Biochemistry [JOURNAL]
Sun
200 papers
Aromatic Cage-Directed Azide-Methyllysine Photochemistry for Profiling Nonhistone Interacting Partners of the MeCP2 Methyl-CpG-Binding Domain.
Padhan J, Ghosh S, Barman S … +1 more , Sudhamalla B
Biochemistry · 2026 Jul · PMID 42397176 · Publisher ↗
Methyl-CpG-binding protein 2 (MeCP2) is a canonical reader of DNA methylation and a key chromatin regulator implicated in neurodevelopment and cancer. Beyond DNA binding, MeCP2 recognizes trimethylated histone H3 lysine...
Methyl-CpG-binding protein 2 (MeCP2) is a canonical reader of DNA methylation and a key chromatin regulator implicated in neurodevelopment and cancer. Beyond DNA binding, MeCP2 recognizes trimethylated histone H3 lysine 27 (H3K27me3) through an aromatic cage within its methyl-CpG-binding domain (MBD). Whether this methyllysine-binding interface also mediates interactions with nonhistone methyllysine-containing proteins remains unknown. Here, we developed an aromatic cage-directed chemoproteomic strategy to capture methyllysine-dependent MeCP2 interactions in cells. By site-specifically incorporating the photocrosslinkable unnatural amino acid 4-azido-l-phenylalanine (AzF) into the MeCP2-MBD, we enabled covalent capture of transient MeCP2-binding partners. Engineered MeCP2 variants efficiently crosslinked to methylated histone ligands, validating preserved methyllysine recognition. Proteomic analysis of crosslinked complexes from human cell lysates uncovered previously unrecognized MeCP2-associated proteins involved in chromatin regulation, RNA processing, translation, and metabolism. Integration with single-cell transcriptomic data revealed coordinated expression of and its interacting partners in defined cellular populations. Imaging and chromatin-based analyses further demonstrated nuclear colocalization and shared genomic occupancy with selected interactors at transcriptionally relevant loci. Together, these findings establish the MeCP2 aromatic cage as a versatile interaction interface and expand the functional landscape of MeCP2 beyond DNA methylation reading to encompass chromatin-associated regulatory networks.
Differential Hydroxypyruvate Processing by and DXP Synthases Reveals Preferential Xylulose 5-Phosphate Formation by the Enzyme.
Henriquez S, Nosal CR, Kramer LJ … +4 more , Toci EM, Trauth SA, Lott AM, Freel Meyers CL
Biochemistry · 2026 Jul · PMID 42390953 · Publisher ↗
The thiamine diphosphate (ThDP)-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to the essential branchpoint metabolite, DXP. DXPS exhibits a lig...
The thiamine diphosphate (ThDP)-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to the essential branchpoint metabolite, DXP. DXPS exhibits a ligand-gated mechanism where the predecarboxylation intermediate derived from pyruvate persists until d-GAP triggers decarboxylation. Together with its substrate/catalytic promiscuity and other features of multifunctionality, ligand gating on DXPS may poise this enzyme to catalyze alternate activities that aid in bacterial adaptation. We previously discovered alternative donors for DXPS, with donor processing and acceptor specificity influenced by the donor. Here, we demonstrate species differences in DXPS substrate usage and processing which could signify that DXPS homologues support bacterial adaptation in distinct ways. We showed that hydroxypyruvate (HPA) is a donor for DXPS (DXPS), similar to DXPS, yet HPA processing and donor-acceptor specificity differs on these homologues; HPA reacts with ThDP on DXPS to form a longer-lived intermediate in the absence of added trigger, relative to DXPS. Notably, DXPS preferentially catalyzes xylulose 5-phosphate formation from HPA and d-GAP, in contrast to DXPS, suggesting a potential connection between DXPS activity and pentose phosphate pathway metabolism in . Finally, both DXPS and DXPS were found to generate a 5-carbon β-ketoacid via a one-substrate reaction of HPA, distinguishing DXPS from transketolase (TK) which produces erythrulose under similar conditions. This study illustrates species differences in DXPS donor usage and processing, and donor-acceptor specificity that supports distinct multifunctionality across bacterial pathogens and the plausibility of species-specific targeting.
Structural and Functional Characterization of Heterologous Nitrogenase Complexes.
