The utility of intrinsically disordered regions (IDRs) in protein function has become an area of broad interest in recent years given their known roles in signaling, biomolecular condensates, and protein activity regulation. The central challenge, however, is that disorder is difficult to characterize, and thus, there is a need to develop approaches to monitor how IDRs influence proteins. Here, we employ a strategy to restrict the freedom of the intrinsically disordered N-terminal tail (ID-tail) of human thymidylate synthase (hTS), which, together with tail truncation, enables testing aspects of how the ID-tail modulates enzyme function. The highly flexible hTS tail is known to impact allosteric substrate cooperativity and conformational switching dynamics, although how the tail brings about these effects has not been determined. We attempted to restrict native tail dynamics by chemically linking the dimer tails and tested linked forms with activity, binding, and nuclear magnetic resonance heteronuclear single quantum coherence (NMR HSQC) spectra. Truncating four N-terminal residues eliminated cooperativity in dUMP binding to apo-hTS, but because a similar result was obtained from the linked full-length protein, complete tail freedom appeared to be important for intersubunit communication. NMR analysis was aided by referencing the inactive M190K mutant, which roughly defined regions of active-inactive conformational switching that were disrupted by tail linkage. Collectively, these results point toward a role of the ID-tail to influence the ordered core of the protein. Moreover, while the natural ID-tail is extremely flexible, it likely possesses specific physiochemical movement dynamics and positioning attributes that facilitate its impact on allosteric cooperativity and conformational dynamics.

Bitter taste receptors (TAS2Rs) are GPCRs functionally expressed in extraoral organs including the lung, heart, brain, and gastrointestinal system and are candidate targets for novel drugs. It is unclear whether TAS2R5 can be stabilized by structurally distinct agonists resulting in pathway selectivity (biasing) to achieve optimal outcomes. We screened TAS2R5 agonists for signaling toward or away from β-arrestin or G protein ([Ca2+]i). Agonists T5-1 (1,10-phenanthroline) and T5-6 (4,7-dimethyl-1,10-phenanthroline) coupled equally to G protein but showed marked differences in homologous desensitization (∼21% and ∼91%, respectively). This desensitization was due to dissimilar degrees of β-arrestin engagement compared to the reference agonist T5-7: T5-6 evoked up to ∼86% enhancement of β-arrestin1 or β-arrestin2 recruitment, while T5-1 was biased in the opposite direction, up to ∼78% less, compared to T5-7. T5-1 elicited little receptor internalization compared to T5-6 consistent with the β-arrestin findings. For the initial response, T5-1 and T5-6 represent one-pathway differences (decreased or increased β-arrestin) leading to bias in different directions, while G protein signaling was equivalent. However, under desensitizing conditions T5-6 was decoupled from G protein representing two-pathway biasing in opposing directions for T5-6 (increased β-arrestin and decreased G protein), imposing extreme biasing of β-arrestin over G protein. Our finding that agonists can affect β-arrestin biasing in either direction suggests that TAS2R5 is sufficiently pliable for specific signals to be engineered depending on the desired therapeutic outcome. The dynamic nature of the directionally contrasting changes of the two pathways over time accentuated bias and is due to the interaction of the two signals.

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as l-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics (MD) simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding and catalysis and the roles of the active-site residues involved in these processes. Our experimental results confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the l-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid via a possible superoxide radical anion intermediate, while generating H2O2 and NH3 as reaction byproducts. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. Structural modeling, MD simulation, and site-directed mutagenesis reveal that residues from both protomers contribute to cofactor and substrate stabilization, including Lys232 and His31 as key catalytic residues as well as additional residues forming an extended active-site interaction network. Moreover, MD simulations uncovered a dimer-mediated alternating-lid mechanism in which coordinated motions at the dimer interface regulate substrate access and product release through transient tunnels while maintaining a protected catalytic environment. This seesaw-like dynamics provides a mechanistic framework for understanding how dimerization controls enzyme function, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into substrate/cofactor stabilization and the catalytic mechanism of MilM and expand our understanding of the remarkable PLP-dependent oxidases and hydroxylases in natural product biosynthesis.

