{"title":"Recently Discovered Heteromeric Enzymes in Natural Product Biosynthesis.","authors":"Zhongtian Yu,Ikuro Abe","doi":"10.1016/j.jbc.2025.108516","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108516","url":null,"abstract":"The abundant diversity and elegant complexity in the chemical structures of natural products have attracted vigorous investigations of the chemistry and enzymology underlying their biosynthetic processes over the past few decades. Among the biochemical events, the formation of complexes of heteromeric enzymes has been observed in the biosynthesis of several natural products and metabolic pathways. In this review, we aim to consolidate the recently discovered cases of heteromeric enzymes in natural product biosynthesis and metabolism, in order to clarify the genetic and structural bases leading to the formation of these heteromeric complexes and provide insights for the rational redesign of proteins in biosynthetic machineries.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"9 1","pages":"108516"},"PeriodicalIF":4.8,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Benjamin R Duffus,Benedict J Elvers,Christian Teutloff,Carola Schulzke,Silke Leimkühler
{"title":"In vitro sulfuration of Rhodobacter capsulatus formate dehydrogenase.","authors":"Benjamin R Duffus,Benedict J Elvers,Christian Teutloff,Carola Schulzke,Silke Leimkühler","doi":"10.1016/j.jbc.2025.108511","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108511","url":null,"abstract":"Metal-dependent formate dehydrogenases (FDHs) are of considerable interest as a bioinspired metalloenzyme target to efficiently reduce the greenhouse gas CO2 into the portable energy carrier formate under physiological conditions. These enzymes were shown to harbor an active site sulfido ligand that is essential for the formate oxidation and CO2 reduction activity and contributes to the oxygen sensitivity of the enzyme, since the ligand is rapidly lost in the presence of O2. Inhibitors like azide or nitrate are routinely employed to protect the active site from oxidative damage. The demonstrated unitary in vitro sulfido ligand incorporation to the active site bis metal-binding pterin guanine dinucleotide (bis-MGD) cofactor in FDH from Rhodobacter capsulatus of this study also completely reactivates the enzyme. Reductive treatment with either sulfide or bisulfite, or with sodium dithionite under weakly acidic conditions in the strict absence of O2 resulted in comparable enzymatic activity to FDH purified after heterologous expression in Escherichia coli. Confirmation of the inserted sulfido ligand was afforded by EPR spectroscopy of a MoV intermediate species associated with MoS6 coordination. Specific insertion of a 33S sulfido ligand to the bis-MGD Mo evidenced the chemical insertion of the sulfido ligand and confirmed its role to serve in defining the electronic character of the sulfurated bis-MGD MoV-SH state. The relevance of these results, in relation to known in vitro sulfuration assays described for other molybdoenzymes, is discussed.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"49 1","pages":"108511"},"PeriodicalIF":4.8,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Cooperative inhibition in cytochrome P450 between a substrate and an apparent non-competitive inhibitor.","authors":"Yu Hirano,Sachiyo Yoneda,Kaori Yasuda,Noriyuki Kurita,Fumihiro Kawagoe,Bunzo Mikami,Teisuke Takita,Kiyoshi Yasukawa,Shinichi Ikushiro,Midori Takimoto-Kamimura,Atsushi Kittaka,Toshiyuki Sakaki,Taro Tamada","doi":"10.1016/j.jbc.2025.108513","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108513","url":null,"abstract":"Cytochrome P450 (CYP) enzymes bind a heme group that acts as a catalytic center. Inhibition mechanisms in CYP enzymes have been studied extensively by biochemical and structural analyses. Non-competitive inhibitors are generally believed to bind to allosteric sites remote from the active site to form enzyme-substrate-inhibitor (ESI) complexes. Docking simulations predict the binding sites of non-competitive inhibitors to CYP enzymes, but to date there has been no experimental structural verification of ESI complexes