Li Y, Narehood SM, Cook BD … +3 more , McGuire KL, Herzik MA, Tezcan FA
Biochemistry · 2026 Jul · PMID 42390130 · Publisher ↗
Nitrogenase is the only known enzyme that catalyzes the reduction of dinitrogen to ammonia. The most prevalent isozyme, molybdenum nitrogenase, comprises the catalytic molybdenum-iron protein (MoFeP) and the ATP-dependen...
Nitrogenase is the only known enzyme that catalyzes the reduction of dinitrogen to ammonia. The most prevalent isozyme, molybdenum nitrogenase, comprises the catalytic molybdenum-iron protein (MoFeP) and the ATP-dependent reductase iron protein (FeP). Although Mo-nitrogenases are widespread across bacteria and archaea and appear to share conserved mechanistic and structural features, FeP and MoFeP show considerable sequence variability across diazotrophs. This raises questions about the conservation of chemomechanical mechanisms coupling FeP-dependent ATP hydrolysis and electron transfer to MoFeP, and about the functional compatibility of nitrogenase components from divergent species. Previous studies showed that some heterologous FeP-MoFeP pairs can functionally complement each other, whereas other pairs lack catalytic activity, but the absence of structural information on such heterologous pairs has limited mechanistic understanding. To this end, we investigated the functional and structural compatibility of FeP and MoFeP from () and (), two phylogenetically and ecologically distinct species. Building on our prior work with -nitrogenase and recently developed cryogenic electron microscopy (cryoEM) protocols, we determined the ADP·BeF-trapped structure of the homologous FeP-MoFeP complex and showed that it adopted the same geometry as its counterpart. Activity measurements showed that heterologous combinations retained 60-80% of homologous catalytic activities despite 30-50% sequence divergence in FeP and MoFeP. High-resolution cryoEM structures of FeP-MoFeP and FeP-MoFeP corroborated these activities and revealed that functional complementation tolerates substantial sequence variation when the core structural elements supporting ATP binding/hydrolysis, protein-protein interaction, electron transfer, and substrate reduction are conserved.
Discovery of Bacterial Unspecific Peroxygenases.
Lopez-Tavera E, Stepnov AA, S Ersdal N … +6 more , Barros-Reguera M, Sandholm RM, La Rosa SL, Sørlie M, Eijsink VGH, Vaaje-Kolstad G
Biochemistry · 2026 Jun · PMID 42374716 · Publisher ↗
Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds because of their vast repertoire of catalyzed...
Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds because of their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have been reported in prokaryotic organisms. Here, we describe the discovery of a diverse family of bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial proteins, termed bacterial UPOs (BUPOs), display clear structural homology to family I (short) fungal UPOs despite low sequence identity (<20%). Expression of one of these proteins (BUPO) in its native host ( . A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in . In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, as well as two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol, and 16-hydroxypalmitic acid, using hydrogen peroxide as a cosubstrate. These enzymes thus represent a previously unknown group of bacterial heme-thiolate peroxygenases that share key structural and functional features with eukaryotic UPOs, offering potential candidates for the development of industrially relevant biocatalysts.
Lactate Biology: Subcellular Routing and Chemical Form Define Function.
Offei NA, Cluntun AA
Biochemistry · 2026 Jun · PMID 42367133 · Publisher ↗
Lactate has undergone a major conceptual shift, from a glycolytic waste product to a circulating metabolic currency and, more recently, a multifunctional regulator of physiology. It serves as a mitochondrial fuel, an epi...
Lactate has undergone a major conceptual shift, from a glycolytic waste product to a circulating metabolic currency and, more recently, a multifunctional regulator of physiology. It serves as a mitochondrial fuel, an epigenetic modifier via protein lactylation, a ligand for the G-protein-coupled receptor HCAR1, and a precursor for endocrine-like N-lactoyl amino acids. This convergence raises a central question: how can a single metabolite support such distinct roles without functional conflict? We propose that the resolution lies not in lactate concentration alone but in two complementary organizing principles: subcellular routing and chemical form. Transporter localization, enzyme compartmentalization, donor formation, and metabolic competition bias lactate toward distinct biochemical fates, while conversion into chemically distinct intermediates─including lactyl-CoA, lactoyl-glutathione, d-lactate, and N-lactoyl amino acids─further constrains the biological outcomes that lactate can support. Lactate's fate is influenced by the cellular compartments it accesses, a process constrained by specific monocarboxylate transporters at the plasma and mitochondrial membranes, isoform-specific localization of lactate dehydrogenases, and compartmentalized enzymatic machinery that converts lactate into distinct biochemical donors. Mitochondrial oxidation, protein lactylation, extracellular signaling, and N-lactoyl amino acid synthesis should therefore not be viewed as parallel consequences of elevated lactate concentration. Instead, they represent interconnected metabolic fates that draw from shared lactate pools and are influenced by compartmental access and local enzymatic context. Here, we integrate evidence from metabolism, epigenetics, and signaling into a spatial framework in which lactate function depends on where it is routed. In this view, lactate is not a promiscuous metabolite but a compartmentalized intermediate whose biological effects are shaped by spatial context. We further distinguish between established, emerging, and speculative aspects of this compartmentalized view to highlight key gaps and prioritize future experimental testing.
