Phosphorylation-Dependent Activation of the XRCC1 Intrinsically Disordered Region: A Computational Structural Analysis

Phosphorylation-Dependent Activation of the XRCC1 Intrinsically Disordered Region: A Computational Structural Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Phosphorylation-Dependent Activation of the XRCC1 Intrinsically Disordered Region: A Computational Structural Analysis Mateen Ur Rehman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9455541/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The X-ray repair cross-complementing protein 1 (XRCC1) is a central scaffold protein involved in DNA single-strand break repair (SSBR), coordinating the assembly of multi-protein repair complexes. Its activity is regulated by post-translational modifications, particularly phosphorylation within intrinsically disordered regions (IDRs), although the structural consequences of these modifications remain poorly understood. In this study, an in silico approach is used to investigate the structural and electrostatic effects of a phosphomimetic mutation at residue S518. The full-length structure of human XRCC1 was predicted using AlphaFold2 via ColabFold, followed by structural and electrostatic analyses in UCSF ChimeraX. Predicted Local Distance Difference Test (pLDDT) scores revealed a heterogeneous structural profile, with well-defined folded domains and a low-confidence inter-domain linker region encompassing S518, consistent with intrinsic disorder. Introduction of the phosphomimetic mutation (S518D) did not result in substantial alterations to the global structure of XRCC1. However, electrostatic surface analysis indicated the emergence of a localized negatively charged region at the mutation site. Additional structural inspection suggested localized perturbations without global destabilization. These findings suggest that residue-level modifications at S518 may modulate the local electrostatic environment of XRCC1 and potentially influence its interactions with downstream DNA repair factors. This study highlights the role of electrostatic tuning within intrinsically disordered regions as a possible regulatory mechanism in scaffold proteins. However, as these observations are based on static computational models, further validation using molecular dynamics simulations and experimental approaches is required. Bioinformatics XRCC1 Intrinsically Disordered Regions (IDRs) Phosphorylation Post-translational Modification AlphaFold Structural Dynamics DNA Repair Figures Figure 1 Figure 2 Introduction Genomic stability relies on the rapid detection and repair of DNA single-strand breaks (SSBs) (Caldecott, 2022 ). The XRCC1 protein functions as the master architectural scaffold in this pathway, arriving early at the damage site to coordinate the assembly of the repair machinery (Tang & Çağlayan, 2021 ). Structurally, XRCC1 consists of distinct, rigid functional domains (an N-terminal domain and two BRCT domains) separated by long, highly flexible linker sequences known as Intrinsically Disordered Regions (IDRs) (Kruswick et al., 2024 ; Ghediri et al., 2025 ). Recent advances in computational biology, particularly structure prediction using AlphaFold2, have enabled detailed investigation of protein structure and function in the absence of experimental data (Yang et al., 2023 ). Additionally, molecular visualization tools enable analysis of structural features, residue interactions, and the potential impact of mutations (Verma et al., 2025 ). Recent advances in computational biology, particularly structure prediction using AlphaFold2, have enabled detailed investigation of protein structure and function in the absence of experimental data (Philipp et al., 2024 ). Additionally, molecular visualization tools enable analysis of structural features, residue interactions, and the potential impact of mutations (Tvaroška et al., 2023 ). However, biochemical assays have established that XRCC1 must be phosphorylated at specific residues within this disordered linker—most notably at Serine 518—to successfully recruit downstream enzymes like DNA Ligase III (Almohdar et al., 2024 ). While the necessity of this phosphorylation event is biochemically proven, the exact biophysical mechanism of how a single phosphate group alters a massive, disordered protein loop remains visually uncharacterized (Nosella et al., 2024 ). In this study, we investigate the structural and electrostatic consequences of a phosphomimetic mutation at residue S518 within XRCC1. We hypothesize that substitution at this position may alter the local electrostatic environment and potentially influence protein interactions. Methodology 2.1. Sequence Retrieval and Mutagenesis The canonical wild-type amino acid sequence for Human XRCC1 was retrieved from the UniProt database (Accession: P18887) (Lazar et al., 2019 ). To simulate the constitutive phosphorylation of the structurally disordered linker region, in silico site-directed mutagenesis was performed. A phosphomimetic mutant sequence (S518D) was generated by substituting the uncharged serine residue at position 518 with a negatively charged aspartic acid residue (Suanes-Cobos et al., 2025 ). 