{"paper_id":"075ea8f7-cd7d-4b30-b26e-c8dafcd3de4f","body_text":"Structure-Based Discovery of a Cryptic Druggable Pocket in TP53 C238Y: Implications for Targeted Therapy | 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 Structure-Based Discovery of a Cryptic Druggable Pocket in TP53 C238Y: Implications for Targeted Therapy hoosdally shakeel This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6370188/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 Mutations in the TP53 gene are frequently found in many different types of human cancers These mutations interfere with important functions that normally prevent tumors, like controlling cell growth and causing programmed cell death. When TP53 can't do its job, cells start multiplying without control, and the cell's genetic material becomes unstable. Even though TP53 has long been known to be a key player in cancer it's been very difficult to develop drugs that target it. This is largely because of its flexible structure and the lack of clear binding sites for drugs. But, recent studies indicate that specific mutations can cause structural changes in TP53, creating new potential binding sites that could be useful for drug development. In this study, I used computer modeling and structural biological analysis to examine the c238y tp53 mutation . The results showed that this mutation dramatically reshapes the protein in the vicinity — it exposes a hidden pocket that could be a promising target for drugs. These results pave the way to conceptualising and designing therapies that are mutationally specific with the end goal being to disrupt or restore the default function of malfunctioning TP53 in cancer. This structural study lays the foundation for a follow-up phase involving virtual screening and drug-binding validation targeting the revealed cryptic pocket. Computational Biology TP53 C238Y mutation Hidden binding site Drug target potential Molecular dynamics Protein pocket reshaping Solvent accessibility Hydrogen bond analysis Cancer drug discovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 SIMPLE SUMMARY TP53 gene is really important for keeping our cells healthy. It does this by fixing damaged DNA, managing cell growth, and telling cells to die if they're too damaged to repair. But when TP53 has mutations,these defenses stop working, and cells start growing out of control. One of the big problems in trying to create drugs that target TP53 is that it's very flexible and doesn't have obvious places where drugs can easily attach. This has led to it being called \"undruggable”. However, some recent findings suggest that certain mutations can change the protein's shape, revealing hidden areas where drugs might be able to bind. In this research, I used computer-based methods to study the C238Y mutation in TP53 and see how it changes the protein's structure. The results showed that this mutation causes some reshaping of the protein's surface, exposing a previously hidden pocket that could be a target for drugs. These results suggest that we should take another look at TP53 as a potential target for cancer drugs, which could lead to the development of drugs that work specifically against certain TP53 mutations . Introduction The TP53 gene plays a key role in maintaining genetic material stability and is one of the most frequently mutated genes in human cancers [1]. The protein it encodes, p53, plays a key role as a tumor suppressor by coordinating multiple protective responses in cells. This includes pathogens of DNA repair, apoptosis, and cell cycle regulation under stress or damage [2, 3]. However, TP53 missense mutations can significantly impair these protective mechanisms [4]. These types of mutations are often linked to more aggressive tumor growth, lower response to treatment and worse outcomes for patients. The C238Y mutation in the DNA binding core domain is the most structurally and functionally relevant of these mutations. In my previous investigation of uterine leiomyosarcoma (ULMS) [5], I described a case of a 48-year-old female diagnosed with uterine leiomyosarcoma that contained this particular C238Y alteration in TP53. In addition, this mutation was also identified in other malignancies, such as osteosarcoma [6], further supporting the clinical importance of this mutation. Here, I present a detailed structural and rigid-body perspective comparison of the wild-type TP53 and the C238Y mutant, which allows us to characterize a mutation-induced remodeling of the binding pockets[7,8], solvent accessibility, and flexibility[9]. Combining structure-based predictions from multiple sources with molecular dynamics simulations [10] and analysis of solvent exposure[11,12], the