Nature's Anaerobic Toolkit: Glycyl Radical Enzymes and Their Expanding Functional and Mechanistic Diversity.
Imrich CN, Chakraborty A, Backman LRF … +2 more , Andorfer MC, Drennan CL
Biochemistry · 2026 Jun · PMID 42361324 · Publisher ↗
Nature has devised an array of enzymatic cofactors that enable challenging chemical transformations to occur on a time scale compatible with biological life. The glycyl radical enzyme (GRE) superfamily, for example, high...
Nature has devised an array of enzymatic cofactors that enable challenging chemical transformations to occur on a time scale compatible with biological life. The glycyl radical enzyme (GRE) superfamily, for example, highlights the catalytic power of protein-based amino acid radicals. GREs utilize a persistent radical housed on the α-carbon of a C-terminally conserved glycine residue to accomplish diverse chemical reactions in anaerobic environments. This radical cofactor is post-translationally installed on the mature, folded protein by a radical -adenosyl-L-methionine (RS) activating enzyme (GRE-AE; activase) specific to each GRE. The catalytic repertoire of GREs is diverse, spanning ribonucleotide reduction for DNA synthesis, acetyl-CoA formation in anaerobic primary metabolism, dehydration reactions in secondary metabolism, decarboxylation reactions in aromatic amino acid fermentation, and hydrocarbon activation in anaerobic bacteria, enabling their survival on crude oil as a carbon source. Pyruvate formate lyase (PFL), the founding member of the GRE superfamily, was first identified several decades ago. In recent years, sequencing technology has enabled the annotation of thousands of unique GRE-encoding sequences. Several dozen of the identified GREs have been characterized further, providing insight into the structure, mechanism, and regulation of this multitudinous and diverse enzyme family. Here, we introduce the superfamily, summarize what is known about their activation and catalysis, note conserved structural features, expand on the previously identified GRE classes, highlight GRE-associated bacterial microcompartments, and discuss the future and outlook of the GRE superfamily.
Structural Bases for the Unconventional Activity of a Viroporin Channel.
Wiley B, Largo E, Nabais L … +2 more , Nieva JL, Domene C
Biochemistry · 2026 Jun · PMID 42345276 · Publisher ↗
Viroporins alter the permeability of cell membranes and regulate the initiation/progression of the viral infection cycle. However, the "unconventional" membrane channel behavior displayed by many members of the family ch...
Viroporins alter the permeability of cell membranes and regulate the initiation/progression of the viral infection cycle. However, the "unconventional" membrane channel behavior displayed by many members of the family challenges their general validation as therapeutic targets. The reported capacity of the Classical Swine Fever Virus p7 viroporin for establishing ion-conducting channels of different sizes exemplifies that behavior. Using all-atom molecular dynamics (MD) simulations, we attempted to elucidate the structural basis and mechanisms underlying the atypical activity of p7. Based on AlphaFold-predicted CSFV p7 hexamer structures with folded-back helical hairpins, we first generated monomers that spanned the entire thickness of the lipid bilayer. Next, we assembled oligomers of varying stoichiometry (pentamers, hexamers, and heptamers) based on those extended transmembrane hairpin (TMH) protomers. We focused on two hexameric models: TMH1, preserving the helix-helix packing interactions observed in the initial model, and TMH2, generated using TMH1 as a template for ColabFold. In line with experimental evidence, the simulations revealed that both structural architecture and oligomeric state determine pore organization in ER-like membranes. TMH1 hexamers and TMH2 heptamers adopted wide pore geometries with extensive hydration and ion accessibility, whereas TMH2 hexamers sampled narrower, more compact hydrated pore states. TMH2 pentamers remained predominantly nonconductive. Dynamic interactions between transmembrane helices and the number of protomers incorporated into the membrane-embedded structure appeared to be instrumental for this capacity. These findings reveal how variation in oligomeric state and helix packing can generate pores with different dimensions and hydration properties, providing a structural framework for interpreting p7's experimentally observed capacity to induce multiple conductance states and size-selective membrane permeabilization.