2.2. Structure Prediction and Validation Three-dimensional protein structures for both the wild-type and the S518D mutant were generated using the AlphaFold2 algorithm via the ColabFold computational framework (Kim et al., 2025 ). Global structural confidence and local topological mapping were assessed using the predicted Local Distance Difference Test (pLDDT) scores extracted during the modeling pipeline (Torres et al., 2024 ). 2.3. Structural Alignment and Electrostatic Topology Models were visualized and analyzed using UCSF Chimera (Meng et al., 2023 ). Global structural stability was confirmed by superimposing the wild-type and mutant structures using the MatchMaker tool (Khan et al., 2023 ). To evaluate the biophysical consequences of the mutation, the Coulombic surface potential was calculated and mapped onto the solid surface topologies of both models to highlight regions of high positive (blue) and negative (red) charge. Results 3.1. Topological mapping of the XRCC1 linker region Analysis of the wild-type pLDDT scores revealed a dichotomous structural landscape. While the N-terminal and BRCT domains exhibited high structural confidence (pLDDT > 90), the linker region encompassing position 518 demonstrated significantly lower confidence scores (pLDDT < 50). This mathematically confirms that S518 resides within a highly flexible IDR, acting as a free-moving "tentacle" in the nucleoplasm ( Fig. 1 ) . 3.2. S518D mutagenesis maintains global architectural stability The ab initio folding of the S518D phosphomimetic mutant yielded a global structural confidence score (pLDDT ≈ 66) comparable to the wild-type. Structural superposition in UCSF Chimera showed high alignment across the rigid BRCT domains. This indicates that introducing the negatively charged aspartic acid did not induce catastrophic misfolding, and the IDR retained its necessary flexibility. 3.3. Electrostatic mapping reveals a phosphorylation-dependent recruitment patch Comparative electrostatic surface topology analysis was conducted to determine the biophysical mechanism of recruitment. In the wild-type model, the IDR encompassing S518 exhibited a predominantly neutral to slightly electropositive surface charge landscape. Conversely, the S518D phosphomimetic mutant exhibited a fundamentally altered local topology, revealing the generation of a highly localized, intense region of negative electrostatic potential ( Fig. 2 ) . The per-residue confidence scores (pLDDT) for XRCC1 are plotted against amino acid positions. The blue line represents the predicted structural confidence across the protein sequence. The red dashed line indicates the disorder threshold (pLDDT < 50), below which residues are considered intrinsically disordered. Shaded regions highlight predicted intrinsically disordered regions (IDRs). Prominent disordered segments are observed between residues ~ 160–310 and ~ 400–520, while the N-terminal (~ 1–150) and C-terminal (~ 530–620) regions exhibit high confidence scores, suggesting well-defined structural domains. 3D structural modeling and Coulombic surface analysis (calculated via UCSF Chimera) reveal a localized region of intense negative electrostatic potential (red) resulting from the S518D substitution. This negatively charged pocket in the intrinsically disordered region serves as the theoretical recruitment interface for downstream DNA repair complexes. Discussion The results of this in silico study provide a clear structural rationale for the biochemical dependence on XRCC1 phosphorylation. The flexible nature of the IDR allows the S518 region to scan the local environment. When unphosphorylated (wild-type), the region remains electrostatically neutral and inactive (Allen et al., 2024 ). However, our S518D model demonstrates that the addition of a negative charge acts as a powerful biophysical switch. The resulting intense electronegative pocket (red surface) operates effectively as a localized "magnet." We propose that this region specifically attracts and anchors the complementary electropositive surfaces of downstream repair factors, such as the BRCT domain of DNA Ligase III. By keeping the global structure intact while drastically altering the local surface charge, the S518 phosphorylation event transforms a random, floppy loop into a highly specific docking interface. This study is based on static