Discovered ∼60years ago, the lipid metabolite 4-hydroxynonenal (HNE) is linked to a plethora of macromolecular targets and biological functions. For a molecule that weighs 156 Da and possesses a single H-bond donor, this is quite a feat. Despite its chemical simplicity, HNE contains an α,β-unsaturated aldehyde system, endowing it with the capability to react covalently with numerous biological functional groups and bestowing on it pleiotropic properties. Regardless of the specific entity engaging with HNE, it is covalent bond formation that has dominated thought on HNE behavior. Indeed, cells possess a flurry of detoxifying enzymes that convert HNE to less reactive chemicals lacking the α,β-unsaturated aldehyde. For instance, the cell can either reduce or oxidize the aldehyde within HNE, deactivating HNE's chemical reactivity. Here, we discuss one of our recent papers that discovered that HNE can modify the detoxification enzyme, Cyp-33e1, in Caenorhabditis elegans, using a customized tissue-specific screen for HNE-sensor proteins. Consistent with the concepts of active site partitioning, HNE also emerged as a substrate of Cyp-33e1. We next discovered that HNE changed lipid storage in worms in a Cyp-33e1-dependent manner. We proposed that the product of Cyp-33e1 detoxifying HNE was responsible for this change in lipid storage and were able to show that 4-hydroxynonenoic acid (HNA), the product of Cyp-33e1 oxidation of HNE, causes this phenotype. We have dubbed this new signaling mode, "deactivation signaling". It sets an important precedent for how the bioactivity of HNE is considered, and we discuss the ramifications of this result in this perspective.

Macromolecular crowding is a defining feature of cellular environments and plays an important role in modulating protein aggregation. Here, we examine how mixed macromolecular crowders influence the aggregation of Huntingtin exon-1 protein with a pathogenic polyglutamine length (HD39Q). Using thioflavin-T (ThT) kinetics, we show that the binary mixtures of polyethylene glycol (PEG8), Dextran (40 and 70), and Ficoll 70 produce biphasic aggregation kinetics that deviate from classical single-step behavior. Confocal imaging using labeled proteins and crowders reveals that not only do the binary combinations of crowders give rise to phase separated conditions, but also these drive the aggregation in subtle but distinct ways based on the respective combinations. Moving beyond binary mixtures for a better in-cell mimic, ternary crowder systems were also studied. These generated hierarchically organized droplet architectures thereby further modulating aggregation pathways and associated protein localization. Comparative imaging of the aggregation of a few model proteins (bovine serum albumin, β-lactoglobulin, and lysozyme) demonstrates that aggregation and partitioning behavior depend strongly on both the properties of the biomolecule and phase composition. These findings highlight how mixed macromolecular crowding can reshape the aggregation landscape by adding complexity at both the spatial and temporal levels, thus making these more relevant for physiologically relevant processes.

Nonheme Fe(II)/2-oxoglutarate (Fe/2-OG) dioxygenases carry out a broad range of oxidative reactions, yet α-hydroxylation of glycyl residues remains exceedingly rare. Here, we demonstrate that the Fe/2-OG enzyme MysH from Nostoc linckia performs this unusual chemistry during mycosporine-like amino acid biosynthesis, converting mono- and di-substituted precursors into palythines and revealing unexpected substrate tolerance. Kinetic isotope effects, detection of a transient hydroxylated intermediate, and glyoxylate byproduct formation support an α-hydroxylation-initiated mechanism. High-resolution crystal structures, complemented by molecular docking, molecular dynamics simulations, and site-directed mutagenesis, define an active-site architecture that positions the glycyl substrate in a near–transition-state geometry. Hybrid QM/MM calculations reveal a low-barrier hydrogen-atom-transfer step followed by hydroxyl rebound and implicate a conserved Trp125 in an electron-transfer network that lowers the activation barrier. Together, these findings establish a mechanistic framework for protein-directed α-glycine C–H activation by nonheme iron enzymes and provide a blueprint for engineering Fe/2-OG dioxygenases to expand the chemical diversity of mycosporines and related natural products.