Targeting the WASF3 Regulatory Complex in Pancreatic Cancer Using Stapled Peptides.
Dill TC, Alger EJ, Gambale S … +1 more , Kennedy EJ
Biochemistry · 2026 Jun · PMID 42342237 · Publisher ↗
Wiskott-Aldrich Syndrome Protein Family (WASF) members form a heteropentameric complex along with ABI1/2, NCKAP1, CYFIP1/2, and BRK1, termed the WASF Regulatory Complex (WRC), which regulates actin cytoskeletal remodelin...
Wiskott-Aldrich Syndrome Protein Family (WASF) members form a heteropentameric complex along with ABI1/2, NCKAP1, CYFIP1/2, and BRK1, termed the WASF Regulatory Complex (WRC), which regulates actin cytoskeletal remodeling. Upregulated WASF3 has been identified in pancreatic cancer and is associated with a poor prognosis, increased invasion, and metastasis. In this study, we sought to determine whether disruption of the WRC could suppress WASF3-mediated actin polymerization and subsequent cellular invasion and motility in pancreatic cancer cells. Here, we screened constrained peptides designed to disrupt the formation of the WRC at multiple protein-protein interfaces. The WASF3-derived constrained peptide WAHM1 was found to suppress cell motility and invasion in PANC-1 and BxPC-3 cells. Further, WAHM1 was found to permeate pancreatic cancer cell lines, bind to its protein targets in the WRC, and reduce WRC protein levels. WAHM1 may serve as a complementary strategy to downregulate WASF3-mediated migration and invasion in pancreatic cancer models.
Thermodynamic and Allosteric Drivers of Stilbene-Mediated Noncompetitive Inhibition of Firefly Luciferase.
Sr Nair A, Dhankhar K, Das A … +2 more , Samanta A, Hazra S
Biochemistry · 2026 Jun · PMID 42339762 · Publisher ↗
luciferase (Ppy luci) is widely used as a reporter molecule in high-throughput assays for biochemical, cell viability, and cellular pathway studies. Resveratrol is a naturally occurring stilbene compound known to inhibit...
luciferase (Ppy luci) is widely used as a reporter molecule in high-throughput assays for biochemical, cell viability, and cellular pathway studies. Resveratrol is a naturally occurring stilbene compound known to inhibit luminescence emission catalyzed by Ppy luci. Although it has been identified as an inhibitor, its detailed mechanism has not been investigated. In the present work, three stilbene compounds (piceatannol, resveratrol, and pterostilbene) were studied for their luminescence inhibition properties to determine the factors underlying inhibition and the role of functionalization. QSAR analysis, followed by steady-state kinetics, suggested an allosteric mode of inhibition by stilbene inhibitors, with piceatannol showing the highest potency. Isothermal titration calorimetry, docking, and MMPBSA/MMGBSA supported the inhibition assay, wherein the piceatannol have a higher binding affinity compared to pterostilbene, driven by enthalpic contributions. In contrast, the melting temperature (Tm) comparison showed that resveratrol causes a higher shift in the global protein conformation despite having reduced interactions with binding site residues. Stability studies using molecular dynamics simulation also highlighted a greater effect of resveratrol on global conformational stability compared to piceatannol. Density functional theory (DFT) analysis indicated higher reactivity of piceatannol, underscoring its ability to hinder the oxidative microenvironment to a greater extent and its inhibition potency. The study indicate that stilbene inhibition is the result of a synergistic effect of enthalpic contributions, localized allosteric perturbations, and changes in the oxidative microenvironment and is less dependent on global conformational effects. Overall, this study highlights the importance of carefully evaluating functionalization-driven, multifactorial inhibition to mitigate assay artifacts.
Gut Bacterial 20-Hydroxysteroid Dehydrogenases Modify Endogenous Glucocorticoids and Corticosteroid Drugs.