computational models and does not account for protein dynamics or direct binding interactions. Future work involving molecular dynamics simulations and experimental validation will be necessary to confirm these findings Conclusion This study provides a computational framework for understanding the phosphorylation-dependent regulation of XRCC1 within its intrinsically disordered region. Using AlphaFold-based structural modeling and electrostatic surface analysis, we demonstrate that a phosphomimetic substitution at residue S518 does not significantly alter the global architecture of XRCC1 but induces localized changes in electrostatic potential. Specifically, the introduction of a negatively charged residue generates a distinct electrostatic patch within the disordered linker region, which may contribute to modulating interactions with downstream DNA repair proteins. These findings support the concept that post-translational modifications within intrinsically disordered regions can function as regulatory elements by altering local physicochemical properties without requiring large-scale conformational changes. Such mechanisms may be particularly relevant for scaffold proteins involved in dynamic cellular processes such as DNA repair. However, it is important to note that these observations are based on static computational models and do not account for protein dynamics or direct binding interactions. Future studies incorporating molecular dynamics simulations and experimental validation will be necessary to further elucidate the functional implications of S518 phosphorylation in XRCC1. References Caldecott KW (2022) DNA single-strand break repair and human genetic disease. Trends Cell Biol 32(9):733–745 Tang Q, Çağlayan M (2021) The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair. J Biol Chem 297(3):101025 Kruswick A, Lam FC, Kong YW, Smerdon SJ, Yaffe MB (2024) BRCT domains as chromatin readers: Structure, function, and clinical implications. Chromatin Readers in Health and Disease. Academic, pp 31–56 Ghediri S, Sarma PA, Saravanan V, Abbadie C, Blossey R, Cleri F (2025) Mechanisms of DNA Damage Recognition by UDG and PARP1 in the Nucleosome. Biomolecules 15(5):649 Yang Z, Zeng X, Zhao Y, Chen R (2023) AlphaFold2 and its applications in the fields of biology and medicine. Signal Transduct Target Therapy 8(1):115 Verma VV, Vimal S, Mishra MK, Sharma VK (2025) A comprehensive review on structural insights through molecular visualization: tools, applications, and limitations. J Mol Model 31(6):173 Philipp M, Moth CW, Ristic N, Tiemann JKS, Seufert F, Panfilova A, Staritzbichler R (2024) MutationExplorer: a webserver for mutation of proteins and 3D visualization of energetic impacts. Nucleic Acids Res 52(W1):W132–W139 Tvaroška I, Kozmon S, Kóňa J (2023) Molecular modeling insights into the structure and behavior of integrins: a review. Cells 12(2):324 Almohdar D, Gulkis M, Ortiz A, Tang Q, Sobol RW, Çağlayan M (2024) Impact of polβ/XRCC1 interaction variants on the efficiency of nick sealing by DNA ligase IIIα in the base excision repair pathway. J Mol Biol 436(4):168410 Nosella ML, Kim TH, Huang SK, Harkness RW, Goncalves M, Pan A, Kay LE (2024) Poly (ADP-ribosyl) ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates. Mol Cell 84(3):429–446 Lazar IM, Karcini A, Ahuja S, Estrada-Palma C (2019) Proteogenomic analysis of protein sequence alterations in breast cancer cells. Sci Rep 9(1):10381 Suanes-Cobos L, Aguilera-Ventura I, Torres-Ramos M, Serrano-Yubero A, Fernández-Aliseda M, Fernández C, Calzado S, M. A (2025) A novel feedback loop between DYRK2 and USP28 regulates cancer homeostasis and DNA damage signaling. Cell Death Differ, 1–15 Kim G, Lee S, Levy Karin E, Kim H, Moriwaki Y, Ovchinnikov S, Mirdita M (2025) Easy and accurate protein structure prediction using ColabFold. Nat Protoc 20(3):620–642 Torres J, Pervushin K, Surya W (2024) Prediction of conformational states in a coronavirus channel using Alphafold-2 and DeepMSA2: Strengths and limitations. Comput Struct Biotechnol J 23:3730–3740 Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, Ferrin TE (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci 32(11):e4792 Khan A, Waqas M, Tufail M, Halim SA, Murad W, Ahmad SU, Al-Harrasi A (2023) In silico scanning of structural and functional deleterious nsSNPs in Arabidopsis thaliana’s SOG1 protein, using molecular dynamic simulation approaches. J Biomol Struct Dynamics 41(21):11629–11646 Allen MC, Karplus PA, Mehl RA, Cooley RB (2024) Genetic encoding of phosphorylated amino acids into proteins. Chem Rev 124(10):6592–6642 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9455541","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625355839,"identity":"50e064ab-efd8-419b-af31-f5a31b2b3241","order_by":0,"name":"Mateen