Journal of Biological Chemistry最新文献_第10页

CpgD is a phosphoglycerate cytidylyltransferase required for ceramide diphosphoglycerate synthesis CpgD是合成二磷酸甘油酸神经酰胺所需的磷酸甘油酸胞基转移酶

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-16 DOI: 10.1016/j.jbc.2025.110386

Tanisha Dhakephalkar, Ziqiang Guan, Eric A. Klein

{"title":"CpgD is a phosphoglycerate cytidylyltransferase required for ceramide diphosphoglycerate synthesis","authors":"Tanisha Dhakephalkar, Ziqiang Guan, Eric A. Klein","doi":"10.1016/j.jbc.2025.110386","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110386","url":null,"abstract":"LPS is essential in most Gram-negative bacteria, but mutants of several species have been isolated that can survive in its absence. <ce:italic>Caulobacter crescentus</ce:italic> viability in the absence of LPS is partially dependent on the anionic sphingolipid ceramide diphosphoglycerate (CPG2). Genetic analyses showed that <ce:italic>ccna_01210</ce:italic>, which encodes a nucleotidyltransferase, is required for CPG2 production. Using purified recombinant protein, we determined that CCNA_01210 (CpgD) is a phosphoglycerate cytidylyltransferase which uses CTP and phosphoglycerate to produce CDP-glycerate, which we hypothesize is the phosphoglycerate donor for CPG2 synthesis. CpgD had optimum activity at pH 7.5-8 in the presence of magnesium. CpgD exhibited Michaelis-Menten kinetics with respect to 3-phosphoglycerate, D-2-phosphoglycerate, and L-2-phosphoglycerate. By contrast, CTP followed Michaelis-Menten kinetics in the presence of 3-phosphoglycerate and L-2-phosphosglycerate but exhibited cooperativity with D-2-phosphoglycerate. Overall, D-2-phosphoglycerate was the preferred substrate <ce:italic>in vitro</ce:italic>. The characterization of this enzyme uncovers another step in the pathway towards CPG2 synthesis.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"6 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144305069","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}

引用次数: 0

A kinetic model of copper homeostasis in Saccharomyces cerevisiae. 酿酒酵母菌铜稳态的动力学模型。

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-16 DOI: 10.1016/j.jbc.2025.110368

Cade Dulaney,Jay R Walton,Paul A Lindahl

{"title":"A kinetic model of copper homeostasis in Saccharomyces cerevisiae.","authors":"Cade Dulaney,Jay R Walton,Paul A Lindahl","doi":"10.1016/j.jbc.2025.110368","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110368","url":null,"abstract":"Rather than inhibiting copper entry when grown on high Cu, yeast cells import excessive Cu while simultaneously increasing expression of metallothionein CUP1 which then sequesters the excess Cu. An ordinary-differential-equations-based kinetic model was developed to investigate this unusual behavior. The assumed reaction network included 25 reactions and 10 components in the cytosol of yeast cells growing in media supplemented with increasing nutrient COPPER concentrations. Published concentrations of copper proteins and coordination complexes that constitutes the low-molecular-mass (or labile) Cu pool were assumed. Other components included transcription factors MAC1 and ACE1, the MAC1-dependent copper importer CTR1, and other copper proteins considered collectively. A second MAC1-independent importer was required for sufficient Cu to enter the cell under Cu-excess conditions. The mathematical system was initially solved at steady-state for each condition in the series. The null-space of the stoichiometric matrix was evaluated using the Basic Pathways approach. Steady-state rates and rate-constants were calculated for each reaction and each condition of the series. Four rate-constants trended higher across the series indicating that the cell regulates those reactions in ways that were not included in the assumed rate-law expressions. This behavior was simulated by augmenting those expressions with logistical functions that sensed labile Cu and/or nutrient COPPER. The resulting integrated dynamical system approximately generated observed component concentrations over the series and was stable to both intracellular and extracellular perturbations. The MAC1-independent importer is predicted to be FET4, a nonspecific importer of both Cu and Fe. Cells likely tolerate excessive Cu import to import sufficient iron.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"101 1","pages":"110368"},"PeriodicalIF":4.8,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144320323","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}

引用次数: 0

Molecular arrangements that accompany binding of rice xylanase inhibitor protein OsXIP and the Rhizopus oryzae GH11 xylanase RXyn2. 水稻木聚糖酶抑制蛋白OsXIP与米根霉GH11木聚糖酶RXyn2结合的分子排列

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-16 DOI: 10.1016/j.jbc.2025.110385