Coyne S, Ghergurovich R, Sacco F … +5 more , Lombardi F, Paar K, DeMartino J, Kenfack AA, Stack TMM
Biochemistry · 2026 Jun · PMID 42338389 · Publisher ↗
Bacterial 20-hydroxysteroid dehydrogenases (20-HSDHs) from the human gut are known to reduce the C-20 carbonyl group of cortisol, producing either 20α- or 20β-dihydrocortisol. Two 20-HSDHs from L2-32 and ATCC 43058, bo...
Bacterial 20-hydroxysteroid dehydrogenases (20-HSDHs) from the human gut are known to reduce the C-20 carbonyl group of cortisol, producing either 20α- or 20β-dihydrocortisol. Two 20-HSDHs from L2-32 and ATCC 43058, both members of the short-chain dehydrogenases/reductases superfamily, are known to produce 20β-dihydrocortisol, while the 20-HSDH from ATCC 35704, belonging to the zinc-containing alcohol dehydrogenase family, produces 20α-dihydrocortisol. These three enzymes were characterized for their activity toward cortisol and structurally related therapeutic corticosteroids. Kinetic analyses revealed narrow substrate specificity, with all enzymes preferring cortisol but maintaining significant activity for prednisone and prednisolone (/ values between 10-10 M s for the 20β-HSDH enzymes), and some detectable activity of the 20β-HSDHs to reduce triamcinolone. The 20β-HSDHs exhibited pH-dependent substrate inhibition, influencing their activity profile. Structural docking studies indicated that suitable substrates occupy a single productive binding mode within the 20β-HSDH enzyme active site. Our findings show that enzymes in the gut microbiome can metabolize corticosteroid drugs by reducing the 20-keto group, which could have implications for drug efficacy and side effects. This work highlights the importance of gut microbial enzymes in the biotransformation of both endogenous and therapeutic steroids, informing future research into drug-microbiome interactions and personalized medicine.
Single-Residue Mutation Switch Reconfigures the Hierarchical Structure and Assembly of Amphiphilic Protein Block Copolymers for Hydration Layer-Dominated Water-Responsive Actuation.
Sun JW, Sun C, Kim S … +7 more , Wang AL, Huang I, Haq-Siddiqi N, Hedaya Z, Tu RS, Chen X, Montclare JK
Biochemistry · 2026 Jun · PMID 42330110 · Publisher ↗
Protein folding dynamics emerge from subtle changes in intramolecular interactions, where hydration shapes free energy landscapes and influences conformational ensembles and supramolecular assembly. While these shape-shi...
Protein folding dynamics emerge from subtle changes in intramolecular interactions, where hydration shapes free energy landscapes and influences conformational ensembles and supramolecular assembly. While these shape-shifting principles have been primarily explored in well-behaved systems with sharp transitions, their applicability to multidomain protein assemblies, particularly those containing partially folded or disordered regions, remains unclear. Here, we investigate how a single leucine-to-alanine mutation modulates the hierarchical interplay between folding, micelle formation, and macroscopic biomaterial behavior using two diblock protein block copolymers (BCPs): CE and CE. In solution, both BCPs exhibit spectral features consistent with their C/C monoblocks, with CE showing α-helical features and CE more disordered ones. Upon dehydration, however, both systems display increased structural order, with CE favoring β-sheets and CE adopting α-helical content. At the mesoscale, both BCPs assemble into amphiphilic micelles, but CE forms smaller, densely packed micelles, whereas CE generates larger micelles that template film networks of varying pore size depending on their packing density. These differences lead to distinct solvation behavior, with CE exhibiting greater uptake of predominantly mobile water, approximately twice that of CE, culminating in an actuation energy density of 2,043 kJ/m, the highest reported for protein BCPs to date. Despite this increased uptake, CE shows reduced mechanical relaxation and a rougher DSC/TGA thermogram, indicative of a broader conformational ensemble compared to CE. Comparisons to monoblocks (C, C, E) and previously reported triblocks (CEC, CEC) further highlight the importance of asymmetric diblock architectures in promoting hydration-sensitive assemblies, laying the rational design groundwork for future high-performance, water-responsive protein biomaterials.
A Thymine Dimer Stalls a High-Fidelity DNA Polymerase by Providing No Template Information in the Same Manner as an Abasic Site.
