Journal of Biological Chemistry最新文献

{"title":"Phosphatidylinositol 3-phosphate metabolism impacts cellular α-Synuclein localisation in Saccharomyces cerevisiae.","authors":"Timo Löser,Fazilet Bekbulat,Christian Behl,Jana Schepers","doi":"10.1016/j.jbc.2025.110666","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110666","url":null,"abstract":"Alpha-Synuclein (αSyn), a hallmark protein of synucleinopathies such as Parkinson's disease, is likely to be involved in neuronal membrane trafficking and synaptic vesicle dynamics at axon terminals. Its specific binding to anionic phospholipids, such as phosphatidylinositol phosphates (PIPs) that are essential for intracellular signaling and membrane trafficking, suggests an involvement in vesicular transport processes. In Saccharomyces cerevisiae, a model organism for cell biological PD research, human αSyn localises to the plasma membrane via the secretory machinery. Employing this yeast model, we investigated the impact of αSyn on cellular quality control mechanisms. Additionally, we focused on the effect of αSyn expression in yeast mutants impaired in specific phospholipid biosynthesis and transport pathways, including endo-vacuolar trafficking and autophagy. In the deletion strains vps34Δ and vps15Δ, lacking PI3P biosynthesis, αSyn mislocalises in the cytosol, and significantly reduces cell viability. In these strains, αSyn species containing an intact lipid-binding N-terminus also form large peri-vacuolar, lipid-rich accumulations. In wild-type cells, αSyn expression alters the morphology of PI3P-rich membrane structures and upregulates transcription of SEC4, which encodes a key regulator of the late secretory pathway. Moreover, αSyn co-localises with overexpressed Sec4 at the emerging cell bud. Our findings demonstrate that PI3P is critical for the targeting of αSyn to the yeast plasma membrane via the secretory pathway, revealing a potential entry point into this complex machinery. Understanding the relationship between αSyn and vesicular trafficking in this system will enhance our knowledge of αSyn-trafficking in mammalian cells and, eventually, in PD, offering new research avenues.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"36 1","pages":"110666"},"PeriodicalIF":4.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144960303","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":"Deciphering the unique autoregulatory mechanisms and substrate specificity of the understudied DCLK3 kinase linked to neurodegenerative diseases.","authors":"Jason D Lu,Peng Zhao,Anup Prasad,Neha Gupta,Nathan Gravel,Tej Shidhaye,Lance Wells,Samiksha Katiyar,Natarajan Kannan","doi":"10.1016/j.jbc.2025.110664","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110664","url":null,"abstract":"Protein kinases represent one of the largest and most druggable protein families. Despite considerable progress in their understanding, approximately one-third of human kinases remain poorly characterized, known as the \"dark\" kinome. Doublecortin-like kinase 3 (DCLK3), a member of this elusive group, has emerged for its involvement in neuroprotection in Huntington's disease and other neurodegenerative disorders. Still, its cellular substrates and regulatory functions remain unknown, hindering progress in therapeutic intervention. Unlike its paralog, DCLK1, whose regulation involves a C-terminal segment docking into the ATP binding pocket, DCLK3 lacks such a tail, suggesting divergent regulatory mechanisms. Through computational and experimental analyses, we discovered that DCLK3 autophosphorylates its truncated tail, tethering it to the catalytic domain in a manner distinct from DCLK1. Using a deep learning model trained on peptide-library datasets, we predicted Tau, a microtubule-associated protein, as a putative DCLK3 substrate, which we validated using in vitro assays and mass spectrometry. Additionally, DCLK3 exhibits a relatively fast turnover with a cellular half-life of approximately 15 hours that can be rescued by MG132-mediated proteasomal inhibition, which results in DCLK3 polyubiquitination and cellular accumulation. Collectively, these results provide the first structural and functional insights into DCLK3, revealing a unique autoregulatory mechanism and a potential therapeutic target for neurodegenerative disorders.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"19 1","pages":"110664"},"PeriodicalIF":4.