JACS Au

Protein aggregation and inactivation upon surface immobilization are major limiting factors for analytical applications in biotechnology related fields. Protein immobilization on solid surfaces often requires multi-step surface passivation which is time consuming and inefficient. Herein, we have discovered that biomolecular condensates of biologically active human serum transferrin (Tf) can effectively prevent surface-induced fibrillation and preserve the native-like conformation of phase separated Tf over a period of 30-days. It has been observed that macromolecular crowding promotes homotypic liquid-liquid phase separation (LLPS) of Tf through enthalpically driven multivalent hydrophobic interactions possibly via the involvement of its low complexity domain (residue 3-20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated reentrant phase separation in a broad range of salt concentrations (0-3 M) solely via the involvement of hydrophobic interactions. Notably, no liquid-to-solid-like phase transition has been observed over a period of 30-days, suggesting the intact conformational integrity of phase separated Tf as revealed from single droplet Raman, circular dichroism, and Fourier transform infrared spectroscopy measurements. More importantly, we discovered that the phase separated condensates of Tf completely inhibit the surface-induced fibrillation of Tf, illustrating the protective role of these liquid-like condensates against denaturation and aggregation of biomolecules. The cell mimicking aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of biomolecules. Our present study highlights an important functional aspect of biologically active protein condensates and may have wide-ranging implications in cell physiology and biotechnological applications.

Atypical Chemokine Receptor 3 (ACKR3) is a G protein-coupled receptor that does not signal through G proteins. It is known as a chemokine scavenger involved in various pathologies, making it an appealing yet intriguing therapeutic target. Indeed, the structural properties that govern ACKR3 functional selectivity and the overall conformational dynamics of ACKR3 activation are poorly understood. Here we combined Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics simulations to examine the binding mode and mechanism of action of various small-molecule ACKR3 ligands of different efficacy for {beta}-arrestin recruitment. Our results show that activation or inhibition of ACKR3 is largely governed by intracellular conformational changes of helix 6, intracellular loop 2 and helix 7, while the DRY motif becomes protected during both processes. Moreover, HDX-MS identifies the binding sites and the allosteric modulation of ACKR3 upon {beta}-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small-molecule ligands, the overall activation dynamics of ACKR3, the binding mode of {beta}-arrestin 1 and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

HMGB1 (high-mobility group box 1) protein is a nonhistone chromatin-associated protein that has been widely reported to be a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in proinflammatory process once it is in an extracellular location. Accumulating evidence has shown HMGB1 undergoes extensive PTMs that remarkably regulated its conformation, localization, and intermolecular interaction. However, the PTMrelated study has been dramatically hindered by the difficulty to access to homogenous proteins with site-specific PTMs of interest. Here, we introduce a protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation, STL/CPL). This methodology has enabled us to generate N-terminal acetylated HMGB1 proteins in high purity. Further studies revealed that the acetylation on N-terminus regulates its interaction with heparin and modulates its stability, representing a regulatory switch to control the HMGB1s activity.