Ur Rehman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDADAyBm/FABJJmZGwioZUZoYZY4AyIZSdDCwNsGIglo0W0/f/DBxx335MzFDj97IDmvNpq/HajlR8U2nFrMziQzG848U2xsOTvN3KBw2/HcGYcZGxh7ztzGreVAMps0b1tC4obbCWYSktuO5TYAtTAztuHRcv4x+++/bQn1G26nf5PgnXMsdz5BLTeS2YAKEhIMbueYSfA21ORuIKzlsbFkb1uC4YbbOWXSEscO5G4EajmI1y/nEx9++NmWIG9wO32b5Ieautx55w8ffPCjArcWdHAYTB4gWj0Q1JGieBSMglEwCkYIAADhR15ebJ0iKAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0000-4253-6442","institution":"The University of Lahore","correspondingAuthor":true,"prefix":"","firstName":"Mateen","middleName":"Ur","lastName":"Rehman","suffix":""}],"badges":[],"createdAt":"2026-04-18 08:43:12","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-9455541/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9455541/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107402134,"identity":"d6fce1c3-35d8-4d19-b9c5-593d7bafcad9","added_by":"auto","created_at":"2026-04-21 07:43:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrediction of intrinsically disordered regions in XRCC1 using AlphaFold pLDDT scores.\u003c/strong\u003e\u003cbr\u003e\nThe per-residue confidence scores (pLDDT) for XRCC1 are plotted against amino acid positions. The blue line represents the predicted structural confidence across the protein sequence. The red dashed line indicates the disorder threshold (pLDDT \u0026lt; 50), below which residues are considered intrinsically disordered. Shaded regions highlight predicted intrinsically disordered regions (IDRs). Prominent disordered segments are observed between residues ~160–310 and ~400–520, while the N-terminal (~1–150) and C-terminal (~530–620) regions exhibit high confidence scores, suggesting well-defined structural domains.\u003c/p\u003e","description":"","filename":"pldtt.png","url":"https://assets-eu.researchsquare.com/files/rs-9455541/v1/de5196422fca9aa8f69e743e.png"},{"id":107402131,"identity":"c137be0a-2722-4d55-94e6-77149d0b1c96","added_by":"auto","created_at":"2026-04-21 07:43:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":191987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrostatic Surface Topology of the XRCC1 S518D Phosphomimetic Mutant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3D structural modeling and Coulombic surface analysis (calculated via UCSF Chimera) reveal a localized region of intense negative electrostatic potential (red) resulting from the S518D substitution. This negatively charged pocket in the intrinsically disordered region serves as the theoretical recruitment interface for downstream DNA repair complexes.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3dmodification.png","url":"https://assets-eu.researchsquare.com/files/rs-9455541/v1/aa9e552c54bfe71abd942126.png"},{"id":107402135,"identity":"0e378dc1-8bb2-4f7e-80ee-b33d846aeba4","added_by":"auto","created_at":"2026-04-21 07:43:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":543322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9455541/v1/2d4dd40b-0b17-4fde-be3d-208e4341fd49.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePhosphorylation-Dependent Activation of the XRCC1 Intrinsically Disordered Region: A Computational Structural Analysis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGenomic stability relies on the rapid detection and repair of DNA single-strand breaks (SSBs) (Caldecott, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The XRCC1 protein functions as the master architectural scaffold in this pathway, arriving early at the damage site to coordinate the assembly of the repair machinery (Tang \u0026amp; \u0026Ccedil;ağlayan, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Structurally, XRCC1 consists of distinct, rigid functional domains (an N-terminal domain and two BRCT domains) separated by long, highly flexible linker sequences known as Intrinsically Disordered Regions (IDRs) (Kruswick et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ghediri et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent advances in computational biology, particularly structure prediction using AlphaFold2, have enabled detailed investigation of protein structure and function in the absence of experimental data (Yang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, molecular visualization tools enable analysis of structural features, residue interactions, and the potential impact of mutations (Verma et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent advances in computational biology, particularly structure prediction using AlphaFold2, have enabled detailed investigation of protein structure and function in the absence of experimental data (Philipp et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, molecular visualization tools enable analysis