formed by CYP enzymes. We performed biochemical and structural analyses of CYP105A1 using the imidazole-containing inhibitors ketoconazole, lanoconazole, and miconazole. Spectroscopic analyses showed that ketoconazole and miconazole act as competitive inhibitors, whereas lanoconazole acts as a non-competitive inhibitor of CYP105A1. The obtained X-ray structures of enzyme-inhibitor (EI) complexes showed that lanoconazole can bind in various orientations to the heme iron compared with ketoconazole and miconazole. We also determined the X-ray structure of an ESI complex comprising CYP105A1, diclofenac, and lanoconazole. This structure shows that lanoconazole binds to the heme iron and that diclofenac closely interacts with the bound lanoconazole but it is positioned distant from the heme group. Quantum mechanical calculations indicate that Cl-π and electrostatic interactions between diclofenac and lanoconazole, and electrostatic interactions between diclofenac and positively charged arginine residues, stabilize formation of the ESI complex. Based on these results, we propose a mechanism for cooperative inhibition between a substrate and an apparent non-competitive inhibitor.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"50 1","pages":"108513"},"PeriodicalIF":4.8,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Diego Pilati,Eugene Kusi Agyei,Marwa Elkhashab,Elisabeth Fuchs,Ian Helstrup Nielsen,Tobias Wang Bjerg,Aina Karen Anthi,Anaïs Jiménez-Reinoso,Marie Beck Iversen,Layla Pohl,Ryo Narita,Susana Frago,Martin R Jakobsen,Jan Terje Andersen,Søren E Degn,Søren R Paludan,Luis Alvarez-Vallina,Kenneth A Howard
{"title":"Exploiting FcRn engagement of an albumin-CpG oligonucleotide covalent conjugate for potent TLR9 immune induction.","authors":"Diego Pilati,Eugene Kusi Agyei,Marwa Elkhashab,Elisabeth Fuchs,Ian Helstrup Nielsen,Tobias Wang Bjerg,Aina Karen Anthi,Anaïs Jiménez-Reinoso,Marie Beck Iversen,Layla Pohl,Ryo Narita,Susana Frago,Martin R Jakobsen,Jan Terje Andersen,Søren E Degn,Søren R Paludan,Luis Alvarez-Vallina,Kenneth A Howard","doi":"10.1016/j.jbc.2025.108508","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108508","url":null,"abstract":"CpG-oligodeoxynucleotide (CpG ODN)-based toll-like receptor (TLR) agonists are promising immunostimulatory adjuvants, however, low entry into TLR-rich cellular endosomal compartments and poor lymphatic accumulation limit clinical translation. In this work, we introduce a recombinant human serum albumin (rHA)-CpG ODN covalent conjugate (rHA-CpG) designed to exploit the neonatal Fc receptor (FcRn)-driven albumin cellular sorting pathway to maximise CpG delivery into TLR9-rich endosomes and accumulate in lymph nodes. Site-selective conjugation of CpG to albumin cysteine 34, distant from its main FcRn binding interface, resulted in a retained pH dependent human FcRn binding, and receptor-driven endosomal trafficking in a cellular recycling assay. Induction of tumour necrosis factor (TNF) secretion in THP-1 cells and interferon alpha (IFN-α) in human hematopoietic stem and progenitor cell (HSPC)-derived plasmacytoid dendritic cells (pDCs), in contrast, to a myeloid differentiation primary response 88 (MyD88) and TLR9 knockout cells, respectively, support TLR9-engagement. The rHA-CpG construct induced greater TNF-α than free CpG ODN in mouse RAW 264.7 cells, and in human peripheral blood mononuclear cells (PBMCs) and expansion of classical (CD14+CD16-) monocytes. Furthermore, greater accumulation of Cy5.5-labelled CpG in the inguinal (>3-fold) and axillary (>18-fold) lymph nodes was observed when conjugated to rHA compared to an unconjugated rHA/CpG mix following subcutaneous injection in mice. Moreover, increased LN accumulation of an rHA variant engineered with high FcRn-binding affinity supports an FcRn-driven mechanism. Demonstration of FcRn-mediated albumin targeting at intra- and extracellular sites provides the mechanistic basis for potent immune induction observed using the novel rHA-CpG conjugate design class introduced in this work.