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144960305","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":"In situ metabolomics reveals intra-islet metabolite changes upon in vivo stimulation of insulin secretion.","authors":"Yan Zhou,Nicolas Baez,Tingting Fu,Thierry Brun,Pierre Maechler","doi":"10.1016/j.jbc.2025.110661","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110661","url":null,"abstract":"Upon glucose stimulation, metabolic pathways of pancreatic beta-cells promptly adapt metabolite levels inducing insulin secretion fine-tuned by mitochondrial glutamate dehydrogenase (GDH). Although well described in vitro, these responses cannot yet be captured in vivo due to the intrinsic nature of the islets scattered throughout the pancreas. Tested first in vitro, glutamate precursor glutamine enhanced glucose-stimulated insulin secretion without eliciting oxidative catabolism, as opposed to glucose. Then, to be as close as possible to the in vivo state, we collected the pancreas of mouse models in fasted versus fed states and at the peak of a glucose tolerance test, immediately followed by snap freezing before in situ analysis of metabolic pathways. On the same series of pancreatic cryosections, islets were identified by dithizone beta-cell staining for metabolic analyses combining spatial in situ redox enzymatic assay with targeted metabolomics using time-of-flight secondary ion MS high-resolution imaging. Direct measurements in cryopreserved pancreatic sections of control and beta-cell specific GDH knockout mice showed tight coupling between glycolysis and mitochondrial pathways favored by low lactate dehydrogenase activity and strong succinate dehydrogenase velocity. In response to regular feeding, intra-islet glutamate and glutamine levels were elevated, an effect dependent on beta-cell GDH. Acute in vivo glucose stimulation increased both alanine and glutamate intra-islet levels. Lack of beta-cell GDH abrogated the rise in glutamate and reduced insulin secretion without impacting alanine levels. Overall, the hallmark of in vivo beta-cell stimulation was a strong mitochondrial activity and GDH-dependent elevation of glutamate required for the full development of insulin secretion.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"19 1","pages":"110661"},"PeriodicalIF":4.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144960221","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":"Dissecting the Cdc37 Co-chaperone Code: Functional Roles in Chaperone-Mediated Stress Adaptation.","authors":"Megan M Mitchem,Ashley Choi,Duhita A Mirikar,Rajlekha Deb,Andrew W Truman","doi":"10.1016/j.jbc.2025.110672","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110672","url":null,"abstract":"Cdc37 is a kinase-specific co-chaperone that scaffolds protein kinase clients to the Hsp90 chaperone system. Although phosphorylation at residues S14 and S17 is known to regulate Cdc37 function, the broader role of phosphorylation across the protein remains unclear. To systematically investigate this, we created a \"Cdc37 code collection,\" a set of 46 yeast strains expressing single phospho-site mutants of Cdc37, and performed phenotypic profiling across a wide panel of environmental and chemical stressors. While canonical sites like S14 and S17 were essential for stress tolerance, 34 additional phospho-mutants exhibited distinct phenotypes, often in a stress-specific manner. Notably, the mutations displayed little overlap in their stress responses, suggesting a modular and context-dependent regulation of Cdc37. Our data reveal that Cdc37 function is intricately modulated by site-specific phosphorylation, which shapes its capacity to maintain proteostasis under diverse cellular conditions. This study provides a comprehensive resource for dissecting the functional landscape of Cdc37 post-translational regulation and highlights new regulatory sites with potential relevance to chaperone-kinase network dysregulation in disease.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"32 1","pages":"110672"},"PeriodicalIF":4.