Angewandte Chemie International Edition

This study introduces SMART (Single Molecule Assay on Ribonucleic Acid by Translated Product), an innovative in vitro platform integrating mRNA display, next-generation sequencing, and bioinformatics to address several key challenges in enzyme engineering. The characteristic feature of SMART is its auxiliary unit, attached to the mRNA-displayed enzyme library to mediate the biotinylation of reactive enzyme variants and enable their enrichment by streptavidin beads pull-down. The unit comprises a hairpin single-stranded DNA that hybridizes with the mRNA and anchors a DNA-binding protein, single-chain Cro, which carries functional auxiliary molecules specific to the enzyme of interest. Here, we report on the establishment of SMART for the engineering of oxidases, specifically D-amino acid oxidase (DAAO) from Schizosaccharomyces pombe, with engineered ascorbate peroxidase 2 as its auxiliary enzyme. In a single selection round of a DAAO library with randomized catalytic residue Y232 and D-Alanine as the substrate, we identified several catalytically active enzyme variants with altered substrate specificity in a matter of days. This surpasses traditional enzyme engineering methods in speed, cost, library capacity, and precision. This research underscores the potential of SMART for the engineering of various oxidases, and other enzymes after the corresponding adjustment of the auxiliary unit.

Molecular switches that respond to a biochemical stimulus in cells have proven utility as a foundation for developing molecular sensors and actuators that could be used to address important biological questions. Developing a molecular switch unfortunately remains difficult as it requires elaborate coordination of sensing and actuation mechanisms built into a single molecule. Here, we rationally designed a molecular switch that changes its subcellular localization in response to an intended stimulus such as an activator of protein kinase A (PKA). By arranging the sequence for Kemptide in tandem, we designed a farnesylated peptide whose localization can dramatically change upon phosphorylation by PKA. After testing a different valence number of Kemptide as well as modulating the linker sequence connecting them, we identified an efficient peptide switch that exhibited dynamic translocation between plasma membranes and internal endomembranes in a PKA activity dependent manner. Due to the modular design and small size, our PKA switch can have versatile utility in future studies as a platform for visualizing and perturbing signal transduction pathways, as well as for performing synthetic operations in cells.

Phenylalanine ammonia-lyases (PALs) deaminate L-phenylalanine to trans-cinnamic acid and ammonium and have idespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis (Trichormus variabilis) has garnered significant attention as the active ingredient in Pegvaliase(R), the only FDA-approved drug treating classical phenylketonuria (PKU). Although an extensive body of literature exists on structure, substrate-specificity, and catalytic mechanism, protein-wide sequence determinants of function remain unknown, which limits the ability to rationally engineer these enzymes. Previously, we developed a high-throughput screen (HTS) for PAL, and here, we leverage it to create a detailed sequence-function landscape of PAL by performing deep mutational scanning (DMS). Our method revealed 79 hotspots that affected a positive change in enzyme fitness, many of which have not been reported previously. Using fitness values and structure-function analysis, we picked a subset of residues for comprehensive single- and multi-site saturation mutagenesis to improve the catalytic activity of PAL and identified combinations of mutations that led to improvement in reaction kinetics in cell-free and cellular contexts. To understand the mechanistic role of the most beneficial mutations, we performed QM/MM and MD and observed that different mutants confer improved catalytic activity via different mechanisms, including stabilizing first transition and intermediate states and improving substrate diffusion into the active site, and decreased product inhibition. Thus, this work provides a comprehensive sequence-function relationship for PAL, identifies positions that improve PAL activity when mutated and assesses their mechanisms of action.

Glycoside hydrolases (GHs) play central roles in carbohydrate metabolism and are widely exploited for industrial and biomedical applications. However, they are often not optimal for applications due to their constrained function and strict stereochemical specificity, necessitating the discovery and optimization of distinct enzymes for each glycosidic configuration. Members of glycoside hydrolase family 1 (GH1) are archetypal retaining {beta}-glycosidases, while -specific activity is rare within this family. Here, I demonstrate that a retaining GH1 enzyme can be engineered to hydrolyze both {beta}- and -configured substrates without altering its canonical catalytic residues. Using a well-characterized {beta}-glycosidase and computational protein design strategies targeting second-shell residues surrounding the active site, a bifunctional {beta}-/-glycosidase containing 45 mutations was generated. The engineered variant acquired the ability to hydrolyze the -configured substrate 4-nitrophenyl--D-glucopyranoside while retaining activity toward the originals {beta}-substrates, with reduced catalytic efficiency and thermostability. Structural modeling and docking analyses reveal that the engineered enzyme preserves the original fold and accommodates substrates within the catalytic pocket in a similar manner to the wild type. These findings provide direct evidence that stereochemical constraint in retaining GH is more flexible than previously appreciated and can be modulated through targeted engineering.

Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects.1,2 Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health.1 However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study.3 Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-Map method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics.4 We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-Map improves our fundamental understanding of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

Non-invasive, real-time, longitudinal imaging of protein functions in living systems with unprecedented specificity is one of the critical challenges of modern biomedical research. Despite several advancements, it is estimated that nearly 35% of the human proteome is not completely characterized. Therefore, the development of new technologies is imperative for shining more light on so-called "dark proteomes". Towards that goal, here we report a platform fusion technology called activity-based protein profiling-bioluminescence resonance energy transfer (ABPP-BRET). This method provides an opportunity to study the post-translational modification of a target protein in real-time in living systems in a longitudinal manner with a high spatio-temporal resolution. This semi-synthetic BRET biosensor method is used for target engagement studies and further for inhibitor profiling in live cells. The simplicity of this method coupled with the critical physical distance dependent BRET read-out turned out to be a powerful method, thus pushing the activity-based protein profiling technology to the next level.

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

Phage therapy is a promising treatment for multidrug-resistant bacterial infections. Due to their high host specificity, phages must be matched to the target clinical strains. Efficiently identifying appropriate phages and producing sufficient titres for clinical use requires comprehensive phage libraries and multiple propagation hosts. An idealised system would use a highly promiscuous bacterial host to isolate a broader range of phages and streamline optimised phage production. Anti-phage defences constrain bacterial host promiscuity, such as restriction-modification systems that recognise and cleave foreign DNA. Here, the type I restriction endonuclease, HsdR, was deleted from Pseudomonas aeruginosa PAO1 to make a more promiscuous phage isolation and propagation host. Removal of this endonuclease more than doubled the efficiency of phage propagation and yielded seven times more phages from freshwater samples than wildtype PAO1 - an important step in producing an optimised P. aeruginosa strain for isolating and propagating phages for clinical phage therapy. Graphical abstract(Created in BioRender.com) [A] The type I R-M system comprises three subunits HsdS, HsdM and HsdR. One HsdS and two HsdM subunits form the methyltransferase, and the addition of two HsdR subunits to this complex forms the restriction endonuclease. [B] In the wildtype PAO1, the type I R-M system destroys phage DNA while protecting the host. The HsdS subunit of the restriction endonuclease binds to unmethylated recognition sequences. Then, HsdR translocates the DNA, pulling it together in both directions until it collides with another restriction endonuclease, cleaving the DNA and preventing phage proliferation. The methyltransferase protects the host DNA through the methylation of recognition sequences, preventing the restriction endonuclease from binding. [C] In {Delta}hsdR, the restriction endonuclease cannot form, preventing phage DNA cleavage and increasing phage proliferation. The methyltransferase is still active, so both host and phage DNA are methylated, providing phage progeny with protection from R-M systems in future hosts. O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/656992v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@9418f8org.highwire.dtl.DTLVardef@f3703dorg.highwire.dtl.DTLVardef@4dcde6org.highwire.dtl.DTLVardef@8d6fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Global detection and identification of protein post-translational modification (PTM) is a major bottleneck due to its dynamic property and rather low abundance. Tremendous efforts have been since made to develop antibody-based immunoaffinity enrichment or bioorthogonal chemistry-based chemical reporter approach but both suffer from inherent limitations. Following our previously reported steric-free tagging strategy, we hereby report the invention of selenol as a new generation of fluorine-displacement probe. The fluorine-selenol based displacement reaction enabled us to efficiently label and image acetylation and glycosylation at cellular level. We further pursued FSeDR in tandem with SILAC based quantitative proteomics to globally profile acetylation substrate proteins in a representative prostate cancer cell line PC3. Our results unraveled the fluorine-based toolbox for powerful chemical biology probing and allow for the future study of PTMs in a systemic manner.

The human kinome contains >500 protein kinases, and regulates up to 30% of the proteome. Kinase study is currently hindered by a lack of in vivo analysis approaches due to two factors: our inability to distinguish the kinase reaction of interest from those of other kinases in live cells and the cell impermeability of the ATP analogs. Herein, we tackled this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the method, was used as our initial prototype. Briefly, enlargement of the ATP binding pocket, by mutating the gate-keeper Methionine residue to a Glycine, prompted the mutant AKT1 to preferentially use the bulky ATP analog N6-Benzyl-ATP-{gamma}-S (A*TP{gamma}S) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The lipid/calcium/phosphate (LCP) nanoparticle was used for efficient intracellular delivery of A*TP{gamma}S, overcoming the cell impermeability issue. The mutant, but not wild-type, AKT1 used the delivered A*TP{gamma}S for autophosphorylation and phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.