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"27 1","pages":"108508"},"PeriodicalIF":4.8,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143831731","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"All intrinsically active Erk1/2 mutants autophosphorylate Threonine207/188, a plausible regulator of the TEY motif phosphorylation.","authors":"Alexey Baskin,Nadine Soudah,Nechama Gilad,Neriah Halevy,Ilona Darlyuk-Saadon,Hanan Schoffman,David Engelberg","doi":"10.1016/j.jbc.2025.108509","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108509","url":null,"abstract":"The extracellular-activated kinases 1 & 2 (Erk1/2) are catalytically active when dually phosphorylated on a TEY motif located at the activation loop. In human patients with cardiac hypertrophy Erk1/2 are phosphorylated on yet another activation loop's residue, T207/188. Intrinsically active variants of Erk1/2, mutated at R84/65, are also (auto)phosphorylated on T207/188. It is not known whether T207/188 phosphorylation is restricted to these cases nor how it affects Erks' activity. We report that T207/188 phosphorylation is not rare, as we found that: 1) All known auto-activated Erk1/2 variants are phosphorylated on T207/188. 2) It occurs in various cell lines and mouse tissues. 3) It is extremely high in patients with skeletal muscle atrophies or myopathies. We propose that T207/188 controls the permissiveness of the TEY motif for phosphorylation because T207/188-mutated Erk1/2 and the yeast Erk/Mpk1 were efficiently dually phosphorylated when expressed in HEK293 or yeast cells, respectively. The T207/188-mutated Mpk1 was not TEY-phosphorylated in cells knocked out for MEKs, suggesting that its enhanced phosphorylation in wildtype cells is MEK-dependent. Thus, as T207/188-mutated Erk1/2 and Mpk1 recruit MEKs the role of T207/188 is to impede MEKs' ability to phosphorylate Erks. T207/188 also impedes autophosphorylation as recombinant Erk2 mutated at T188 is spontaneously autophosphorylated, although exclusively on Y185. The role of T207/188 in regulating activation loop phosphorylation may be common to most Ser/Thr kinases as 86% of them (in the human kinome) possess T207/188 orthologs, and 160 of them were already reported to be phosphorylated on this residue.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"76 1","pages":"108509"},"PeriodicalIF":4.8,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143831723","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Structural and biochemical characterization of the 3'-5' tRNA splicing ligases.","authors":"Sebastian Chamera,Weronika Zajko,Mariusz Czarnocki-Cieciura,Marcin Jaciuk,Łukasz Koziej,Jakub Nowak,Krzysztof Wycisk,Małgorzata Sroka,Andrzej Chramiec-Głąbik,Mirosław Śmietański,Filip Gołębiowski,Marcin Warmiński,Jacek Jemielity,Sebastian Glatt,Marcin Nowotny","doi":"10.1016/j.jbc.2025.108506","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108506","url":null,"abstract":"In archaea and metazoa, tRNA exons are ligated by the RNA ligases RtcB and RTCB, respectively. The metazoan RTCB forms a stable complex with four additional subunits, DDX1, FAM98B, CGI99 and ASHWIN. The role and assembly of these four components remain elusive. Furthermore, we lack structural information of how RNA substrates are recognized by 3'-5' tRNA ligases. Here, we use thiol-based chemical cross-linking to confirm the involvement of specific residues of RtcB in RNA binding and we present a cryo-electron microscopy structure of the purified five-subunit D. rerio tRNA ligase complex. The structure implies that the DDX1 helicase module is mobile and can modulate the activity of RTCB. Taken together, the presented results enhance our mechanistic understanding of RNA binding by 3'-5' tRNA splicing ligases and architecture of the metazoan tRNA ligase complex.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"35 1","pages":"108506"},"PeriodicalIF":4.8,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143827119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alexander V Peskin,Flavia C Meotti,Nicholas J Magon,Luiz F de Souza,Armindo Salvador,Christine C Winterbourn