The cat eye syndrome chromosome region candidate 2 (CECR2) protein is an epigenetic regulator involved in chromatin remodeling and transcriptional control. The CECR2 bromodomain (CECR2-BRD) plays a pivotal role in directing the activity of CECR2 through its capacity to recognize and bind acetylated lysine residues on histone proteins. This study elucidates the binding specificity and structural mechanisms of CECR2-BRD interactions with both histone and non-histone ligands, employing techniques such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and a high-throughput peptide assay. The CECR2-BRD selectively binds acetylated histone H3 and H4 ligands, exhibiting a preference for multi-acetylated over mono-acetylated targets. The highest affinity was observed for tetra-acetylated histone H4. Neighboring post-translational modifications, including methylation and phosphorylation, modulate acetyllysine recognition, with significant effects observed for histone H3 ligands. Additionally, this study explored the interaction of the CECR2-BRD with the acetylated RelA subunit of NF-{kappa}B, a pivotal transcription factor in inflammatory signaling. Dysregulated NF-{kappa}B signaling is implicated in numerous pathologies, including cancer progression, with acetylation of RelA at lysine 310 (K310ac) being critical for its transcriptional activity. Recent evidence linking the CECR2-BRD to RelA suggests it plays a role in inflammatory and metastatic pathways, underscoring the need to understand the molecular basis of this interaction. We found the CECR2-BRD binds to acetylated RelA with micromolar affinity, and uses a distinctive binding mode to recognize this non-histone ligand. These results provide new insight on the role of CECR2 in regulating NF-{kappa}B-mediated inflammatory pathways. Functional mutagenesis of critical residues, such as Asn514 and Asp464, highlight their roles in ligand specificity and binding dynamics. Notably, the CECR2-BRD remained monomeric in solution and exhibited differential conformational responses upon ligand binding, suggesting adaptive recognition mechanisms. Furthermore, the CECR2-BRD exclusively interacts with nucleosome substrates containing multi-acetylated histones, emphasizing its role in transcriptional activation within euchromatic regions. These findings position the CECR2-BRD as a key chromatin reader and a promising therapeutic target for modulating transcriptional and inflammatory processes, particularly through the development of selective bromodomain inhibitors. HIGHLIGHTSO_LIThe CECR2 bromodomain recognizes a range of combinatorial PTMs on the histone H3 and H4 N-terminal tails. C_LIO_LIThe CECR2 bromodomain binds to an acetylated RelA ligand with micromolar affinity. C_LIO_LINMR perturbation studies delineate the distinct binding modes driving CECR2-BRD recognition of histone versus non-histone ligands. C_LIO_LISite-directed mutagenesis reveals the specificity determinants of CECR2-BRD ligand binding. C_LIO_LIThe bromodomain of CECR2 exhibits a strong interaction with multi-acetylated nucleosomes. C_LI

Nuclear receptors (NRs) comprise a superfamily of (ligand-)regulated transcription factors that are pivotal in orchestrating gene networks essential for development, metabolism, and cellular homeostasis. Their activity is critical for normal physiology, and consequently, dysregulation of NR signaling is implicated in a wide array of human diseases. Within this superfamily, the orphan nuclear receptor Nur77 and the glucocorticoid receptor (GR) are key regulators that exhibit significant crosstalk, primarily antagonistic, which is crucial for modulating inflammatory and stress responses. Despite the recognized importance of their interplay, the precise molecular mechanisms by which GR modulates Nur77s engagement with DNA remain incompletely defined. This study elucidates the direct impact of GR and its ligand, dexamethasone (Dex), on the DNA binding dynamics of Nur77. Single-molecule DNA tightrope assays revealed that Nur77 employs a three-dimensional diffusion-based search mechanism on non-specific DNA, characterized by transient interactions with two distinct dissociation kinetic profiles. GR significantly stabilizes Nur77-DNA interactions, evidenced by a shift towards longer residence times, primarily achieved by slowing the dissociation of the more transiently interacting Nur77 population. Conversely, single-molecule analysis and biochemical assays demonstrated that Dex alone markedly reduces Nur77s overall DNA binding affinity kinetics and frequency in a sequence-dependent manner, to such an extent that accurate quantification was unfeasible. These findings delineate distinct modulatory effects of the GR protein and its ligand on Nur77-DNA interactions, providing crucial biophysical insights into their complex regulatory interplay and revealing a direct, GR-independent impact of dexamethasone on Nur77s DNA engagement. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/685035v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1207cdorg.highwire.dtl.DTLVardef@1be29eborg.highwire.dtl.DTLVardef@1b23eaaorg.highwire.dtl.DTLVardef@13030b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

A current challenge in the field of life sciences is to decipher, in their native environment, the functional activation of cell surface receptors upon binding of complex ligands. Lack of suitable nanoscopic methods has hampered our ability to meet this challenge in an experimental manner. Here, we use for the first time the interplay between atomic force microscopy, steered molecular dynamics and functional assays to elucidate the complex ligand-binding mechanism of C5a with the human G protein-coupled C5a receptor (C5aR). We have identified two independent binding sites acting in concert where the N-terminal C5aR serves as kinetic trap and the transmembrane domain as functional site. Our results corroborate the two-site binding model and clearly identify a cooperative effect between two binding sites within the C5aR. We anticipate that our methodology could be used for development and design of new therapeutic agents to negatively modulate C5aR activity.