Takayuki Ohnuma,Jun Tanaka,Harutada Ozaki,Keigo Mitsui,Daichi Tsujitsugu,Miki Okugawa,Toru Takeda,Makoto Ihara,Tamo Fukamizo,Daijiro Takeshita

{"title":"Molecular arrangements that accompany binding of rice xylanase inhibitor protein OsXIP and the Rhizopus oryzae GH11 xylanase RXyn2.","authors":"Takayuki Ohnuma,Jun Tanaka,Harutada Ozaki,Keigo Mitsui,Daichi Tsujitsugu,Miki Okugawa,Toru Takeda,Makoto Ihara,Tamo Fukamizo,Daijiro Takeshita","doi":"10.1016/j.jbc.2025.110385","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110385","url":null,"abstract":"Plants have evolved xylanase inhibitor proteins as part of their defense mechanisms against phytopathogens. The rice xylanase inhibitor protein (OsXIP) is structurally similar to GH18 chitinase and homologous to wheat XIP-type inhibitor (XIP-I), which inhibits both GH10 and GH11 xylanases. Various inhibition and interaction analyses showed that OsXIP competitively inhibits the hydrolytic activity of GH11 xylanase RXyn2, but not the activity of GH10 xylanase RXyn1 from Rhizopus oryzae. The crystal structure of the OsXIP/RXyn2 complex showed that OsXIP, which has a (β/α)8-barrel fold, extrudes the loop between α4 and β5 (Lα4β5OsXIP) and inserts the loop into the xylotriose binding site (-3 to -1 subsite) formed by the inner β-sheet (palm) of RXyn2 jelly roll. The guanidyl group of Arg155 in Lα4β5OsXIP was shown to be critical for the inhibitory activity by mutational analysis. Notably, in the complex structure, the cylindrical cavity formed by the palm of RXyn2 jelly roll stacked upright on the loops at the N-terminal ends of the β-strands of OsXIP (I-formation). On the other hand, in the complex structure of XIP-I and GH11 xylanase from Talaromyces funiculosus (XYNC), the cavity of XYNC laid tangentially to the part of the corresponding region of XIP-I through the Lα4β5XIP-I (T-formation). The dissociation constant of the OsXIP/RXyn2 complex was one tenth of that of the XIP-I/XYNC complex (4.2 versus 41.5 nM). OsXIP may have adapted to bind and inhibit GH11 enzymes, which are resistant to the inhibition by XIP-I type proteins, by changing its binding mode.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"23 1","pages":"110385"},"PeriodicalIF":4.8,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144320324","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}

引用次数: 0

Structural biology of Parkinson’s Disease-associated Leucine-Rich Repeat Kinase 2 (LRRK2) 帕金森病相关富亮氨酸重复激酶2 （LRRK2）的结构生物学研究

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-14 DOI: 10.1016/j.jbc.2025.110376

Andres E. Leschziner

引用次数: 0

FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-CBL-NF-κB signaling FAK活性通过VEGFR2-CBL-NF-κB信号加剧了紊乱的血流介导的动脉粥样硬化

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-14 DOI: 10.1016/j.jbc.2025.110383

James M. Murphy, Duyen Thi Kieu Tran, Kyuho Jeong, Ly Nguyen, Mai Thi Nguyen, Dhananjay Tambe, Hanjoong Jo, Eun-Young Erin Ahn, Ssang-Taek Steve Lim

{"title":"FAK activity exacerbates disturbed flow-mediated atherosclerosis via VEGFR2-CBL-NF-κB signaling","authors":"James M. Murphy, Duyen Thi Kieu Tran, Kyuho Jeong, Ly Nguyen, Mai Thi Nguyen, Dhananjay Tambe, Hanjoong Jo, Eun-Young Erin Ahn, Ssang-Taek Steve Lim","doi":"10.1016/j.jbc.2025.110383","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110383","url":null,"abstract":"Atherosclerosis develops at predictable sites in the vasculature where branch points and curvatures create non-laminar disturbed flow. This disturbed flow causes vascular inflammation by increased endothelial cell (EC) barrier permeability and the expression of inflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1). Vascular endothelial growth factor receptor 2 (VEGFR2) regulates flow-induced EC inflammation; however, there are still some gaps in understanding the precise signaling mechanism or pathway. Focal adhesion kinase (FAK) is a protein tyrosine kinase whose expression has been implicated in flow-mediated signaling in ECs. However, the link between FAK and VEGFR2 in flow-mediated inflammation signaling has remained unelucidated. Here we found that priming of VEGFR2 with VEGF was critical for flow-mediated activation of FAK and NF-kB. Mechanistically, FAK activation triggers tyrosine phosphorylation of Casitas B-lineage lymphoma (CBL; an E3 ubiquitin ligase) that interacts with VEGFR2 under flow conditions. However, FAK inhibition reduced VEGFR2-FAK-CBL complex formation, partly due to reduced expression of VEGFR2 on the cell membrane. Further, <ce:italic>Apoe-/-</ce:italic> mice fed a western diet (WD) exhibited increased FAK activity within the atheroprone disturbed flow region of the inner aortic arch compared to the outer arch. Disturbed flow-induced FAK activation is associated with elevated VEGFR2 on the surface of ECs of the inner aortic arch, but not in the outer arch. Taken together, these data suggest that suppression of augmented FAK activity under disturbed flow may prove beneficial in reducing pro-inflammatory signaling of the endothelial layer.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"625 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144305073","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}

