N Nature Chemical Biology · Dec 05, 2025 Accurate single-domain scaffolding of three nonoverlapping protein epitopes using deep learning De novo protein design has seen major success in scaffolding single functional motifs; however, in nature, most proteins present multiple functional sites. Here, we describe an approach to simultaneously scaffold multiple functional sites in a single-domain protein using deep learning. We designed small single-domain immunogens, under 130 residues, that present three distinct and irregular motifs from respiratory syncytial virus. These motifs together comprise nearly half of the designed proteins; hence, the overall folds are quite unusual with little global similarity to proteins in the Protein Data Bank. Despite this, X-ray crystal structures confirmed the accuracy of presentation of each of the motifs and the multiepitope design yields improved cross-reactive titers and neutralizing response compared to a single-epitope immunogen. The successful presentation of three distinct binding surfaces in a small single-domain protein highlights the power of generative deep learning methods to solve complex protein design problems. Machine learning Protein design Proteins Vaccines biology
N Nature Chemical Biology · Nov 21, 2025 Molecular basis of SAM-AMP synthesis and degradation in the type III-B CRISPR–Cas system Upon sensing nonself target RNA, the CorA-associated type III-B CRISPR–Cas system catalyzesS-adenosyl methionine (SAM) and ATP to synthesize SAM-AMP, which activates the effector CorA and triggers immune responses. SAM-AMP can be degraded by NrN and SAM lyase, potentially deactivating the system. Here we find that the type III-B effector complex fromBacteroidesfragilisuses a specific mechanism to recognize nonself target RNA and synthesize SAM-AMP. The 3′ anti-tag of nonself target RNA induces conformational changes in the Cmr2 subunit, triggering SAM-AMP synthesis independently of the stalk loop of Cmr3 subunit. SAM-AMP binding induces NrN to transit from an open to a closed conformation, enabling hydrolysis of the 3′–5′ phosphodiester bond. SAM lyase forms a triangular trimer that specifically degrades SAM-AMP into 5′-methylthioadenosine-AMP and homoserine lactone. These findings unveil unique mechanisms for SAM-AMP synthesis and degradation and provide deeper insights into the molecular basis of type III CRISPR–Cas signaling. Bacteria Biosynthesis Enzyme mechanisms Small molecules Structural biology biology
N Nature Chemical Biology · Nov 21, 2025 Dynamic metal coordination controls chemoselectivity in a radical halogenase The activation of inert C(sp3)–H bonds by nonheme Fe enzymes provides a powerful biocatalytic platform for the chemical synthesis of molecules with increased sp3 complexity. In this context, FeII/α-ketoglutarate-dependent radical halogenases are uniquely capable of carrying out transfer of a diverse array of bound anions following C–H activation. Here, we provide experimental evidence that bifurcation of radical rebound after H-atom abstraction can be driven both by the ability of a dynamic metal coordination sphere to reorganize and by a second-sphere hydrogen-bonding network where only two residues are sufficient. In addition, we present crystallographic data supporting the existence of an early peroxyhemiketal intermediate in the O2 activation pathway of FeII/α-ketoglutarate-dependent enzymes. These data provide a paradigm for understanding the evolution of catalytic plasticity in these enzymes and yields insight into the design principles by which to expand their reaction scope. Radical FeII/α-ketoglutarate-dependent halogenases are powerful biocatalysts for C–H functionalization. Here, the authors reveal the mechanistic basis for chemoselectivity in a lysine halogenase. Biocatalysis Enzyme mechanisms Enzymes biology