ABSTRACTLegumains are cysteine proteases that, in addition to their canonical hydrolase function, can act as peptide ligases or transpeptidases. In humans, this activity becomes particularly relevant under pathophysiological conditions, where legumain relocalizes to near-neutral pH compartments favoring ligation/transpeptidation over hydrolysis. Here, we combined in vitro positional scanning with in silico substrate profiling to elucidate the substrate determinants governing human legumain-mediated peptide cyclization. We identified glycine residues at P1'' and P1' and basic residues at P2'/P2'' as key determinants of human legumain-mediated peptide cyclization. Guided by these insights, we designed an optimized substrate exhibiting substantially enhanced cyclization efficiency. Computational analysis not only recapitulated the experimental observations but also predicted a covalent inhibition mechanism involving a P1' cysteine, revealed a kcat-tuning switch embedded within the substrate, and highlighted its potential for developing high-performance fluorogenic substrates. Collectively, these findings advance the mechanistic understanding of legumains transpeptidase activity and provide a framework for developing selective probes and inhibitors across the legumain family and related cysteine proteases. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/693912v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@121ffcborg.highwire.dtl.DTLVardef@120a587org.highwire.dtl.DTLVardef@536fc7org.highwire.dtl.DTLVardef@1cfaa67_HPS_FORMAT_FIGEXP M_FIG C_FIG

ROR{beta} is an understudied nuclear receptor recently implicated in numerous pathologies. Using immunoprecipitation mass spectrometry, the transcription factor coregulatory protein and histone acetyltransferase p300 (EP300) was identified as a direct interacting protein of ROR{beta}. Crosslinking mass spectrometry (XL-MS) confirmed that p300 interacts with the DNA binding domain (DBD), hinge region, and ligand binding domain (LBD) of ROR{beta}. Both p300 and SIRT1 impact the turnover rate and transcriptional activity of ROR{beta}. p300-dependent acetylation sites were found to be constrained to the DBD and hinge region of ROR{beta} and were deacetylated by SIRT1. Sites that were ubiquitinated in the presence of p300 and SIRT1 were also discovered, with some sites sensitive to proteasome inhibitor. A constitutively acetylated mimic at K176 in the hinge prevented ubiquitination of ROR{beta} at distal sites. Uncovering regulatory mechanisms of ROR{beta} via protein interactions and PTMs will provide strategies for development of therapeutics targeting the receptor. Teaserp300 and SIRT1 work in concert to coordinate posttranslational modifications of the nuclear receptor ROR{beta}. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/621067v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1c95ed8org.highwire.dtl.DTLVardef@171f144org.highwire.dtl.DTLVardef@196d643org.highwire.dtl.DTLVardef@1ccd3c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Platinum anticancer drugs tend to target DNA whereas certain ruthenium and osmium organometallic compounds, including those with known anticancer activity, preferentially bind histone proteins in chromatin. We earlier found that Ru/Os arene 2-pyridinecarbothioamide antitumor agents display unique or partially overlapping profiles of histone protein binding in the nucleosome compared to Ru arene phosphaadamantane (RAPTA) antimetastasis drugs, but the basis for this difference is unclear. Here we structurally characterized the nucleosome binding effects of arene ligand substitutions and carried out a multiscale simulation analysis, which reveals that the interplay between metal cation and non-leaving ligand identity dictates adduct stability and whether complexes target electronegative surface patches, internal crevices, or both. We show that the nucleosome superhelical crevice acts as a small molecule selectivity filter and that multi-site binding profiles can be expanded or reduced through defined ligand substitutions, which modulate dynamic and steric attributes. Our findings suggest new avenues for rationally developing Ru/Os organometallics that could help expand the scope of chromatin-targeting therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/688318v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@188bc1dorg.highwire.dtl.DTLVardef@1f619d5org.highwire.dtl.DTLVardef@1a0ebaorg.highwire.dtl.DTLVardef@bd0abb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the major neglected diseases globally, killing 12,000 people per year and silently infecting an estimated 7 million people worldwide. Current diagnostic methods are limited by cost, complexity and cold-chain requirements. The T. cruzi flagellar protein Tc24 is a promising antigen for serological tests but suffers of poor solubility and heat stability. Here, we computationally engineered and produced three variants of Tc24, which exhibit remarkable heat stability, up to 69{degrees}C, and express with higher solubility in E. coli compared to the wild-type protein, reducing production costs, eliminating the need for a cold chain and therefore facilitating cost-effective production and storage without refrigeration and even in lyophilised form. These variants remained remarkably stable for 70 days in solution at 25{degrees}C and successfully detected antibodies in human sera samples from Chagas disease patients from Northern and Southern regions of Latin America, demonstrating the preservation of their antigenicity. The best-performing engineered variant was incorporated into a prototype of lateral flow test, demonstrating potential for rapid, affordable and accessible Chagas disease diagnostics in resource-limited settings.