Obesity and its associated metabolic syndrome present significant therapeutic challenges, with current pharmacological interventions often falling short of replicating the multifaceted benefits of bariatric surgery. Recent advances in incretin-based therapies, particularly GLP-1 and GIP coagonists, have demonstrated substantial improvements in glycemic control and weight management, yet residual cardiovascular risk and lipid abnormalities persist. Fibroblast growth factor 21 (FGF21) has emerged as a promising protein to complement incretin pharmacology due to its potent lipid-lowering effects and favorable safety profile. This study describes the engineering of a balanced, long-acting triple agonist that simultaneously targets FGF21, GLP-1, and GIP receptors. Through strategic N- and C-terminal modifications and lipid conjugation, a novel optimized FGF21 analogue is engineered to exhibit enhanced potency, stability, and sustained pharmacokinetics compared to native protein. In Diet-Induced Obesity (DIO) mice, this FGF21 analogue achieves near normalization of body weight, superior to benchmark GLP-1 agonists, and demonstrates additive efficacy when combined with a GLP-1/GIP receptor coagonist. Based on this additivity, a unimolecular triagonist is engineered, and mechanistic studies confirm balanced receptor activity at the FGF21 and incretin receptors to achieve combinational pharmacology, with significant reductions in body fat, improved glucose tolerance, and extended duration of action. These findings position the FGF21/GLP-1/GIP triagonist as a first-in-class candidate for next-generation metabolic disease therapy, potentially approximating the efficacy of surgical intervention while addressing lipid disorders inadequately managed by current incretin therapies.

Mitochondria are increasingly recognized as signaling organelles that coordinate cell-fate decisions during stress. Because outer mitochondrial membrane (OMM) proteins are exposed to the cytosol, they are prominent substrates for ubiquitination, a dynamic post-translational modification that encodes information through diverse chain architectures and linkage types. In this review, we examine how ubiquitination of OMM proteins functions as a molecular switch that integrates mitochondrial stress signals and engages three major, often antagonistic, stress-response mechanisms: mitophagy, cell death, and innate immune signaling. We highlight an emerging concept that a stress-responsive "ubiquitin code" is written on OMM substrates, in which pathway selection is coordinated by the identity of ubiquitinated OMM proteins together with the linkage type and branching of attached polyubiquitin chains. We provide an updated overview of the E3 ubiquitin ligases and deubiquitinases (DUBs) that write and erase this code and summarize ubiquitin linkage types reported on key OMM substrates across these pathways. For mitophagy, we cover both PARKIN-dependent and PARKIN-independent mechanisms mediated by other E3 ligases and counteracted by DUBs. For innate immunity, we discuss how ubiquitination of OMM proteins regulates the MDA5/RIG-I-MAVS axis and NF-κB signaling. For cell death, we describe how ubiquitination of anti- and pro-apoptotic BCL-2 family proteins can either lower or increase the threshold for the induction of apoptosis. We also highlight the newfound role of PARKIN to drive apoptosis through a BAX/BAK-independent mechanism. Finally, we discuss therapeutic opportunities to reprogram OMM ubiquitination by targeting E3 ligases or DUBs directly, or by using PROTAC- and DUBTAC-based strategies.

Fidelity of DNA polymerases, defined as their ability to incorporate a correct dNMP opposite an undamaged template nucleotide, is a key factor in ensuring genome stability. Many DNA polymerases have been extensively investigated to understand the structural and biochemical basis of their fidelity. However, the great majority of these studies employed simple primer-template substrates, disregarding the possible effects of more complex DNA structures. Here, we have estimated the fidelity of a single dNMP incorporation by the Klenow fragment of Escherichia coli DNA polymerase I (KF) and bacteriophage RB69 DNA polymerase (RBpol) during the strand displacement synthesis, a situation common in lagging strand replication and DNA repair. Although KF was more efficient than RBpol in the presence of a downstream strand, both polymerases demonstrated a 3-fold (up to an order of magnitude) increase in the fidelity compared with the primer-template system. To assess the dependence of fidelity on the energy spent on the disruption of the base pair ahead, we varied its strength using mismatches and synthetic base analogs. Both KF and RBpol generally traded efficiency for fidelity, making fewer errors when they had to disrupt stronger base pairs. Thus, the energetic penalty imposed by the downstream strand acts as a fidelity checkpoint, enhancing discrimination against incorrect nucleotides.