{"title":"Mechanism of glutathionylation of the active site thiols of peroxiredoxin 2.","authors":"Alexander V Peskin,Flavia C Meotti,Nicholas J Magon,Luiz F de Souza,Armindo Salvador,Christine C Winterbourn","doi":"10.1016/j.jbc.2025.108503","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108503","url":null,"abstract":"Peroxiredoxin 2 (Prdx2) undergoes ready glutathionylation, and glutaredoxin-catalyzed deglutathionylation provides an alternative mechanism to thioredoxin/thioredoxin reductase for recycling the reduced protein (Peskin et al JBC 216, 3053, 2016). To elucidate the mechanism of glutathionylation, we have carried out kinetic studies using stopped flow and SDS PAGE plus product analysis by mass spectrometry. Kinetic modelling shows a mechanism in which exchange of Prdx2 disulfide with physiological concentrations of GSH occurs over seconds to minutes, initially at one active site to produce glutathionylated dimers linked by one disulfide. Exchange with GSH yields glutathionylation at both the peroxidatic (CP) and resolving cysteines (CR), the former predominating. Rate constants of 1.5 M-1s-1 and 0.021 s-1 were determined for exchange-mediated glutathionylation and deglutathionylation. Similar exchange reactions subsequently occur at the second active site. The rate of reaction of the CP sulfenic acid of wildtype Prdx2 with GSH (k = 10 M-1s-1) is 8-30 fold slower than when CR is mutated to Ser, Trp or Asp and this reaction cannot effectively compete with intramolecular condensation. Consequently, when H2O2 reacts with reduced Prdx2 in the presence of GSH, the initial product is predominately the Prdx disulfide and glutathionylation subsequently occurs by exchange. However, glutathionylation of CR in the presence of H2O2 facilitates condensation of CP sulfenic acid with GSH to give diglutathionylated products and suppresses hyperoxidation. This displaces equilibria and accelerates the conversion of Prdx2 to monomeric species. These results have implications for understanding the mechanism of relays between Prdx2 and other thiol proteins.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"112 1","pages":"108503"},"PeriodicalIF":4.8,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143827117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Charles L Mitchell,Mikhail Matveyenka,Dmitry Kurouski
{"title":"Neuroprotective Properties of Transition Metal Dichalcogenide Nanoflowers Alleviate Acute and Chronic Neurological Conditions Linked to Mitochondrial Dysfunction.","authors":"Charles L Mitchell,Mikhail Matveyenka,Dmitry Kurouski","doi":"10.1016/j.jbc.2025.108498","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108498","url":null,"abstract":"Mitochondrial dysfunction is an expected cause of etiology and progression in numerous human neurological pathologies, including stroke, Alzheimer's, and Parkinson's diseases. Therefore, a neuroprotective treatment is an urgent and unmet need. Transition metal dichalcogenide nanoflowers (TMD NFs) exhibit unique biological properties. However, neuroprotective properties of these nanomaterials remain poorly understood. In the current study, the biological effect of molybdenum disulfide (MoS2) and molybdenum diselenide (MoSe2) TMD NFs on neurons and astrocytes was investigated. It was found that both nanomaterials lowered reactive oxygen species (ROS) levels, reduced mitochondrial impairment, and increased mitochondrial biogenesis. Neuroprotective effects of both TMD NFs resulted from upregulation of the PGC-1α pathway, the biological system responsible for mitochondrial biogenesis. Furthermore, administration of TMD NFs to C. elegans extended lifespan of the nematodes. These results indicate that TMD NFs can be used as novel neuroprotective therapeutic agents against acute and chronic neurological condition linked to mitochondrial dysfunction.