Affinity purification is a essential technique for isolating highly purified proteins; however, generating affinity ligands require significant time and financial investment. To address these limitations, this study proposes a novel affinity chromatography method utilizing in silico-designed cyclic peptides as ligands. Targeting Complement C1q (C1q), a plasma protein that plays crucial roles in classical complement pathway, we employed the biomolecular structure prediction model, AlphaFold2, to design specific binding cyclic peptides. Based on these designs, we synthesized lariat-type cyclic peptides characterized by disulfide cyclization and biotinylation, which were subsequently immobilized on streptavidin carriers. Performance tests confirmed that the resulting column specifically captured C1q, allowing for elution via a standard NaCl concentration gradient. Notably, high selectivity was preserved even in the presence of plasma, underscoring the ligands practical robustness. By overcoming traditional constraints through (1) rapid and simple design, (2) high specificity, and (3) universal versatility without genetic modification, this de novo design strategy represents a potential breakthrough in protein purification technologies. HighlightsO_LIAI-driven de novo design generated a specific cyclic peptide ligand for Complement C1q C_LIO_LIThe synthetic ligand enabled one-step purification of Complement C1q directly from human plasma C_LIO_LIMild elution conditions preserved the targets oligomeric structure and native interactome C_LIO_LIThis label-free strategy offers a rapid, low-cost alternative to antibody-based chromatography C_LI

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of six novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Among the candidate enzymes, u17, u10, and u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Building on these results, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. Collectively, these findings highlight amidase signature urethanases as valuable scaffolds for advancing sustainable and scalable biocatalytic recycling of polyurethanes. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/705263v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@127bf23org.highwire.dtl.DTLVardef@75c29corg.highwire.dtl.DTLVardef@13bbf30org.highwire.dtl.DTLVardef@18504a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cross-linking mass spectrometry (XL-MS) has emerged as an attractive technology for investigating protein complexes and protein-protein interactions (PPIs). However, commonly used cross-linking strategies present significant challenges for precise analysis of protein complexes and dynamic PPIs in native biological environments. Here we present the visible-light-controlled lysine-selective cross-linking (VL-XL) strategy for in-depth analysis of protein complexes and dynamic PPIs both in vitro and in live cells, building on light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) chemistry. We demonstrate that the VL-XL strategy effectively explores the dynamic dimerization of PD-L1 stimulated by exogenous modulators. Moreover, the VL-XL strategy successfully profiles the time-resolved EGF-stimulated EGFR interactome, providing valuable insights into the regulatory mechanisms of EGFR signaling and intracellular trafficking. Importantly, the VL-XL strategy efficiently deciphers the molecular glue (MG) induced dynamic PPIs and substrate profile of MG degrader, opening an innovative avenue for identifying neo-substrates. By harnessing the advantages of temporal controllability, good biocompatibility, and lysine selectivity, the VL-XL method simplifies MS data analysis and facilitates the acquisition of accurate structural information of protein complexes and the elucidation of elusive PPIs in live cells. Overall, the VL-XL strategy expands the XL-MS toolbox, and realizes in-depth analysis of protein complexes and dynamic PPIs, which will inspire innovative solutions for protein interactomes research and structural systems biology.

A robust technology is critically needed for identifying preferred protein targets of glycosaminoglycans (GAGs), and synthetic mimetics thereof, in biological milieu. We present a robust 10-step strategy for identification and validation of preferred protein targets of highly sulfated, synthetic, small, GAG-like molecules using diazirine-based photoaffinity labeling- proteomics approach. Our work reveals that optimally designed, homogeneous probes based on minimalistic photoactivation and affinity pulldown groups coupled with rigorous proteomics, biochemical and orthogonal validation steps offer excellent potential to identify preferred targets of GAG mimetics from the potentially numerous possible targets that cloud GAG interaction studies. Application of this 10-step strategy for a promising highly sulfated, small GAG mimetic led to identification of only a handful of preferred targets in human plasma. This new robust strategy will greatly aid drug discovery and development efforts involving GAG sequences, or sulfated small mimetics thereof, as leads.