Walsh AR, Kim HR, Beese LS … +1 more , Wu EY
Biochemistry · 2026 Jun · PMID 42329088 · Publisher ↗
Cyclobutane pyrimidine dimers are photolesions that form when UV-B irradiation causes adjacent thymine or cytosine bases to covalently bond and can arrest DNA synthesis by replicative polymerases. To gain insight into th...
Cyclobutane pyrimidine dimers are photolesions that form when UV-B irradiation causes adjacent thymine or cytosine bases to covalently bond and can arrest DNA synthesis by replicative polymerases. To gain insight into the effects of cis-syn cyclobutane thymine dimers (T=T) on DNA replication in a model polymerase, we conducted solution and crystallographic studies of the Bacillus DNA polymerase I large fragment (BF) in complex with a T=T-containing template and an incoming dNTP. A thymine dimer lesion blocked nucleotide addition by BF in solution. The crystal structure of the BF-T=T-dATP complex indicated that the thymine dimer is too large to enter the template insertion site, preventing the lesion from providing any information to copy. We compare the T=T ternary complex with a BF ternary complex with an abasic site analogue and show that both lesions are noninstructional and induce nearly identical conformations in the polymerase and the substrate dATP. The incoming dATP inserts too far into the active site in the absence of a template base and distorts into a structure that is not conducive for catalysis with the primer 3'-hydroxyl. Together, our results show that thymine dimers and abasic sites can stall DNA synthesis by providing an absence of information through similar mechanisms.
The Ascent of Miniproteins.
Yoo Y, Nanda V
Biochemistry · 2026 Jun · PMID 42314113 · Publisher ↗
Miniproteins are present across all branches of life and play central roles in diverse biological processes, but their existence fundamentally challenges our assumptions about the minimal sequence, structural, and energe...
Miniproteins are present across all branches of life and play central roles in diverse biological processes, but their existence fundamentally challenges our assumptions about the minimal sequence, structural, and energetic requirements necessary for protein stability and function. More than molecular curiosities, miniproteins may offer a window into the earliest catalysts that emerged at the origin of life. Here, we focus on metal-containing miniproteins, where coordination chemistry can impart both structural stability and redox activity essential for metabolism. We ask whether primordial peptides could have evolved into complex proteins through miniprotein intermediates by surveying natural miniproteins as snapshots along an evolutionary trajectory, and examining engineered miniproteins that can model the gaps between these snapshots. Finally, we explore whether miniproteins continue to evolve today─either recapitulating early evolutionary processes or giving rise to entirely new folds and functions.
A Systematic Analysis of Lipid-Protein Interactions in the Protein Data Bank.
Puri N, McShan AC
Biochemistry · 2026 Jun · PMID 42313607 · Publisher ↗
Lipid-protein interactions are ubiquitous in biology, where they are fundamental to membrane structure, cell signaling, immunology, and metabolism. Despite the availability of thousands of experimentally determined lipid...
Lipid-protein interactions are ubiquitous in biology, where they are fundamental to membrane structure, cell signaling, immunology, and metabolism. Despite the availability of thousands of experimentally determined lipid-protein structures, the molecular basis for lipid recognition and specificity across the lipid-protein interactome remains incompletely understood. Here, we report a systematic analysis of 113,782 annular and nonannular lipid-protein complexes spanning the eight lipid classes. Pairwise atomic interactions are linked to lipid and protein physicochemical properties and binding geometries. Hydrophobic contacts, hydrogen bonds, and salt bridges contributed to over 99% of lipid-protein interactions. Lipid class-, protein sublocalization-, protein function-, and protein fold-dependent trends were identified. Protein pockets were finely tuned for lipid size, shape, and polarity: fatty acyls associated with narrow, moderately hydrophobic pockets; saccharolipids and glycerophospholipids bound to larger, polar cavities; and sterols and prenols preferentially occupied compact hydrophobic sites. Global analysis across different protein families identified similarities in interaction profiles, while also highlighting protein-specific recognition adapted to biochemical function. Lipid-protein interaction maps were projected onto lipid structures to uncover conserved and divergent hotspots and coldspots across lipid classes. The heatmaps imply that recognition and specificity are mediated by tailored anchoring of polar head groups and varying interaction with hydrophobic tails. Together, the data establish nature's principles governing lipid binding, lipid selectivity, and complex stability, and collectively provide a molecular atlas of the lipid-protein interactome. The work enables the elucidation of lipid biology at scale and establishes guiding principles for the rational design of chemical probes and therapeutics targeting lipid biology.