of structural features, residue interactions, and the potential impact of mutations (Tvaroška et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, biochemical assays have established that XRCC1 must be phosphorylated at specific residues within this disordered linker\u0026mdash;most notably at Serine 518\u0026mdash;to successfully recruit downstream enzymes like DNA Ligase III (Almohdar et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While the necessity of this phosphorylation event is biochemically proven, the exact biophysical mechanism of how a single phosphate group alters a massive, disordered protein loop remains visually uncharacterized (Nosella et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, we investigate the structural and electrostatic consequences of a phosphomimetic mutation at residue S518 within XRCC1. We hypothesize that substitution at this position may alter the local electrostatic environment and potentially influence protein interactions.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cp\u003e \u003cb\u003e2.1. Sequence Retrieval and Mutagenesis\u003c/b\u003e The canonical wild-type amino acid sequence for Human XRCC1 was retrieved from the UniProt database (Accession: P18887) (Lazar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To simulate the constitutive phosphorylation of the structurally disordered linker region, \u003cem\u003ein silico\u003c/em\u003e site-directed mutagenesis was performed. A phosphomimetic mutant sequence (S518D) was generated by substituting the uncharged serine residue at position 518 with a negatively charged aspartic acid residue (Suanes-Cobos et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003e2.2. Structure Prediction and Validation\u003c/b\u003e Three-dimensional protein structures for both the wild-type and the S518D mutant were generated using the AlphaFold2 algorithm via the ColabFold computational framework (Kim et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Global structural confidence and local topological mapping were assessed using the predicted Local Distance Difference Test (pLDDT) scores extracted during the modeling pipeline (Torres et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3. Structural Alignment and Electrostatic Topology\u003c/b\u003e Models were visualized and analyzed using UCSF Chimera (Meng et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Global structural stability was confirmed by superimposing the wild-type and mutant structures using the MatchMaker tool (Khan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To evaluate the biophysical consequences of the mutation, the Coulombic surface potential was calculated and mapped onto the solid surface topologies of both models to highlight regions of high positive (blue) and negative (red) charge.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e3.1. Topological mapping of the XRCC1 linker region\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnalysis of the wild-type pLDDT scores revealed a dichotomous structural landscape. While the N-terminal and BRCT domains exhibited high structural confidence (pLDDT\u0026thinsp;\u0026gt;\u0026thinsp;90), the linker region encompassing position 518 demonstrated significantly lower confidence scores (pLDDT\u0026thinsp;\u0026lt;\u0026thinsp;50). This mathematically confirms that S518 resides within a highly flexible IDR, acting as a free-moving \"tentacle\" in the nucleoplasm \u003cem\u003e(\u003c/em\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cem\u003e)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2. S518D mutagenesis maintains global architectural stability\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe ab initio folding of the S518D phosphomimetic mutant yielded a global structural confidence score (pLDDT\u0026thinsp;\u0026asymp;\u0026thinsp;66) comparable to the wild-type. Structural superposition in UCSF Chimera showed high alignment across the rigid BRCT domains. This indicates that introducing the negatively charged aspartic acid did not induce catastrophic misfolding, and the IDR retained its necessary flexibility.