N Nature Chemical Biology · Nov 21, 2025 SINE compounds activate exportin 1 degradation through an allosteric mechanism Overexpression of exportin 1 (XPO1/CRM1) in cancer cells mislocalizes numerous cancer-related nuclear export cargoes. Covalent selective inhibitors of nuclear export (SINEs), including the cancer drug selinexor, restore proper nuclear localization by blocking XPO1–cargo interaction. These inhibitors also induce XPO1 degradation through the Cullin–RING E3 ligase (CRL) substrate receptor ASB8. Here we present cryo-electron microscopy structures revealing ASB8 binding to a cryptic XPO1 site that is exposed upon SINE conjugation. Unlike typical molecular glue degraders that directly bridge CRLs and substrates, SINEs bind XPO1 independently of ASB8, triggering an allosteric mechanism that enables high-affinity ASB8 recruitment, leading to XPO1 ubiquitination and degradation. ASB8-mediated degradation is also triggered by the endogenous itaconate derivative 4-octyl itaconate, suggesting that synthetic XPO1 inhibitors exploit a native cellular mechanism. This allosteric XPO1 degradation mechanism expands known modes of targeted protein degradation beyond molecular glue degraders and proteolysis-targeting chimeras of CRL4. Cancer therapy Small molecules Structural biology biology
N Nature Chemical Biology · Nov 18, 2025 Temporal photoproximity labeling of ligand-activated EGFR neighborhoods using MultiMap Photoproximity labeling proteomics (PLP) methods have recently shown that cell surface receptors can form lateral interactome networks. Here, we present a paired set of PLP workflows that dynamically track neighborhood changes for oncogenic epidermal growth factor receptor (EGFR) over time, both outside and inside of cells. We achieved this by augmenting the multiscale PLP workflow we call MultiMap, where three photoprobes with different labeling ranges were photoactivated by one photocatalyst, eosin Y, anchored extracellularly and intracellularly on EGFR. We identified hundreds of neighboring proteins that changed within minutes to over 1 h after the addition of EGF. These neighborhoods reveal dynamic interactomes during early, middle and late signaling that drive phosphorylation, internalization, degradation and transcriptional regulation. This rapid ‘molecular photographic’ labeling approach provides snapshots of signaling neighborhoods, revealing their dynamic nature and potential for drug targeting. Chemical tools Membrane proteins Proteomics Target identification biology
N Nature Chemical Biology · Nov 14, 2025 Dynamics driving the precursor in NifEN scaffold during nitrogenase FeMo-cofactor assembly Nitrogenase catalyzes atmospheric nitrogen fixation, a critical biological process that depends on an intricate organometallic cofactor assembled by a dedicated multiprotein system. Here we uncover the structural basis for the function of NifEN, the scaffold protein that mediates the final stages of cofactor biosynthesis before its incorporation into nitrogenase. High-resolution structural analyses reveal that the cofactor precursor initially binds at a surface docking site before being transferred into a specialized cavity for further maturation. This process involves dynamic structural rearrangements, including coordinated domain motions and partial unfolding, enabling the scaffold to alternate between open and closed states. Additionally, a rear channel extends to the precursor-binding cavity, likely facilitating the entry of the modifying components molybdenum and homocitrate. These findings illuminate the dynamic mechanisms underlying FeMo-cofactor assembly and underscore the functional divergence between NifEN, the biosynthetic scaffold, and NifDK, the catalytic component of nitrogenase. Enzyme mechanisms Metals Proteins X-ray crystallography biology
N Nature Chemical Biology · Nov 13, 2025 Chemical richness and diversity of uncultivated ‘Entotheonella’ symbionts in marine sponges Marine sponges are the source of numerous bioactive natural products that serve as chemical defenses and provide pharmaceutical leads for drug development. For some of the compounds, symbiotic bacteria have been established as the actual producers. Among the known sponge symbionts, ‘CandidatusEntotheonella’ members stand out because of their abundant and variable biosynthetic gene clusters (BGCs). Here, to obtain broader insights into this producer taxon, we conduct a comparative analysis on eight sponges through metagenomic and single-bacterial sequencing and biochemical studies. The data suggest sets of biosynthetic genes that are largely unique in 14 ‘Entotheonella’ candidate species and a member of a sister lineage named ‘CandidatusProxinella’. Four biosynthetic loci were linked in silico or experimentally to cytotoxins, antibiotics and the terpene cembrene A from corals. The results support widespread and diverse bacterial roles in the chemistry of sponges and aid the development of sustainable production methods for sponge-derived therapeutics. Bacteria Biosynthesis Natural products biology
