Artificial metalloenzymes (ArMs) are proteins engineered to contain metal cofactors that often catalyze chemical reactions rarely or never observed in natural biological processes. They hold promise for applications incl...Artificial metalloenzymes (ArMs) are proteins engineered to contain metal cofactors that often catalyze chemical reactions rarely or never observed in natural biological processes. They hold promise for applications including fine-chemical production, control of cellular function and therapeutics. Many of these applications are difficult to achieve due to cofactor inactivation in complex biological environments and cofactor-induced cellular stress. In this study, I explore the most recent strategies for developing robust, biocompatible ArMs that function in cell lysates, on cell surfaces or intracellularly. The pros and cons of developing and using ArMs in these three environments are described. I also examine how active ArMs might tolerate their environment, and the outstanding challenges and opportunities, including the need for simple methods of construction, improved catalytic performance and exploration of other reactions and microorganisms.
Dalwadi U, Subramanian A, Deal A
… +27 more, Conrad JE, Nadjsombati T, Venkatesh M, Boone M, Egea PF, He L, Jain N, Lee DJ, Liu Y, Reineke LC, Saito K, Talledge N, Toutkoushian H, Le Vasseur M, Zappa F, de Groot RJ, Acosta-Alvear D, Arthur CP, Nunnari J, Marqusee S, Lawrence RE, Costa-Mattioli M, Crawford JJ, van Kuppeveld FJM, Croll TI, Walter P, Frost A
The ternary complex, composed of eIF2, GTP and initiator methionyl-tRNA, delivers the first amino acid to the ribosome to initiate protein synthesis. Eukaryotic initiation factor 2B (eIF2B) catalyzes GDP to GTP exchange...The ternary complex, composed of eIF2, GTP and initiator methionyl-tRNA, delivers the first amino acid to the ribosome to initiate protein synthesis. Eukaryotic initiation factor 2B (eIF2B) catalyzes GDP to GTP exchange on eIF2, thereby setting the ternary complex level. Stress-induced phosphorylation converts eIF2 from the substrate of eIF2B into an inhibitor (eIF2-P). This conversion reduces ternary complex levels and induces the integrated stress response (ISR). Here we chart an allosteric axis running through eIF2B, revealing the importance of an α-helix in its β-subunit, the 'latch-helix', that hooks onto the α-subunit to induce eIF2B activity. eIF2-P binding promotes latch-helix unhooking, opening eIF2B, which inhibits its activity. Convergently evolved viral proteins stabilize this latch-helix-binding active state of eIF2B. Using these insights, we generated ISR-activating compounds that stabilize eIF2B in its inhibited, unlatched state. Our study thus highlights how long-range eIF2B allostery can be pharmacologically manipulated to sustain or attenuate the ISR.
Image-guided pharmacotherapy, in which drug activity is strictly confined to disease areas revealed by medical imaging modalities, holds great promise for the development of personalized medicine. A key step in realizing...Image-guided pharmacotherapy, in which drug activity is strictly confined to disease areas revealed by medical imaging modalities, holds great promise for the development of personalized medicine. A key step in realizing this potential is the development of safe and precise methods for drug activation. Electromagnetic radiation, spanning the energy ranges from γ-rays to ultraviolet-visible and infrared light, has emerged as a method of choice for this purpose. Because many imaging modalities rely on electromagnetic radiation to generate signals (for example, optical and optoacoustic imaging, computed tomography and positron emission tomography), this phenomenon can be used to both activate a drug and monitor tissue response, creating a theranostic synergy. This Perspective outlines the molecular basis for drug activation with electromagnetic radiation, offers a clinical perspective in discussing the most suitable applications and advantages available and discusses the challenges, such as the delivery of light at the site of interest, and steps needed for successful translation into daily clinical practice.