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"5 1","pages":"108498"},"PeriodicalIF":4.8,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143824786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Loss of sex-determining region Y-box 2 (SOX2) captures embryonic stem cells in a primed pluripotent state.","authors":"Min Qi,Bowen Wang,Huaqi Liao,Yuzhuo Xu,Lixia Dong,Lijun Xu,Yin Xia,Xiaochun Jiang,Shizhang Ling,Jinzhong Qin","doi":"10.1016/j.jbc.2025.108501","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108501","url":null,"abstract":"Two main pluripotent cell lines can be established from the preimplantation and postimplantation mouse embryo as naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. Although the two pluripotent states are interconvertible, the molecular mechanism controlling the transition between naïve and primed pluripotency remains to be fully elucidated. Here, by performing a CRISPR-based loss-of-function screen in ESCs, we identify Sox2 involved in the repression of lineage-specification marker brachyury (T). Upon Sox2 ablation in ESCs, two populations of cells mutually exclusive for CDX2 (trophectoderm marker) and T expression can be observed. T-positive cells display features resembling the salient characteristics of EpiSCs including molecular and functional properties. By using genetic ablation approach, we show that acquisition and maintenance of primed pluripotency in Sox2 null T-positive cells heavily depend on fibroblast growth factor (Fgf) and Nodal, which is produced in an autocrine manner in these cells. We further demonstrate that Sox3 compensates for the absence of Sox2 in maintaining the primed state of Sox2-null pluripotent cells. Establishment of Sox2-deficient pluripotent cells will enable the elucidation of the mechanisms controlling the transition of cells between different states of pluripotency.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"108 1","pages":"108501"},"PeriodicalIF":4.8,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143824824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ivana Nemčovičová,Juraj Kóňa,Monika Poláková,Tomáš Klunda,Andrej Bitala,Mário Benko,Simona Lenhartová,Marek Nemčovič
{"title":"Small glycomimetic antagonists of the cytomegalovirus glycoprotein UL141 prevent binding to TRAIL death receptor.","authors":"Ivana Nemčovičová,Juraj Kóňa,Monika Poláková,Tomáš Klunda,Andrej Bitala,Mário Benko,Simona Lenhartová,Marek Nemčovič","doi":"10.1016/j.jbc.2025.108490","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108490","url":null,"abstract":"Human cytomegalovirus (HCMV) UL141 inhibits immune recognition of virally infected cells by natural killer (NK) cells and cytotoxic T cells through modulation of cellular receptors (e.g., TRAIL-R2/-R1, CD155, CD112). Recent findings suggest that UL141 is also a critical component of the HCMV virion, that further emphasizing its significance. In this study, we aimed to develop a small synthetic compound as a UL141 antagonist. Building on our crystal structure analysis, we designed compounds to specifically bind viral UL141, thereby blocking its interaction with target receptors thus inhibiting its immunoevasive functions. We evaluated a small library of synthesized compounds composed of diverse saccharide units conjugated with non-saccharide moieties, such as non-ionic glycolipids, pyrrolidines, and 'click' conjugates. An ELISA-like TMB binding assay, coupled with dynabeads coating, was employed to assess the ability of these compounds to inhibit TRAIL-R2 binding in vitro. The most promising compounds capable of inhibiting complex formation were further analyzed using surface plasmon resonance (SPR). Compound 18 exhibited the strongest binding affinity to UL141, with KD of 2.93 μM. Molecular docking studies identified specific binding sites on UL141, and the fragmented molecular orbital (FMO) method was applied to evaluate interaction energy patterns between the antagonist and the UL141 protein. Mutational analysis was conducted to validate the identified binding sites on UL141. Additionally, cellular cytotoxicity assays were performed to confirm the non-toxic properties of these compounds. Collectively, our findings suggest that synthetic glycomimetics represent promising candidates for targeting the viral glycoprotein HCMV UL141, thereby disrupting TRAIL death receptor signaling thus mitigating viral activity.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"27 1","pages":"108490"},"PeriodicalIF":4.8,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143824785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}