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3. Electrostatic mapping reveals a phosphorylation-dependent recruitment patch\u003c/b\u003e Comparative electrostatic surface topology analysis was conducted to determine the biophysical mechanism of recruitment. In the wild-type model, the IDR encompassing S518 exhibited a predominantly neutral to slightly electropositive surface charge landscape. Conversely, the S518D phosphomimetic mutant exhibited a fundamentally altered local topology, revealing the generation of a highly localized, intense region of negative electrostatic potential \u003cem\u003e(\u003c/em\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cem\u003e)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe per-residue confidence scores (pLDDT) for XRCC1 are plotted against amino acid positions. The blue line represents the predicted structural confidence across the protein sequence. The red dashed line indicates the disorder threshold (pLDDT\u0026thinsp;\u0026lt;\u0026thinsp;50), below which residues are considered intrinsically disordered. Shaded regions highlight predicted intrinsically disordered regions (IDRs). Prominent disordered segments are observed between residues\u0026thinsp;~\u0026thinsp;160\u0026ndash;310 and ~\u0026thinsp;400\u0026ndash;520, while the N-terminal (~\u0026thinsp;1\u0026ndash;150) and C-terminal (~\u0026thinsp;530\u0026ndash;620) regions exhibit high confidence scores, suggesting well-defined structural domains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e3D structural modeling and Coulombic surface analysis (calculated via UCSF Chimera) reveal a localized region of intense negative electrostatic potential (red) resulting from the S518D substitution. This negatively charged pocket in the intrinsically disordered region serves as the theoretical recruitment interface for downstream DNA repair complexes.\u003c/em\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this \u003cem\u003ein silico\u003c/em\u003e study provide a clear structural rationale for the biochemical dependence on XRCC1 phosphorylation. The flexible nature of the IDR allows the S518 region to scan the local environment. When unphosphorylated (wild-type), the region remains electrostatically neutral and inactive (Allen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, our S518D model demonstrates that the addition of a negative charge acts as a powerful biophysical switch.\u003c/p\u003e \u003cp\u003eThe resulting intense electronegative pocket (red surface) operates effectively as a localized \"magnet.\" We propose that this region specifically attracts and anchors the complementary electropositive surfaces of downstream repair factors, such as the BRCT domain of DNA Ligase III. By keeping the global structure intact while drastically altering the local surface charge, the S518 phosphorylation event transforms a random, floppy loop into a highly specific docking interface. This study is based on static computational models and does not account for protein dynamics or direct binding interactions. Future work involving molecular dynamics simulations and experimental validation will be necessary to confirm these findings\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides a computational framework for understanding the phosphorylation-dependent regulation of XRCC1 within its intrinsically disordered region. Using AlphaFold-based structural modeling and electrostatic surface analysis, we demonstrate that a phosphomimetic substitution at residue S518 does not significantly alter the global architecture of XRCC1 but induces localized changes in electrostatic potential. Specifically, the introduction of a negatively charged residue generates a distinct electrostatic patch within the disordered linker region, which may contribute to modulating interactions with downstream DNA repair proteins.\u003c/p\u003e \u003cp\u003eThese findings support the concept that post-translational modifications within intrinsically disordered regions can function as regulatory elements by altering local physicochemical properties without requiring large-scale conformational changes. Such mechanisms may be particularly relevant for scaffold proteins involved in dynamic cellular processes such as DNA repair.\u003c/p\u003e \u003cp\u003eHowever, it is important to note that these observations are based on static computational models and do not account for protein dynamics or direct binding interactions. Future studies incorporating molecular dynamics simulations and experimental validation will be necessary to further elucidate the functional implications of S518 phosphorylation in XRCC1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCaldecott KW (2022) DNA single-strand break repair and human genetic disease. Trends Cell Biol 32(9):733\u0026ndash;745\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang Q, \u0026Ccedil;ağlayan M (2021) The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair. J Biol Chem 297(3):101025\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKruswick A, Lam FC, Kong YW, Smerdon SJ, Yaffe MB (2024) BRCT domains as chromatin readers: Structure, function, and clinical implications. Chromatin Readers in Health and Disease. Academic, pp 31\u0026ndash;56\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhediri S, Sarma PA, Saravanan V, Abbadie C, Blossey R, Cleri F (2025) Mechanisms of DNA