct selectivity profiles based on sequence-to-function analyses. We discovered >200 LanMs with stronger selectivity against low-value La relative to the prototypical LanM. This includes a LanM that can perform a challenging one-stage separation of Pr from La with up to >99.9 mol% purity and 83% yield. SpyCI-LAMBS is a powerful tool that can rapidly collect high-fidelity selectivity data to inform metal ion separations and machine-learning-assisted metalloprotein design.
Aslan M, d'Amico EA, Cho NH
… +8 more, Taheri A, Perez-Bertoldi JM, Zhao Y, Zhong X, Blaauw M, Carter AP, Dumont S, Yildiz A
Nat Chem Biol
· 2026 Mar · PMID 41840068
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During cell division, the nuclear mitotic apparatus protein (NuMA) orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein moto...During cell division, the nuclear mitotic apparatus protein (NuMA) orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein motors, microtubules and other cellular factors. Here we used in vitro reconstitution, cryo-electron microscopy and live-cell imaging to understand the mechanism and regulation of NuMA. We determined the structure of the processive dynein-dynactin-NuMA complex (DDN) and showed that the NuMA N terminus drives dynein motility in vitro and facilitates dynein-mediated transport in live cells. The C terminus of NuMA directly binds and suppresses the dynamics of the microtubule minus-end. Full-length NuMA is autoinhibited for its interactions with dynein and microtubules, whereas mitotically phosphorylated NuMA activates dynein in vitro and interphase cells. Together with dynein, activated full-length NuMA focuses microtubule minus-ends into aster-like structures. These results provide critical insights into the activation of NuMA and dynein for their mitotic functions.
Reactive oxygen species (ROS) dynamics exhibits rhythmic oscillations in cancer cells but how this rhythm influences tumorigenesis and therapeutic responses remains unclear. Here we found coexistence of ROS rhythmicity a...Reactive oxygen species (ROS) dynamics exhibits rhythmic oscillations in cancer cells but how this rhythm influences tumorigenesis and therapeutic responses remains unclear. Here we found coexistence of ROS rhythmicity and rhythm loss in tumor samples. Under low-ROS conditions, indoleamine 2,3-dioxygenase 1 (IDO1), an immune-checkpoint molecule, binds to KEAP1 for proteasomal degradation in the nucleus. In contrast, elevated ROS levels drive IDO1 translocation into the cytosol, where it binds mitochondria-released heme to form an active holoenzyme. This holoenzyme catalyzes tryptophan to kynurenine that allosterically activates glucose-6-phosphate dehydrogenase, enhancing NADPH production and promoting ROS clearance. However, in hypoxic tumor microenvironments, ROS rhythmicity is lost. Compensating for this, hypoxic tumor cells mobilize the sulfenylated aryl hydrocarbon receptor (AhR)-mediated glycogenolysis pathway to manage disordered ROS accumulation, maintaining elevated ROS levels that favor tumor growth. Dual inhibition of IDO1 and AhR significantly prolongs survival of NSG mice, highlighting enforced disruption of ROS rhythm as a common therapeutic strategy.
The biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) leverages iterative catalysis to enhance structural and biological diversity. Traditionally, iterative enzymes install posttra...The biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) leverages iterative catalysis to enhance structural and biological diversity. Traditionally, iterative enzymes install posttranslational modifications on linear peptides, rather than mature RiPPs with intricate three-dimensional structures, which require complex changes in substrate binding. Here we present a prolific class of GCN5-related N-acetyltransferases (GNATs) that iteratively and consecutively acylate two Lys residues within the loop and ring motifs of lasso peptides, diverging from the typical iterative modification of linear peptides. Utilizing high-resolution cryogenic-electron microscopy and enzymatic reconstitution, we define the lasso peptide-binding pocket of IatT and pinpoint key residues that distinguish its two distinct acetylation steps. Structure-based engineering of IatT's acetyl-recognition site expands the cavity to accommodate longer-chain acyl groups, enabling the creation of lipolasso peptides, a class of ribosomal lipopeptide. This engineering strategy can be applied to any RiPP biosynthetic gene cluster encoding GNAT, facilitating the efficient diversification of ribosomal lipopeptides.
Ng MY, Nix MN, Du G
… +18 more, Davidek I, Burger N, Shin S, Toenjes S, Takeda H, Cheah Xin Yan M, Zhang B, Xiao H, Wei SM, Seo HS, Dhe-Paganon S, Wales TE, Engen JR, Mills EL, Che J, Zhang T, Gray NS, Chouchani ET
Nat Chem Biol
· 2026 Jul · PMID 41820548
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Immunological proteins are major disease targets, yet most remain undrugged. Post-translational redox modification of cysteine residues has emerged as an important mode of immune cell regulation, particularly in macropha...Immunological proteins are major disease targets, yet most remain undrugged. Post-translational redox modification of cysteine residues has emerged as an important mode of immune cell regulation, particularly in macrophage cytokine responses. Here we develop a strategy for systematic discovery and small-molecule functionalization of redox-regulated cysteines on immunological proteins. Using deep redox proteomics, we annotate 788 in vivo redox-regulated cysteines across diverse immune-relevant protein domains. We demonstrate how these sites enable cysteine-directed pharmacology through discovery of a novel cysteine activation site on the immune regulator SHP1. Targeting C102, we develop a highly selective covalent agonist, SCA, which binds the N-SH2 domain to relieve autoinhibition and activate SHP1. In mouse and human macrophages, SCA selectively engages SHP1 C102, antagonizing interleukin-1 receptor-associated kinase signaling and lipopolysaccharide-induced proinflammatory cytokine production. Together, this work identifies a druggable cysteine redox switch controlling macrophage cytokine responses and provides a compendium of redox-regulated sites for therapeutic development.