Cells cope with salt stress, hypoxia or elevated cytosolic Ca by regulating their mitochondrial Na levels. The discovery of the mitochondrial Na/Ca exchanger and its disease relevance has revealed the need to map mitocho...Cells cope with salt stress, hypoxia or elevated cytosolic Ca by regulating their mitochondrial Na levels. The discovery of the mitochondrial Na/Ca exchanger and its disease relevance has revealed the need to map mitochondrial Na in situ. Here we describe a ratiometric fluorescent reporter for Na, denoted MitRatiNa, that reports mitochondrial Na levels independent of membrane potential and in diverse cell lines. Na in individual mitochondria varies greatly and, depending on cell type, can be as low as 1-5 mM or as high as 40 mM on average. We demonstrate that mitochondrial Na increases during cytosolic Ca elevation, inhibition of glycolysis or respiration. Mitochondria in skin fibroblasts from healthy humans show a high Na population that disappears in fibroblasts of persons with mitochondrial diseases. The newfound ability to map absolute Na at the resolution of single mitochondria enables the dissection of regulatory mechanisms for mitochondrial Ca and Na and potential identification of new therapeutic avenues.
Cytochrome P450s (CYPs) constitute a superfamily of thiolate-ligated heme metalloenzymes principally responsible for the hydroxylation of unactivated C-H bonds. The proximal cysteine is an obligatory and universally cons...Cytochrome P450s (CYPs) constitute a superfamily of thiolate-ligated heme metalloenzymes principally responsible for the hydroxylation of unactivated C-H bonds. The proximal cysteine is an obligatory and universally conserved residue for the CYP enzyme class. Herein, we challenge this paradigm by systematically identifying noncanonical CYPs (ncCYPs) that do not harbor a proximal cysteine ligand. Our bioinformatic search revealed 20 distinct ncCYP families encoded in diverse microbial genomes with alternative residues at this position. We characterize a native serine-ligated CYP with a high-spin ferric resting state that catalyzes azide reduction and nitrene insertion reactions. Its crystal structure clearly shows a typical CYP fold and a serine alkoxide as a proximal heme ligand. In addition, we report the discovery and characterization of the first native selenocysteine-ligated CYP in nature. Our findings expand the CYP metalloenzyme family and provide opportunities for future enzymatic and biocatalytic discoveries.
The TIM barrel is the most prevalent fold in natural enzymes, supporting efficient catalysis of diverse reactions. While de novo TIM barrels have been designed, their minimalistic architecture lacks structural elements e...The TIM barrel is the most prevalent fold in natural enzymes, supporting efficient catalysis of diverse reactions. While de novo TIM barrels have been designed, their minimalistic architecture lacks structural elements essential for substrate binding and catalysis. Here, we present CANVAS, a computational workflow that introduces a structural lid into a minimal de novo TIM barrel to anchor catalytic residues and form an active site. Starting from two scaffolds, we designed nine variants with tailored lids for the Kemp elimination. Four showed measurable activity, with the most active reaching a catalytic efficiency of 21,000 M s. A cocrystal structure with a transition-state analog confirmed the accuracy of the designed lid and active site. Using the structure of a lower-activity variant, we applied ensemble-based design, increasing catalytic efficiency >1,600-fold to 32,000 M s. These results demonstrate that de novo TIM barrels can be endowed with efficient catalytic function, establishing a platform for building enzymes from minimal protein scaffolds.
Yu J, Sheng X, Fan Z
… +18 more, Wang Z, Cao D, Tang Y, Hao Y, Zhang Y, Shao P, Ma H, Cao T, Zeng C, Rao J, Chen M, Chen K, Li X, Teng D, Luo X, Wang M, Zhang S, Zheng M
Accelerating molecular probe discovery and lead optimization requires accurate and efficient binding affinity prediction. Here we present PBCNet2.0, a Cartesian tensor-based Siamese neural network for protein-ligand rela...Accelerating molecular probe discovery and lead optimization requires accurate and efficient binding affinity prediction. Here we present PBCNet2.0, a Cartesian tensor-based Siamese neural network for protein-ligand relative binding affinity prediction. Trained on 8.6 million protein-ligand complex pairs, PBCNet2.0 achieves zero-shot accuracy similar to computationally intensive physics-based simulations while remaining highly efficient. Retrospective prioritization experiments show that PBCNet2.0 improves optimization efficiency by 7.18-fold and reduces resource use by 41%. Mechanistic analyses indicate that the model captures intermolecular interactions and encodes spatial geometric constraints, enabling sensitivity to subtle effects such as fluorine orthogonal multipolar interactions. Notably, although not trained on mutation data, PBCNet2.0 exhibits an emergent capability to predict affinity changes induced by binding pocket residue variations, supporting resistance analysis. We prospectively validated these capabilities on ENPP1 and ALDH1B1, accurately resolving affinity shifts from minor interaction and conformational differences and identifying critical binding residues with a hit rate of five out of six selected residues.