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144960304","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":"Annexin A2 interferes with complement regulation within the glomerulus.","authors":"Brandon Renner, Jennifer Laskowski, Felix Poppelaars, Russell Whelan, Kyrie Milliron, Vojtech Petr, Judith Blaine, Yunus Ozekin, Viviana P Ferreira, Matthew C Pickering, Christoph Q Schmidt, Katherine Hajjar, Anne Davidson, Joshua M Thurman","doi":"10.1016/j.jbc.2025.110657","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110657","url":null,"abstract":"<p><p>The alternative pathway of complement is an important pathogenic driver of a variety of glomerular diseases. Factor H is a soluble complement regulatory protein, and it is known to play a critical role in protecting the kidney from alternative pathway-mediated injury. Other proteins, however, can interfere with complement regulation by Factor H, thereby predisposing the kidney to injury. Annexin A2 was previously shown to bind to Factor H and is expressed by several resident cell types in the kidney. In the current study, we show that extracellular annexin A2 binds to the region of Factor H encompassing short consensus repeats 6-8, impairing the ability of Factor H to regulate complement activation on the surface of glomerular endothelial cells and podocytes in vitro and in vivo. Annexin A2 does not, however, impair Factor H function on extracellular matrix or guinea pig erythrocytes. Targeted deletion of annexin A2 in mice attenuates cyclosporine-induced kidney injury in mice, and deficiency of annexin A2 expression reduces complement activation on the surface of extracellular vesicles released from endothelial cells in this model. Review of publicly available kidney transcription datasets revealed that annexin A2 is expressed by several cell types in the kidney, and that expression is increased in multiple different disease states. Annexin A2, therefore, may serve as an intrinsic \"positive regulator\" of complement activation in the kidney, promoting the inflammatory response after various kidney insults.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110657"},"PeriodicalIF":4.0,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955229","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":"A biophysical and molecular characterization of the interaction between the Alzheimer risk factor BIN1 and the neuronal scaffold protein p140Cap.","authors":"Danielle M Blazier, Eric M Lewandowski, Natasha Ram, Xiujun Zhang, Shuai Wang, Om Patel, Lisa Collier, Paola Defilippi, Yu Chen, Gopal Thinakaran","doi":"10.1016/j.jbc.2025.110665","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110665","url":null,"abstract":"<p><p>Bridging Integrator 1 (BIN1) is a genetic risk factor for late-onset Alzheimer disease. BIN1's participation in endocytosis, membrane remodeling, and modulation of actin dynamics is well-characterized in non-neuronal cells. In neurons, BIN1 is enriched at presynaptic sites, where it facilitates excitatory neurotransmitter vesicle release. However, how BIN1 is involved in synaptic vesicle dynamics is not well understood. A C-terminal Src Homology 3 (SH3) domain is invariant in all BIN1 isoforms and promotes protein-protein interactions with proteins harboring proline-rich motifs. While BIN1 interactions with dynamin, synaptojanin, RIN3, and tau have been identified and experimentally validated, the list of BIN1-interacting molecules is not exhaustive. Here, we report the neuronal scaffolding protein p140Cap, encoded by SRCIN1, as a BIN1 SH3 domain-interacting protein. We performed surface plasmon resonance to ascertain the affinity of BIN1-SH3 domain for p140Cap and identified a peptide containing three proline-rich motifs that exhibited biologically relevant affinity (K_D = 7.7 μM). Additional surface plasmon resonance experiments, coupled with alanine-scanning mutagenesis, revealed that two class II motifs, but not a class I motif, in p140Cap facilitated binding. Confocal microscopy and proximity ligation assays confirmed that BIN1 colocalizes with, and is within molecular distance of, p140Cap in cultured cells and in the mouse brain. Co-immunoprecipitation assays validated the interaction, and glutathione S-transferase pull-down revealed that a rare BIN1 coding variant (rs138047593) significantly reduces p140Cap and tau binding, highlighting the impact of this mutant on interacting protein binding efficiency. The functional implications of BIN1:p140Cap interaction for neuronal functions warrant further investigation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110665"},"PeriodicalIF":4.0,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955275","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":"ARHGAP10 is a novel microtubule-associated protein that regulates the