引用次数: 0

Characterization of the activity of KTX-1001, a small molecule inhibitor of multiple myeloma SET domain (MMSET) using surface plasmon resonance (SPR) 利用表面等离子体共振（SPR）表征多发性骨髓瘤SET结构域（MMSET）小分子抑制剂KTX-1001的活性

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-14 DOI: 10.1016/j.jbc.2025.110382

Chad A. Lewis, Charles Schmidt, Lisa Beebe, Terrence J. Connolly

{"title":"Characterization of the activity of KTX-1001, a small molecule inhibitor of multiple myeloma SET domain (MMSET) using surface plasmon resonance (SPR)","authors":"Chad A. Lewis, Charles Schmidt, Lisa Beebe, Terrence J. Connolly","doi":"10.1016/j.jbc.2025.110382","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110382","url":null,"abstract":"KTX-1001 is a small molecule inhibitor of MMSET in early clinical development for multiple myeloma. It was identified as a potent and selective inhibitor of MMSET (also known as NSD2) using a high throughput biochemical assay with LC/MS-MS detection of SAH production as the endpoint. Subsequent evaluation of the binding of KTX-1001 to its target was conducted using surface plasmon resonance (SPR) to quantify on-rate, off-rate, and equilibrium dissociation constant utilizing the SET domain as the immobilized target. In this format, no saturable or specific binding could be observed, despite the potent activity in the assay using full length NSD2 and radiolabeled SAM. To interrogate the discordance between potent activity and the lack of detectable binding in the assay, a series of experiments were designed in which KTX-1001 with a biotin-PEG tether (KTX-1001-3) was immobilized to the chip, with target (NSD2 SET domain) in-flow, with nucleosomes and with and without cofactor. These experiments demonstrated that KTX-1001-3 bound to the SET domain in a specific and saturable manner, with an affinity comparable to the IC<ce:inf loc=\"post\">50</ce:inf> determined in the enzymatic assay. Further, these studies confirmed unique binding properties of KTX-1001 in the presence of nucleosomes, cofactor, and in combination. These data identify the utility of SPR in a “reverse” format, where immobilization of KTX-1001 allowed for the interrogation of binding to a protein target that may be challenging if conformation changes are induced in the coupling of that biologic target to a chip surface. Collectively, this analysis demonstrates the specific potent biochemical activity of KTX-1001 against MMSET and supports the ongoing evaluation of KTX-1001 in the clinic.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"142 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144305071","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}

引用次数: 0

Colchicine inhibits vascular calcification by suppression inflammasome activation through the enhancement of the Sirt2-PP2Ac signaling pathway 秋水仙碱通过增强Sirt2-PP2Ac信号通路抑制炎性体激活，从而抑制血管钙化

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-14 DOI: 10.1016/j.jbc.2025.110381

Shu Yang, Heming Huang, Kewei Jiang, Ying Peng, Zhen Liang, Xinyu Gong, Lixing Li, Yanchun Li, Buchun Zhang, Yuanli Chen, Xiaoxiao Yang