Guanine Exchange Factors (GEF) of the Dbl family are the main activators of RhoA GTPases. GEF and GTPase activity is tightly regulated at the subcellular level with fast kinetics. Therefore, to fully understand the function of Dbl GEFs requires their study in living cells. Towards developing molecular tools that reversibly and rapidly modulate the activity of endogenous GEFs in living cells, here we developed a general platform for engineering inhibitors against members of the Dbl family of GEFs using generative protein design. Engineered proteins showed high affinity and remarkable specificity for the target GEFs and modulated GEF activity both in vitro and in cells. In a proof-of-principle example, a GEF inhibitor was coupled to a light-activated module, enabling the optogenetic control of its activity in cells. These findings show that generative protein design can create modulators of intracellular signaling and broaden the range of tools available for biological research.

Programmable site-specific nucleases have revolutionized the genome editing. However, these systems still face challenges such as guide dependency, delivery issues, and off-target effects. Harnessing the natural functions of structure-guided nucleases offer promising alternatives for generating site-specific double-strand DNA breaks. Yet, structure-guided nucleases require precise reaction conditions and validation for in-vivo applicability. To address these limitations, we developed the PNA-Coupled FokI-(d)RusA (PC-FIRA) system. PC-FIRA combines the sequence-specific binding ability of peptide nucleic acids (PNAs) with the catalytic efficiency of FokI nuclease fused to a structurally-guided inactive RusA resolvase (FokI-(d)RusA). This system allows for precise double-strand DNA breaks without the constraints of existing site-specific nuclease and structure-guided nucleases. Through in vitro optimizations, we achieved high target specificity and cleavage efficiency. This included adjusting incubation temperature, buffer composition, ion concentration, and cleavage timing. Diverse DNA structures, such as Holliday Junctions, linear, and circular DNA, were tested demonstrating the potential activity on different target forms. Further investigation has revealed the PC-FIRA system capacity for facilitating the precise deletion of large DNA fragments. This can be useful in cloning, large-fragment DNA assembly, and genome engineering, with promising applications in biotechnology, medicine, agriculture, and synthetic biology.

Nonribosomal peptide synthetases (NRPSs) assemble bioactive peptides from an enormous repertoire of building blocks. How binding pocket residues of the nonribosomal adenylation domain, the so-called specificity code, determine which building block becomes incorporated has been a landmark discovery in NRPS enzymology. While specificity codes enable the prediction of substrate specificity from protein sequence, design strategies based on rewriting the specificity code have been limited in scope. An important reason for failed NRPS design has been that multispecificity has not been considered, for a lack of suitable assay formats. Here, we employ a multiplexed hydroxamate specificity assay (HAMA) to determine substrate profiles for mutant libraries of A-domain in the termination module the SrfAC of surfactin synthetase. A generalist version of SrfAC is developed and the functional flexibility of the adenylation reaction is probed by fully randomizing 15 residues in and around the active site. We identify mutations with profound impact on substrate selectivity and thus reveal a remarkable evolvability of A-domains. Statistical analysis of the specificity divergence caused by point mutations has determined the impact of each code position on specificity, which will serve as a roadmap for NRPS engineering. The shortness of evolutionary pathways between NRPS specificities explains the rich natural substrate scope and suggests directed evolution guided by A-domain promiscuity as a promising strategy.

Enzymes are instrumental to life and key actors of pathologies, making them relevant drug targets. Most enzyme inhibitors consist of small molecules. Although efficient, their development is long, costly and can come with unwanted off-targeting. Substantial gain in specificity and discovery efficiency is possible using biologicals. Best exemplified by antibodies, these drugs derived from living systems display high specificity and their development is eased by harnessing natural evolution. Aptamers are nucleic acids sharing functional similarities with antibodies while being deprived of many of their limitations. Yet, the success rate of inhibitory aptamer discovery remained hampered by the lack of an efficient discovery pipeline. In this work, we addressed this issue by introducing an ultrahigh-throughput strategy combining in vitro selection, microfluidic screening and bioinformatics. We demonstrate its efficiency by discovering a modified aptamer that specifically and strongly inhibits SPM-1, a beta-lactamase that remained recalcitrant to the development of potent inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/608213v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b15e8forg.highwire.dtl.DTLVardef@b7259forg.highwire.dtl.DTLVardef@6f24borg.highwire.dtl.DTLVardef@d4f48_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lysosomes are central catabolic organelles involved in lipid homeostasis and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions as well as their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photo-crosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and LIMP-2/SCARB2, also directly bind to sphingosine. In addition, we showed that absence of either protein leads to lysosomal sphingosine accumulation which suggests a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.