resorption activity of osteoclasts.","authors":"Laura Jentschel, Anne Blangy, Guillaume Bompard","doi":"10.1016/j.jbc.2025.110668","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110668","url":null,"abstract":"<p><p>Adult-bone homeostasis is maintained through the reciprocal actions of osteoclasts and osteoblasts, which respectively resorb and deposit new bone. Excessive osteoclast activity leads to bone loss and contributes to conditions like osteoporosis. Osteoclasts form a specialized adhesion structure called the actin ring that is crucial for bone resorption and relies on both the actin and microtubule cytoskeletons. Our previous studies identified the β-tubulin isotype TUBB6 as a regulator of actin ring dynamics essential for osteoclast function, and found ARHGAP10, a negative regulator of the GTPases CDC42 and RHOA, as a potential mediator of TUBB6 function. Here we show that ARHGAP10 is a novel microtubule-associated protein critical for osteoclast function. ARHGAP10 directly binds microtubules through its BAR-PH domain, which requires positively-charged lysine residues K37, K41 and K44 within the BAR domain. CRISPR/Cas9 mediated knockout of Arhgap10 affects the morphology of the actin ring and impairs osteoclast resorption activity, correlated with altered actin ring dynamics. Complementation experiments reveal that the ability of ARHGAP10 to bind microtubules and to negatively regulate RHO-GTPases are essential for its role in osteoclast resorption activity. These findings uncover a novel cytoskeletal regulator in osteoclasts and suggest that targeting the microtubule-actin interface via ARHGAP10 could represent a therapeutic strategy in bone loss disorder.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110668"},"PeriodicalIF":4.0,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955186","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":"African swine fever virus pMGF505-9R enhances RIG-I-like Receptor signaling by promoting RING finger protein 125 auto-ubiquitination.","authors":"Jinyu Zhao, Xinyu Zhang, Chunxiao Mou, Kaichuang Shi, Zhenhai Chen","doi":"10.1016/j.jbc.2025.110669","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110669","url":null,"abstract":"<p><p>African swine fever (ASF) is a highly infectious disease that poses a significant threat to the global pig industry. Recent studies have demonstrated that the African swine fever virus (ASFV) infection can cause severe inflammatory responses and promote the production of cytokines, but it is still unclear whether the viral proteins play a role in this process. Therefore, we conducted a genome-wide screening by dual luciferase activity assay. The results showed that six viral proteins of ASFV have a stimulating effect on the promoter activity of IFN-β. Among them, the non-structural protein pMGF505-9R significantly increased the activity of IFN-β promoter and promote the expression of IFN-β. In addition, pMGF505-9R can also increase the promoter activity of NF-κB and promote the expression of IL-1β. Further research found that pMGF505-9R could regulate the RIG-I-like Receptor (RLR) signaling pathways by influencing the protein level of RING finger protein 125(RNF125), ultimately affecting the expression of IFN-β and IL-1β. Mechanistically, pMGF505-9R interacted with the host E3 ubiquitin ligase RNF125. This interaction promoted the auto-ubiquitination and subsequent degradation of RNF125, which in turn reduced the K48 ubiquitination of RIG-I, MDA5, and MAVS, which ultimately promoted the production of IFN-β and IL-1β. The results provide novel insights into the immune regulatory mechanisms of ASFV which would greatly improve our understanding of virus pathogenesis.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110669"},"PeriodicalIF":4.0,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955254","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":"The cation channel Trpa1 and chemokine Cxcl1 mediate axonal degeneration in spared nerve injury-induced neuropathic pain.","authors":"Liu-Qing Ye, Xuan-Xuan Huang, Hua-Feng Liu, Tian Li, Yu Wang, Xiao-Hui Chen, Yu Zheng, Zuo-Ming Chen, Qin-Yu Liu, Fan Yang, Nan-Qi Li, Li Wan","doi":"10.1016/j.jbc.2025.110654","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110654","url":null,"abstract":"<p><p>Demyelination of peripheral nerve injury is a vital cause of neuropathic pain. Schwann cells play an important role in supporting and maintaining