{"title":"Colchicine inhibits vascular calcification by suppression inflammasome activation through the enhancement of the Sirt2-PP2Ac signaling pathway","authors":"Shu Yang, Heming Huang, Kewei Jiang, Ying Peng, Zhen Liang, Xinyu Gong, Lixing Li, Yanchun Li, Buchun Zhang, Yuanli Chen, Xiaoxiao Yang","doi":"10.1016/j.jbc.2025.110381","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110381","url":null,"abstract":"Colchicine (Col) is a traditional herbal medicine derived from the plant <ce:italic>Colchicum autumnale</ce:italic>. With the property of anti-inflammation, Col has been demonstrated certain therapeutic effects in cardiovascular diseases (CVDs). Vascular calcification is positively related to the morbidity and mortality of CVDs. However, the specific cardiovascular conditions for which Col is effective remain unclear, particularly its impact on vascular calcification. In this study, we used high phosphate to induced calcium deposition in vascular smooth muscle cells (VSMCs), and Vitamin D3 plus nicotine or 5/6 nephrectomy along with high phosphate diet to construct vascular calcification mouse models. Our results showed that Col reduced calcium accumulation <ce:italic>in vitro</ce:italic>, and vascular calcification both in <ce:italic>ex-vivo</ce:italic> and <ce:italic>in vivo</ce:italic> models, which was evidenced by the Alizarin red S staining and calcium content determination. <ce:italic>In vitro</ce:italic> results showed that Col inhibited vascular calcification is contributed to the reduction of NLRP3 inflammasome activation through enhanced phosphorylation at Ser 5. In addition, we indicated that phosphorylation of NLRP3 is regulated by the activity of protein phosphatase 2Ac (PP2Ac). Furthermore, we identified that Sirt2 as a master regulator of PP2Ac activation through regulation its acetylation at Lys 136. More importantly, we demonstrated that Col-inhibited vascular calcification is dependent on Sirt2 expression by using the Sirt2 knockout mice. We demonstrate that Col protects vascular calcification. Our study provides novel insight into the clinical application of Col. We also suggest that Sirt2 is a novel target for vascular calcification treatment, and that Col may act as an activator of Sirt2, which could be beneficial in other diseases.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"51 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144305072","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}

引用次数: 0

Cytochrome P450BM-3 and P450 11A1 retain Compound I (FeO3+) chemistry with electrophilic substrates poised for Compound 0 (Fe3+O2¯) reactions 细胞色素P450BM-3和P450 11A1保留了化合物I （FeO3+）与亲电底物的化学反应，准备进行化合物0 （Fe3+O2¯）的反应

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-14 DOI: 10.1016/j.jbc.2025.110378

Kevin D. McCarty, Yasuhiro Tateishi, F. Peter Guengerich

{"title":"Cytochrome P450BM-3 and P450 11A1 retain Compound I (FeO3+) chemistry with electrophilic substrates poised for Compound 0 (Fe3+O2¯) reactions","authors":"Kevin D. McCarty, Yasuhiro Tateishi, F. Peter Guengerich","doi":"10.1016/j.jbc.2025.110378","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110378","url":null,"abstract":"The catalytic cycle of cytochrome P450 (P450) enzymes involves ferric peroxide anion (Fe<ce:sup loc=\"post\">3+</ce:sup>O<ce:inf loc=\"post\">2</ce:inf>¯, Compound 0) and perferryl oxygen (FeO<ce:sup loc=\"post\">3+</ce:sup>, Compound I) intermediates. Compound I is generally viewed as responsible for most P450-catalyzed oxidations, but Compound 0 has been implicated in the oxidation of some carbonyl compounds, particularly deformylation reactions. We considered the hypothesis that Compound 0 could also attack other electrophilic carbon atoms and accordingly positioned keto groups at preferred hydroxylation sites of substrates for two P450s with well-defined catalytic reactions, bacterial P450<ce:inf loc=\"post\">BM-3</ce:inf> (102A1) and human P450 11A1. The predicted products of Compound I and Compound 0 reactions were analyzed. With the normally preferred ω-1 site blocked, P450<ce:inf loc=\"post\">BM-3</ce:inf> oxidized 12-oxotridecanoic acid (12-oxo C13:0) only at the ω-2 position (yielding 11-hydroxy,12-oxotridecanoic acid), indicative of a Compound I oxidation. P450 11A1 is highly selective for catalyzing the 22<ce:italic>R</ce:italic>-hydroxylation of cholesterol (and some other sterols) in the first step of its overall side-chain cleavage reaction. With 22-oxocholesterol as the substrate, P450 11A1 (slowly) generated only 23-hydroxy,22-oxocholesterol, indicative of Compound I oxidation. Neither P450 generated the products expected from nucleophilic Compound 0 reactions. We conclude that the strategic placement of electrophilic oxo substituents at sites of substrate hydroxylation failed to divert the oxidation mechanism to a Compound 0 pathway with either enzyme. Instead, the Compound I mechanism – blocked at the preferred reaction site – was redirected to neighboring carbons, suggesting that the basis for Compound 0-mediated reactions lies in chemical properties of the enzyme rather than those of the substrate.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"14 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144305074","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}

引用次数: 0

Molecular basis for Gβγ-SNARE mediated inhibition of synaptic vesicle fusion. Gβγ-SNARE介导的突触囊泡融合抑制的分子基础。