Improved Stability and Brightness Following Iterative Redesign of a De Novo Biliprotein.
Morey-Burrows FS, Yang T, Ahern W … +6 more , Baker D, Hitchcock A, Leggett GJ, Clark J, Leney AC, Hunter CN
Biochemistry · 2026 Jun · PMID 42295740 · Publisher ↗
Bilins, linear tetrapyrroles derived from heme catabolism, are ubiquitous across biological systems. The constraints imposed by binding to a protein confer valuable optical properties on bilins, which are used for sensin...
Bilins, linear tetrapyrroles derived from heme catabolism, are ubiquitous across biological systems. The constraints imposed by binding to a protein confer valuable optical properties on bilins, which are used for sensing and harvesting light, and offer potential for fluorescence imaging, optogenetics, and biosensing. Recently, bilins have been used to evaluate methods for designing ligand binding sites, and novel biliproteins (BPs) that bind phycoerythrobilin (PEB) emerged from computational design using RoseTTAFold Diffusion All-Atom (RFDiffusionAA) and LigandMPNN. Here, we find much lower stability, extinction coefficients and fluorescence quantum yields for de novo designs compared to the native CpcA-PEB biliprotein, so we used a LigandMPNN-AlphaFold 3 (AF3)-Rosetta Relax pipeline to redesign one BP, C11, which can be implemented quickly and without in-house GPUs. The top three ligand confidence scores emerging from this procedure yielded C11-578, C11-620, and C11-756 redesigns that retain 50%, 52%, and 57% identities, respectively, with C11. AF3-Rosetta modeling of the BP redesigns predicted similar conformations of the PEB, decreased solvent-accessible surface area of the bilin binding site, an additional predicted hydrogen bond in C11-620, and a more rigid protein backbone. The new constraints on bilin binding improved the stability, absorption properties and fluorescence yields for C11-578, C11-620, and C11-756, and the brightness of the redesigns improved up to 13-fold, approaching the values for the native CpcA-PEB BP. Further design iterations can be used to create a range of absorbing and emitting BPs, including oligomeric assemblies housing internal energy cascades.
Advanced Peptide Modifications for Superior Therapeutics.
Bhadra J, De N, Momin MSA … +2 more , Sannigrahi A, Bhunia D
Biochemistry · 2026 Jun · PMID 42290059 · Publisher ↗
Peptides have emerged as a unique class of therapeutics that bridge the gap between small molecules and biologics. Their high specificity, favorable safety profiles, and diverse biological functions make them promising c...
Peptides have emerged as a unique class of therapeutics that bridge the gap between small molecules and biologics. Their high specificity, favorable safety profiles, and diverse biological functions make them promising candidates across therapeutic areas, including metabolic disorders, oncology, infectious diseases, and pain management. Advances in phage display, chemical synthesis, structural modification, and drug delivery technologies have significantly accelerated peptide drug development. This review highlights recent progress in peptide therapeutics, covering their advantages and limitations, strategies for design and modification, use of noncanonical amino acids, and applications in drug delivery systems. In addition, the roles of therapeutic peptides in diabetes, cancer, antiviral and antimicrobial therapies, and peptide-drug conjugates are discussed, with emphasis on emerging opportunities and challenges for future clinical translation.
PAX3-FOXO1 Contacts BRD4 through Its Acetylated Intrinsically Disordered Region.
Fraser OA, Schleicher MN, Pagano ML … +1 more , Showalter SA
Biochemistry · 2026 Jun · PMID 42284071 · Publisher ↗
Intrinsically disordered regions (IDRs) of transcription factors are frequent sites for post-translational modifications (PTMs), which mediate regulation through diverse mechanisms including protein-protein interactions....