This study reports the discovery and characterization of novel CRBN molecular glues that selectively induce the proteasomal degradation of the hematopoietic-specific signaling protein VAV1, a key target in hematological malignancies and autoimmune diseases. Utilizing unbiased global proteomics, we identified phenyl-glutarimide derivatives NGT-201-12, as effective VAV1 degraders, with its C-terminal SH3 domain (SH3-2) being crucial for this interaction. A significant finding is the elucidation of a non-canonical RT-loop degron (RDxS motif, residues 796-799) within VAV1 SH3-2, distinct from previously characterized G-loop degrons. This discovery, supported by advanced computational modeling using the physics- and AI-driven GluePlex workflow and validated by site-directed mutagenesis, highlights versatility of CRBN in recognizing diverse neosubstrate motifs. Furthermore, we demonstrate that applying Free Energy Perturbation (FEP+) calculations to these predicted ternary structures yields cooperativity metrics that correlate with experimental degradation potency, overcoming the limitations of standard molecular docking. This establishes a robust workflow where, once a ternary complex is predicted--even with initial weak binders--FEP+ can be utilized to prospectively rank analogs and optimize molecular glue potency. Additionally, we demonstrate that strategic chemical modifications, particularly conformational restriction via halogen substitution (e.g., NGT-201-18), markedly potentiate VAV1 degradation, a principle supported by density functional theory (DFT) calculations. Comprehensive structure-activity relationship (SAR) studies provided a roadmap for designing next-generation VAV1 degraders. Importantly, dose-response proteomics not only confirmed VAV1 as the primary target but also revealed LIMD1, possessing a canonical G-loop, as an off-target for some analogs, indicating a single molecular glue can engage disparate degron motifs. The identification of the VAV1 RT-loop degron prompted a proteome-wide search, revealing other SH3-containing proteins as potential targets or off-targets. In conclusion, this research unveils a novel non-canonical RT-loop degron in VAV1, demonstrates the utility of conformational restriction in enhancing degrader potency, and underscores the critical role of integrating global proteomics with advanced structural modeling and FEP calculations for understanding degrader potency and selectivity. These findings offer a promising therapeutic strategy for targeting VAV1 and significantly expand the landscape of CRBN neosubstrate recognition and the rational design of molecular glue degraders.

Transient protein-protein interactions are fundamental aspects of many biochemical reactions, but they are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) analysis is a powerful tool to facilitate the analysis of transient interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes--EIN/HPr with a KD of 7 M and EIIAGlc/EIIBGlc with a KD of 25 M--as model systems, we compared the effects of two amine-specific homo-bifunctional cross-linkers of different cross-linking speeds. Protein cross-linking by DOPA2, a di-ortho-phthalaldehyde cross-linker, is 60-120 times faster than that by DSS, an N-hydroxysuccinimide ester cross-linker. We analyzed the differences in the number of cross-links identified that reflected the stereospecific complex (SC), the final lowest-energy conformational state, and that of cross-links that reflected the encounter complexes (ECs), an ensemble of short-lived intermediate conformations mediated by nonspecific electrostatic interactions. We found that the faster DOPA2 cross-linking favored the SC whereas the slower DSS cross-linking favored the ECs. We propose a mechanistic model for this intriguing observation. This study suggests that it is feasible to probe the dynamics of protein-protein interaction using cross-linkers of different cross-linking speeds.