the removal and regeneration of myelin debris from neuronal axons in the peripheral nervous system. Creating a good immune microenvironment would promote the Schwann cells to repair the injured nerve and reverse the allodynia of neuropathic pain. Trpa1, a transient receptor potential ion channel, and Cxcl1, a chemokine, play crucial roles in allodynia in pain pathophysiology. However, their interaction in neuropathic pain remains unclear. This study aimed to elucidate the role of Trpa1 in neuropathic pain and its interaction with Cxcl1. Using a spared nerve injury (SNI) model in mice, mechanical allodynia was observed alongside increased Trpa1 expression in injured nerves. Treatment with a Trpa1 inhibitor alleviated allodynia, suggesting Trpa1 is involved in neuropathic pain. Transcriptome sequencing revealed immune pathway enrichment post-Trpa1 inhibition. Cxcl1 recruited Cxcr2-positive macrophages to injured sites, whereas Trpa1 inhibition reduced Cxcl1 expression and Cxcr2 recruitment in Schwann cells and in neurons. Additionally, Mag(Myelin-associated glycoprotein), crucial for axonal stability, was downregulated in the SNI model but increased post-Trpa1 blockade, indicating Trpa1 plays an important role in Cxcl1-mediated immune cascade in axonal degeneration. In summary, neuronal and dedifferentiated Schwann cell Trpa1 regulates Cxcl1 synthesis, recruiting macrophages to nerve injury sites, mediating neuropathic pain. Collectively, our findings suggest Trpa1 activation weakens axonal resistance to degeneration by inhibiting Mag. This study highlights Trpa1 is multifaceted involvement in neuropathic pain, suggesting a potential therapeutic targets for neuropathic pain management.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110654"},"PeriodicalIF":4.0,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955321","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":"Deciphering Mutational Effects on Inducible NO Synthase Conformational Dynamics via Quantitative Cross-Linking Mass Spectrometry and AlphaFold2 Subsampling.","authors":"Ting Jiang, Haikun Zhang, Gabriel Monteiro Da Silva, Yadav Prasad Gyawali, Changjian Feng","doi":"10.1016/j.jbc.2025.110673","DOIUrl":"10.1016/j.jbc.2025.110673","url":null,"abstract":"<p><p>Mammalian nitric oxide synthases (NOSs) are flavo-hemoproteins that rely on dynamic interdomain interactions for activity. Calmodulin (CaM) facilitates specific, interdomain FMN-heme interactions that enable inter-subunit FMN-heme electron transfer essential for nitric oxide biosynthesis. Our quantitative cross-linking mass spectrometry (qXL MS) results demonstrate that the abundance of inter-subunit cross-links correlates with CaM-induced formation of the docked FMN/heme complex in neuronal NOS [Jiang et al., Biochemistry, 2024, 63, 1395-1411]. Here, we extend this methodology to the human inducible NOS (iNOS) isoform, comparing wild-type (wt) and E546N mutant oxygenase/FMN (oxyFMN) constructs. Using parallel reaction monitoring-based qXL MS, we assessed mutation-induced changes in interdomain dynamics. The E546N mutation substantially reduced abundance of the inter-subunit cross-links between the FMN and heme domains. Cross-links between CaM and iNOS domains were also altered by the mutation, indicating that the changes at the FMN-heme docking interface propagate allosterically throughout the iNOS-CaM complex. Although standard AlphaFold2 structural modeling yielded similar docked architectures for wt and mutant, cross-link-guided AlphaLink2 modeling revealed distinct structural differences. AlphaFold2 subsampling further predicted alternative conformations and their relative populations; consistent with qXL MS data, E546N exhibited a broader distribution of predicted conformations, with a higher undocked state population than wt. Importantly, 90% of cross-links were consistent with an ensemble of representative conformations derived from AlphaFold2 subsampling and cross-link-guided AlphaLink2 modeling, capturing both docked and undocked states alongside multiple orientations. This integrated qXL MS and AlphaFold2 subsampling strategy provides a quantitative framework for mapping mutation-induced alterations in functional dynamics of multidomain proteins.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110673"},"PeriodicalIF":4.0,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955171","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}