Damage Recognition by UDG and PARP1 in the Nucleosome. Biomolecules 15(5):649\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Z, Zeng X, Zhao Y, Chen R (2023) AlphaFold2 and its applications in the fields of biology and medicine. Signal Transduct Target Therapy 8(1):115\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma VV, Vimal S, Mishra MK, Sharma VK (2025) A comprehensive review on structural insights through molecular visualization: tools, applications, and limitations. J Mol Model 31(6):173\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhilipp M, Moth CW, Ristic N, Tiemann JKS, Seufert F, Panfilova A, Staritzbichler R (2024) MutationExplorer: a webserver for mutation of proteins and 3D visualization of energetic impacts. Nucleic Acids Res 52(W1):W132\u0026ndash;W139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTvaroška I, Kozmon S, K\u0026oacute;ňa J (2023) Molecular modeling insights into the structure and behavior of integrins: a review. Cells 12(2):324\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmohdar D, Gulkis M, Ortiz A, Tang Q, Sobol RW, \u0026Ccedil;ağlayan M (2024) Impact of polβ/XRCC1 interaction variants on the efficiency of nick sealing by DNA ligase IIIα in the base excision repair pathway. J Mol Biol 436(4):168410\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNosella ML, Kim TH, Huang SK, Harkness RW, Goncalves M, Pan A, Kay LE (2024) Poly (ADP-ribosyl) ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates. 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J Biomol Struct Dynamics 41(21):11629\u0026ndash;11646\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllen MC, Karplus PA, Mehl RA, Cooley RB (2024) Genetic encoding of phosphorylated amino acids into proteins. Chem Rev 124(10):6592\u0026ndash;6642\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"XRCC1, Intrinsically Disordered Regions (IDRs), Phosphorylation, Post-translational Modification, AlphaFold, Structural Dynamics, DNA Repair","lastPublishedDoi":"10.21203/rs.3.rs-9455541/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9455541/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe X-ray repair cross-complementing protein 1 (XRCC1) is a central scaffold protein involved in DNA single-strand break repair (SSBR), coordinating the assembly of multi-protein repair complexes. Its activity is regulated by post-translational modifications, particularly phosphorylation within intrinsically disordered regions (IDRs), although the structural consequences of these modifications remain poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, an in silico approach is used to investigate the structural and electrostatic effects of a phosphomimetic mutation at residue S518. The full-length structure of human XRCC1 was predicted using AlphaFold2 via ColabFold, followed by structural and electrostatic analyses in UCSF ChimeraX. Predicted Local Distance Difference Test (pLDDT) scores revealed a heterogeneous structural profile, with well-defined folded domains and a low-confidence inter-domain linker region encompassing S518, consistent with intrinsic disorder.\u003c/p\u003e \u003cp\u003eIntroduction of the phosphomimetic mutation (S518D) did not result in substantial alterations to the global structure of XRCC1. However, electrostatic surface analysis indicated the emergence of a localized negatively charged region at the mutation site. Additional structural inspection suggested localized perturbations without global destabilization.\u003c/p\u003e \u003cp\u003eThese findings suggest that residue-level modifications at S518 may modulate the local electrostatic environment of XRCC1 and potentially influence its interactions with downstream DNA repair factors. This study highlights the role of electrostatic tuning within intrinsically disordered regions as a possible regulatory mechanism in scaffold proteins. However, as these observations are based on static computational models, further validation using molecular dynamics simulations and experimental approaches is required.\u003c/p\u003e","manuscriptTitle":"Phosphorylation-Dependent Activation of the XRCC1 Intrinsically Disordered Region: A Computational Structural Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 07:42:37","doi":"10.21203/rs.3.rs-9455541/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6bc4b737-ab77-427c-a7d3-a05a62189b0c","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":66566129,"name":"Bioinformatics"}],"tags":[],"updatedAt":"2026-04-21T07:42:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 07:42:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9455541","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9455541","identity":"rs-9455541","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}