Intensity and duration of biological signals encode a few pathways to direct diverse cellular behaviors, yet quantifying these features in single cells remains difficult. To address this challenge, we developed INSCRIBE,...Intensity and duration of biological signals encode a few pathways to direct diverse cellular behaviors, yet quantifying these features in single cells remains difficult. To address this challenge, we developed INSCRIBE, which uses a CRISPR base editor to mutate genomic targets at rates proportional to signaling activity. Edits are recovered at the endpoint through a new ratiometric readout strategy from images of two fluorescence channels. We engineered human cells to record WNT and BMP activity. Following defined exogenous stimulations, INSCRIBE accurately recovered signal intensity in dose-response experiments and exposure duration in time-course experiments. Applying INSCRIBE revealed a persistent memory in the BMP pathway, where progeny of high-responding cells remained more sensitive to subsequent BMP stimulation for up to 3 weeks. Together, our results establish a scalable platform for genetic recording and in situ readout of signaling activity in single cells, advancing quantitative analysis of cell-cell communication during development and disease.
Asparagine-linked glycans are essential for the maturation and function of most eukaryotic secretory proteins. The biosynthesis and transfer of dolichylpyrophosphate-anchored GlcNAcManGlc glycan is a highly conserved pro...Asparagine-linked glycans are essential for the maturation and function of most eukaryotic secretory proteins. The biosynthesis and transfer of dolichylpyrophosphate-anchored GlcNAcManGlc glycan is a highly conserved process occurring in the endoplasmic reticulum (ER) membrane and involving over a dozen membrane proteins whose dysfunction is linked to congenital disorders of glycosylation (CDGs). Three membrane-integral mannosyltransferases, ALG3, ALG9 and ALG12, mediate four consecutive mannosylation reactions that convert GlcNAcMan to GlcNAcMan. Here, using chemoenzymatically synthesized lipid-linked glycan donor and acceptor analogs, we recapitulated this biosynthetic pathway in vitro. High-resolution cryo-electron microscopy structures of pseudo-Michaelis complexes of each step revealed how the branched glycan is accurately synthesized and unwanted side products are averted. Molecular dynamics simulations and mutagenesis studies uncovered a subtle but precise mechanism selecting the dolichylphosphomannose donor substrate over dolichylphosphoglucose, which is also present in the ER membrane. Our results also provide mechanistic explanations for enzyme dysfunction in CDGs and offer opportunities for N-glycan engineering.
Zimmermann J, Lang L, Malo Pueyo J
… +21 more, Riedel M, Wahni K, Stobbe D, Leiskau L, Aref E, Lux C, Janvier S, Vertommen D, Lenhard S, Hannemann F, Thangamuragan S, Castro H, Helms V, Tomas AM, Herrmann JM, Salvador A, Mühlhaus T, Riemer J, Messens J, Deponte M, Morgan B
Nat Chem Biol
· 2026 Apr · PMID 41807831
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Peroxiredoxins are thiol peroxidases, which detoxify peroxides, relay redox signals and act as chaperones. In eukaryotes, multiple peroxiredoxin-1 (Prx1)/AhpC-type isoforms frequently co-exist in the same subcellular com...Peroxiredoxins are thiol peroxidases, which detoxify peroxides, relay redox signals and act as chaperones. In eukaryotes, multiple peroxiredoxin-1 (Prx1)/AhpC-type isoforms frequently co-exist in the same subcellular compartment, yet have been assumed to assemble only as homo-oligomeric complexes. Here we show that hetero-oligomerization is a conserved and functionally relevant property of Prx1/AhpC-type peroxiredoxins. Using biochemical reconstitution, native mass photometry, electron microscopy and live-cell assays, we demonstrate formation of heterodimers and heterodecamers, with diverse subunit stoichiometries, in peroxiredoxin pairs from different eukaryotic kingdoms. In Saccharomyces cerevisiae, oxidative challenge induces Tsa1-Tsa2 heterodecamerization with substoichiometric Tsa2 incorporation sufficing to stabilize the decameric state. Functional hetero-oligomers are also observed forming among human, plant and Leishmania peroxiredoxins. Our findings provide new insights into peroxiredoxin structural plasticity with broad implications for redox biology, stress responses and cellular adaptation, and also challenge the long-held paradigm of peroxiredoxin homo-oligomerization.