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-14 DOI: 10.1016/j.jbc.2025.110377

Anna R. Eitel, Benjamin K. Mueller, Ali I. Kaya, Montana Young, Jackson B. Cassada, Eric W. Bell, Lauren Schnitkey, Zack Zurawski, Yun Y. Yim, Qiangjun Zhou, Jens Meiler, Heidi E. Hamm

{"title":"Molecular basis for Gβγ-SNARE mediated inhibition of synaptic vesicle fusion.","authors":"Anna R. Eitel, Benjamin K. Mueller, Ali I. Kaya, Montana Young, Jackson B. Cassada, Eric W. Bell, Lauren Schnitkey, Zack Zurawski, Yun Y. Yim, Qiangjun Zhou, Jens Meiler, Heidi E. Hamm","doi":"10.1016/j.jbc.2025.110377","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110377","url":null,"abstract":"Neurotransmitter release is a complex process involving tightly controlled co-factors and protein-protein interactions. G protein coupled receptors negatively regulate exocytosis via the interaction of G-protein βγ (Gβγ) heterodimers with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The neuronal ternary SNARE complex comprises synaptosomal-associated protein-25 (SNAP25), syntaxin-1A, and synaptobrevin-2. The regions of the SNARE complex that are important for interactions with Gβγ have been extensively characterized, but the critical sites on Gβγ are not well understood. Furthermore, the molecular basis for the specificity of different Gβ and Gγ isoforms for SNARE proteins remains elusive. Thus, we holistically probed the entire family of human Gβ and Gγ isoforms for regions critical for the target-SNARE (tSNARE) interaction using a peptide screening approach. Gβ and γ peptides with high affinities for tSNARE were then subjected to alanine scanning mutagenesis to identify the interaction sites. We found that the N-terminal coiled-coil domain of Gβγ as well as the β-propeller domain of Gβ are hotspots for SNARE interactions. Additionally, we found that the N-terminal Gγ2 peptide is a potent inhibitor of interactions between full-length Gβ1γ2 and SNAP25. We discovered that Gβ1γ2 preferentially interacts with ternary SNARE in the pre-fusion, partially zipped conformation, likely due to increased exposure of the C-terminus of SNAP25. Our combined results suggest that specific Gβγ heterodimers bind to ternary SNARE in the docked and primed state via critical residues of the β-propeller and N-terminal coil-coil domains. We propose that Gβγ binding disrupts zippering up the SNARE complex and thereby vesicle fusion.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"43 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144304983","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}

引用次数: 0

CH−π Interactions Confer Orientational Flexibility in Protein–Carbohydrate Binding Sites CH−π相互作用赋予蛋白质-碳水化合物结合位点的取向灵活性

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-06-14 DOI: 10.1016/j.jbc.2025.110379

Allison M. Keys, David W. Kastner, Laura L. Kiessling, Heather J. Kulik

{"title":"CH−π Interactions Confer Orientational Flexibility in Protein–Carbohydrate Binding Sites","authors":"Allison M. Keys, David W. Kastner, Laura L. Kiessling, Heather J. Kulik","doi":"10.1016/j.jbc.2025.110379","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110379","url":null,"abstract":"Protein−carbohydrate binding plays an essential role in biological processes including cellular recognition and immune signaling. However, glycans are hydrophilic with limited hydrophobic surfaces, a challenge for selective recognition by proteins. CH–π stacking interactions are pervasive in protein-carbohydrate binding sites and have emerged as critical drivers of protein–carbohydrate recognition. They are highly favorable and have a broad orientational landscape. However, it is unknown how CH−π stacking interaction orientational dynamics are influenced by the protein environment and what their functional interplay is with hydrogen bonds in protein–carbohydrate binding. Here, we employ well-tempered metadynamics simulations to obtain binding free energy landscapes for a set of protein−β-D-galactoside complexes with CH–π stacking interactions. Our data show that the favored orientation of a CH−π stacking interaction is controlled by the location of hydrogen bonds in the protein binding site. Complexes with extended carbohydrate ligands that form additional hydrogen bonds have more specific orientational dependences, while protein variant complexes with fewer hydrogen bonds have broader free energy landscapes with glycan ligands adopting multiple CH−π stacking interaction orientations. We also show that forming multiple CH−π stacking interactions facilitates the dynamics necessary for the translocation of oligosaccharide ligands within a processive enzyme. Our findings underscore the cooperative nature of hydrogen bonds and CH−π stacking interactions, demonstrating that tuning the number and positions of these interactions through evolution or protein engineering can alter ligand recognition or support ligand movement in protein binding sites.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"40 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144305075","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}

引用次数: 0