Intrinsically disordered regions (IDRs) of transcription factors are frequent sites for post-translational modifications (PTMs), which mediate regulation through diverse mechanisms including protein-protein interactions. In the fusion oncoprotein PAX3-FOXO1, which drives alveolar rhabdomyosarcoma, a lysine-rich region of the FOXO1 IDR is subject to acetylation resulting in stabilization and enhanced transcriptional activity. Here, we leveraged C direct-detect nuclear magnetic resonance (NMR) spectroscopy to characterize acetylation in this system and identified a novel acetylation site corresponding to lysine 233 in endogenous FOXO1. In previous structural characterization of the endogenous FOXO1 DNA binding domain, local structure appears to prevent this site from becoming acetylated, suggesting that it becomes exposed in the context of the fusion protein. In addition, we demonstrate that the first bromodomain of the bromodomain and extraterminal domain-containing protein BRD4 binds to the acetylated region of interest and that this interaction is inhibited through the bromodomain and extraterminal domain inhibitor JQ1. These findings confer molecular mechanistic detail to previous observations that BRD4 and PAX3-FOXO1 colocalize at superenhancers in ARMS, adding to the growing body of literature exploring how BRD4 contacts cancer-relevant transcription factors in ways potentially relevant to the use of bromodomain and extraterminal domain inhibitors in cancer treatment.
Synthetic Substrate Discovery for Hck Kinase via Phage Display.
Kannan A, Heier JL, Motwani DP … +4 more , Meyers HJ, Andrews M, Gonzalez JE, Parker LL
Biochemistry · 2026 Jun · PMID 42283445 · Publisher ↗
Protein tyrosine kinases govern essential cellular processes, including proliferation, survival, and migration. Dysregulation of this enzyme family has been implicated in multiple hallmarks of cancer. These kinases catal...
Protein tyrosine kinases govern essential cellular processes, including proliferation, survival, and migration. Dysregulation of this enzyme family has been implicated in multiple hallmarks of cancer. These kinases catalyze tyrosine phosphorylation in a tightly controlled manner, with substrate recognition dictated by the local chemical environment of surrounding residues. Although several strategies have been developed to define protein tyrosine kinase substrate specificity, these approaches have notable limitations in scale and resolution. To address these challenges, we present a modernized phage display platform integrated with next-generation sequencing, enabling comprehensive and high-throughput profiling of tyrosine kinase substrates. This method enabled the simultaneous assessment of the relative phosphorylation of billions of potential substrates for Hck kinase, followed by analysis of positional and motif enrichment to guide iterative substrate design. The novel substrates developed through this process exhibited robust kinetic behavior (K < 30 μM) when evaluated by HPLC/MS assays, yielding insights into Hck substrate preferences within multipositional sequence contexts.
Correction to " Genistein Arrests Cell Cycle Progression of A549 Cells at the G2/M Phase and Depolymerizes Interphase Microtubules through Binding to a Unique Site of Tubulin".
Mukherjee S, Acharya BR, Bhattacharyya B … +1 more , Chakrabarti G
Biochemistry · 2026 Jun · PMID 42281368 · Publisher ↗
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Mechanistic Insights into the Stimulation of PARP2 Self-Parylation Catalytic Activity by DNA Breaks and PAR.
Greeshma SP, Hanuman DS, Rajakumara E
Biochemistry · 2026 Jun · PMID 42274357 · Publisher ↗
Poly(ADP-ribose) polymerases (PARPs) play a central role in maintaining genome stability and catalyzing the transfer of ADP-ribose units from NAD to target proteins through a process known as poly(ADP-ribosyl)ation (PARy...
Poly(ADP-ribose) polymerases (PARPs) play a central role in maintaining genome stability and catalyzing the transfer of ADP-ribose units from NAD to target proteins through a process known as poly(ADP-ribosyl)ation (PARylation). While mammalian PARPs are extensively characterized, their plant homologues remain less explored. Here, we dissect the mechanistic regulation of PARP2 (atPARP2) through its interactions with DNA and poly(ADP-ribose) (PAR). Biolayer interferometry (BLI) binding studies revealed that atPARP2 recognizes phosphorylated DNA double-strand breaks (p-DSBs) and PAR with a nanomolar affinity. Using the SAP domain of atPARP2, we reported for the first time that it binds both PAR and p-DSBs, thereby significantly enhancing the binding affinity of atPARP2 for PAR and p-DSBs. In addition, isothermal titration calorimetry (ITC) and biochemical assays demonstrated that PAR serves as an allosteric activator of atPARP2, with the WGR domain acting as a key module for PAR binding. Enzymatic assays further established the indispensable role of SAP domains in enhancing DNA- and PAR-dependent catalytic activities. Together, our findings demonstrate that atPARP2 may employ a modular strategy, integrating DNA break sensing and PAR recognition, to fine-tune its catalytic function in response to genotoxic stress.
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