Numerous biomolecular condensates coexist within cells, yet the factors governing their miscibility remain poorly understood. Here, by examining 28 intrinsically disordered regions in 378 pairwise combinations, we identi...Numerous biomolecular condensates coexist within cells, yet the factors governing their miscibility remain poorly understood. Here, by examining 28 intrinsically disordered regions in 378 pairwise combinations, we identify key sequence determinants of condensate miscibility: serine and aromatic residues promote miscibility, while charged amino acids drive immiscibility. Mutagenesis experiments establish these as causal relationships. Protein-protein interaction network analyses and molecular simulations reveal that serine and aromatic residues favor heterotypic interactions, whereas high charge content reinforces homotypic association. Serine phosphorylation acts as a regulatory switch that shifts this balance, altering condensate miscibility. We further show that miscibility between transcription factor (TF) and RNA polymerase II (Pol II) condensates directly influences transactivation, TFs with high overall charge content display reduced Pol II miscibility and impaired transcriptional output, and modulating charged residue content in TFs correspondingly tunes transcription. These findings establish a residue-level grammar for predicting and engineering condensate miscibility.
Protease-mediated activation of immune effectors is an evolutionarily conserved mechanism. This study identifies a widespread trypsin-MBL (metallo-β-lactamase) module as a core effector in diverse antiviral bacterial imm...Protease-mediated activation of immune effectors is an evolutionarily conserved mechanism. This study identifies a widespread trypsin-MBL (metallo-β-lactamase) module as a core effector in diverse antiviral bacterial immune systems, such as Hachiman, AVAST and Argonaute. Focusing on the Hachiman-associated trypsin-MBL system, we show that trypsin•HamAB protease activity is inhibited by ATP, while MBL is an autoinhibited DNase with two insertion loops obstructing its catalytic site. Upon infection, trypsin•HamAB senses foreign DNA and hydrolyzes ATP, activating trypsin-like activity, which specifically cleaves MBL at the insertion loops to release repression. The activated MBL depletes DNA and arrests host cell growth. Cryo-electron microscopy structures of trypsin•HamAB-DNA reveal that DNA binding and ATP hydrolysis trigger HamAB oligomerization and trypsin-like domain release, enabling its activation. Our work elucidates a conserved immune mechanism wherein proteolytic activation of a nuclease enables robust immunity against phage while multilayered controls prevent self-toxicity, expanding the repertoire of immune processes governed by regulatory proteolysis.
Bioorthogonal reactions have revolutionized molecular biology through the conjugation of molecules within cellular environments. However, classical bioorthogonal reagents often suffer from nonspecific reactivity across d...Bioorthogonal reactions have revolutionized molecular biology through the conjugation of molecules within cellular environments. However, classical bioorthogonal reagents often suffer from nonspecific reactivity across diverse physiological contexts, diminishing their precision. This limitation presents considerable challenges in complex biological systems where multiple cell types coexist. Here we demonstrate tetrazine release and activation by cellular enzymes (TRACE), a method enabling cell-type-specific bioorthogonal chemistry. TRACE uses caged dihydrotetrazine derivatives, which remain inert until activated by specific cellular enzymes. Optimizing the electronic properties of the dihydrotetrazine scaffold enables rapid uncaging and activation of tetrazines within minutes. We demonstrate the utility of TRACE for the targeted release of cytotoxic drugs, selectively impacting the viability of enzyme-expressing cells in cocultures. Additionally, our method facilitates the delivery of imaging agents to subcellular structures in an enzyme-activity-dependent manner. TRACE represents a promising approach for programmable bioorthogonal chemistry in therapeutic and imaging applications.