Enzymes offer unparalleled selectivity and sustainability for chemical synthesis, yet their widespread industrial application is often hindered by the slow and uncertain process of discovering and optimizing suitable biocatalysts. While directed evolution remains the gold standard for enzyme optimization, its success hinges on the availability of a starting enzyme with measurable activity, a persistent bottleneck for many desired functions. Designing libraries likely to contain such functional starting points remains a major challenge. In this work, we use the GenSLM protein language model (PLM) along with a series of filters to generate novel sequences of the {beta}-subunit of tryptophan synthase (TrpB) that express in Escherichia coli, are stable, and are catalytically active in the absence of a TrpA partner. Many generated TrpBs also demonstrated significant substrate promiscuity, accepting non-canonical substrates typically inaccessible to natural TrpBs. Remarkably, several outperformed both natural and laboratory-optimized TrpBs on native and non-canonical substrates. Comparative analysis of the most active and promiscuous generated TrpB and its closest natural homolog confirmed that this enhanced functional versatility does not stem from the natural enzyme, highlighting the creative potential of generative models. Our results demonstrate that the model can generate enzymes which not only preserve natural structure and function but also acquire non-natural properties, establishing PLMs as powerful tools for biocatalyst discovery and engineering, with the potential in some cases to bypass further optimization.

The dynamics of protein subcellular localization are intricately regulated, requiring new tools for controlling protein translocation to uncover the biological significance of post-translational regulation of protein localization. Here, we developed a new method, Protein Rerouting via INtein-mediated Trans-Splicing (PRINTS), which enables precise control of protein translocation across diverse subcellular compartments. By reconstituting functional signaling peptides, PRINTS can efficiently relocalize fluorescent proteins to the 26S proteasome, nucleus, mitochondria, plasma membrane, endomembrane organelles, and liquid-liquid phase separation (LLPS) condensates. Furthermore, we incorporated the optically regulated dimerization domain CRY2clust into the PRINTS system, achieving light-mediated control of protein entry into the cell membrane and LLPS membraneless compartments. Strikingly, we observed that HNRNPA1 promiscuously recruit CRY2- or intein-containing proteins into LLPS condensate, whereas FXR1, another LLPS protein showed minimal non-specific activity. PRINTS also allow organelle relocalization, such as LLPS condensates or mitochondria to cell membrane in a light-controllable manner. Overall, PRINTS provides a versatile and robust platform for manipulating protein and organelle subcellular translocations, offering a powerful tool to investigate the regulatory coordination and crosstalk between membranous and membraneless compartments in response to physiological needs.

Modern drug discovery demands efficient strategies for generating structurally diverse compound libraries. Skeletal editing--a transformative paradigm enabling precise atom-level modifications within molecular frameworks, offers a sustainable alternative to traditional synthetic routes. While carbene insertion-mediated approaches have dominated single-carbon insertion strategies, current methodologies are limited by their reliance on hazardous, unstable carbene precursors and harsh reaction conditions. Herein, we report a multicopper oxidase (MCO)-catalyzed skeletal editing that enables the direct, one-step transformation of phenolic and indole derivatives into functionalized tropones and quinoline analogues through exogenous single-carbon insertion. This platform employs stable and safe nitroalkanes as carbon sources and O2 as the sole terminal oxidant. It accommodates a broad substrate scope and yields products with superior antibacterial activity against to multidrug-resistant strains relative to their parent compounds. This work introduces the first biocatalytic platform for exogenous single-carbon insertion skeletal editing. This sustainable and scalable strategy overcomes key limitations of synthetic approaches, offering efficient skeletal remolding and rapid expansion of bioactive compound libraries. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/714988v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@ed9336org.highwire.dtl.DTLVardef@15beeeaorg.highwire.dtl.DTLVardef@a26525org.highwire.dtl.DTLVardef@19e7707_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1, a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer (R)-1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the (S)-1 enantiomer was >100-fold less active. The (R)-1 enantiomer also demonstrated high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of (R)-1 to nsP2hel protein was confirmed by 19F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of (R)-1, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate (R)-1 (RA-NSP2-1) as a high-quality chemical probe and (S)-1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.

SCFSkp2/Cks1 is an E3 ubiquitin ligase, whose substrate specificity is determined by the oncogenic F-box protein Skp2 and the adaptor protein Cks1. A principal target of SCFSkp2/Cks1 is the cyclin-dependent kinase inhibitor p27. Elevated levels of Skp2 and reduced levels of p27 are common in a variety of cancers, and there is consequently a need to develop effective inhibitors of the Skp2-p27 interaction. However, conventional small-molecule approaches are challenging due to the extended bi-molecular interface that spans both Skp2 and Cks1, the lack of suitable binding pockets on this surface, and the intrinsically disordered nature of p27. Here, we develop macrocyclic peptides capable of binding to SCFSkp2/Cks1 with nanomolar affinities, an enhancement of almost two orders of magnitude over the natural p27 peptide. We show that these macrocyclic peptides inhibit p27 ubiquitination in vitro, restore p27 levels in a breast cancer cell line, and reduce cell proliferation.