The SARS-CoV-2 spike protein is highly antigenic, with epitopes in three distinct regions of the receptor binding domain (RBD) alone that have known mechanisms of neutralization. In previous work, we predicted a fourth RBD epitope based on allosteric conformational perturbations measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS) upon complexation with the canonical spike protein target, human angiotensin-converting enzyme 2 (hACE2). We subsequently identified a pan-neutralizing antibody (ICO-hu104) with the predicted epitope, however, as the epitope was somewhat distant from the hACE2 binding interface, and our previous work limited to the spike RBD, the neutralization mechanism was unclear. Using HDX-MS, we investigated the binding of ICO-hu104 to the full-length SARS-CoV-2 spike protein from Wuhan, Delta and Omicron variants. We demonstrate that binding of ICO-hu104 at its epitope results in an increase in deuterium uptake in the distant HR1 domain for variants of concern, which in a biological context could be indicative of destabilisation of the helices within this region, promoting S1 shedding or failure of helical extension during S2-mediated fusion. This is supported by our computational modelling, highlighting propagation of allosteric effects to the S2 coiled-coil region. Collectively, this work demonstrates an alternative neutralization mechanism for ICO-hu104 which is distinct from its first-generation predecessors and thus opens alternative avenues targeting non-RBD epitopes through assessment of allosteric perturbations. HighlightsO_LIHDX-MS reveals decreased deuterium uptake within the HR1 region of S2 for SARS-CoV-2 spike protein for variants of concern when bound to ICO-hu104. C_LIO_LIComputational modelling validates high allosteric coupling between ICO-hu104 epitope and HR1 region. C_LIO_LISuggests an alternative neutralization mechanism from its predecessor ICO-hu23, whereby destabilisation of helices within the HR1 region promotes S1 shedding and/or failure of helical extension during S2-mediated fusion. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/635280v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1175bdborg.highwire.dtl.DTLVardef@8fb17borg.highwire.dtl.DTLVardef@1cd13f7org.highwire.dtl.DTLVardef@d9c121_HPS_FORMAT_FIGEXP M_FIG C_FIG

Magnetic Resonance Imaging (MRI) is a cornerstone of modern clinical diagnostics, often enhanced by contrast agents. Traditionally, these agents are chemically synthesized, which can involve complex, costly, and environmentally unfriendly processes. Here, we report a novel biocatalytic approach for the efficient, safe, and eco-friendly synthesis of 5-methyl-5,6-dihydrothymidine (5-MDHT), a potent Chemical Exchange Saturation Transfer (CEST) MRI probe for imaging in vivo expression of the Herpes Simplex Virus Type-1 Thymidine Kinase (HSV1-TK) reporter gene. We demonstrate that 5-MDHT can be biosynthesized via one- or two-step enzymatic reactions using human purine nucleoside phosphorylase (hPNPase) and the SgvMVAV SAM-dependent methyltransferase. hPNPase catalyzed the base-exchange reaction with catalytic efficiencies (kcat/KM) between 138-316 s-1 M-1, while SgvMVAV methylation of 5,6-dihydrothymidine yielded 5-MDHT with a catalytic efficiency of 26 s-1 M-1. Molecular dynamics simulations supported the enzymatic binding and selectivity observed experimentally. The resulting 5-MDHT was validated using CEST-MRI, showing a distinct exchangeable imino proton signal at 5.3 ppm. These findings highlight the chemo- and regioselectivity of the biocatalysts and establish biocatalysis as a viable platform for producing clinically relevant MRI contrast agents.

Adenylate kinases (AKs) are phosphotransferases that are frequently employed as models to investigate protein structure-function relationships. Prior studies have shown that AK homologs of different stabilities retain cellular activity in cells following circular permutation that split the AMP binding domain into fragments coded at different ends of the primary structure, such that this domain was no longer embedded as a continuous polypeptide within the core domain. Herein, we show mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologs at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30{degrees}C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than native AKs, and they exhibited multi-phasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK, and mass spectrometry suggested that the new termini within the AMP binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/564053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ee9ecdorg.highwire.dtl.DTLVardef@fbb415org.highwire.dtl.DTLVardef@ebdd90org.highwire.dtl.DTLVardef@11f4271_HPS_FORMAT_FIGEXP M_FIG C_FIG

The biocatalytic recycling of plastics, such as polyethylene terephthalate (PET), promises a sustainable alternative to our present open-loop cycles. Engineering of PET-hydrolases for this purpose has focused on improving activity near the glass-transition temperature of the polymer by increasing their thermostability, neglecting other features of the protein-polymer system that affect enzymatic activity. Here, we isolate the effect of electrostatics on the activity of a thermophilic PETase by rationally redesigning its surface charge, while preserving its thermodynamic properties. The enzyme variant, SfInv, shows orders of magnitude improvements in binding affinity and in activity towards untreated plastic films, with inverted morphological preference. When combined, the wildtype enzyme and SfInv act synergistically, revealing an entirely new mechanism for cooperative activities driven by complimentary electrostatic interactions at the PET surface. These findings highlight unexplored avenues in improving PETase function through the control of morphological preference or introduction of protein cooperativity by exploiting protein electrostatics.