study reveals a cryptic, druggable pocket that is specifically exposed by the C238Y mutant, which challenges the long-held view of TP53 as an ‘undruggable’ target [13]. Methods 2.1 Construction of the Molecular Model Used: Wild-Type and Mutant TP53 In order to begin my investigation, I needed an accurate 3D representations of the TP53 protein in both its normal (wild-type) and C238Y mutant forms. I started with a very high-resolution (1.8 Å) crystal structure from the PDB (PDB ID: 2OCJ) a sort of ‘blueprint’ of the wild-type (wt) protein. Next, I launched the PyMOL[14] molecular visualization program to remove the cysteine at position 238, replacing it with tyrosine. This generation leads to a C238Y mutant model. Since mutations can sometimes cause structural distortions,and I minimized the structure using UCSF Chimera[15], which means I ‘relaxed’ the structure to relieve any possible steric clashes among residues. To improve my model further and verify its physical plausibility, I used the CHIRON web server[16] which refined protein structures Lastly, to validate the model, I used the SAVES 6.0 suite[17], multiple tests including topology checks such as proper amino acid geometries Ramachandran plots, atomic clashes, MolProbity scores, and background checks such as root-mean-square deviation and Z-scores to assess overall structural quality model. This was an essential step to ensure that further analysis were based on reliable model structure. 2.2 Binding Sites identification:using different software tools based on different identication methods To identify potential ligand-binding pockets, both wild type WT and mutant TP53 structures were analyzed using seven web-based prediction tools: FTMap[18], CryptoSite[19], PocketMiner[20], DoGSiteScorer[21], P2Rank[22], PockDrug[23], and DREAMM[24]. Each software used different methodologies—ranging from fragment-based mapping , cryptic site prediction , machine learning algorithms and membrane-targeted scoring—to detect hotspots and druggable regions. Binding site residues predicted by each tool were identified and aligned to highlight overlapping or common sites. These shared regions were identified to define consensus binding sites that would be relevant for ligand binding. 2.3 Molecular Dynamics Simulations Proteins are dynamic and they have a conformation. To study the influence of the C238Y mutation on the TP53 flexibility and dynamics, I carried out molecular dynamics (MD) simulations. My initial approach applied coarse-grained simulations using CABS-flex 2.0 to analyse residue-level dynamics and contact maps. Furthermore I then performed simulations with the SimpleRun module in the PlayMolecule suite with the AMBER force field. Those were fine-tuned to track structural changes over time, both pocket motion and radius of gyration. That gave some insight into how the mutation might affect how flexible the protein is, and therefore how it would affect drug binding 2.4 Hydrogen Bonds and Distance Measurements I wanted to know the local effects of the C238Y mutation, in particular in the vicinity of GLU 221, which seemed to be significant for the formation of a possible binding pocket. The hydrogen bond in this region were analyzed by Visual Molecular Dynamics (VMD). UCSF Chimera was also used to measure the distances for important residues, specifically TYR 238 and GLU 221, to measure this structural change caused by the mutation. These measurements yielded actual numbers on how the mutation changes the local environment 2.5 Measuring solvent exposure using solvent accessibility surface area (SASA) The Solvent Accessibility Surface Area (SASA) was calculated by using GetArea server to assess the accessibility of the potential drug-binding pocket of the C238Y mutation to solvents . I studied GLU 221 and the residues surrounding it, such as PRO 219, TYR 220, and PRO 222 from the wild-type and mutant structures. By checking the SASA numbers, I could find out if the mutation made the pocket more hidden or visible. 2.6 Structures Features Comparison using Pocket Volume and Structural Overlays Lastly, to visualize the differences in TP53 structural changes between wild-type and mutant, consensus binding site residues were mapped onto the protein surface in PyMOL. I performed pocket size estimations using DoGSiteScorer to quantify the volume available for drug binding. I constructed structural overlays to compare pocket formation and conformational states directly and illustrate how the C238Y mutation could reveal cryptic binding pockets .1 Validation of the TP53 C238Y Mutant Model The C238Y mutant structure was successfully generated and optimized through a combination of structural modeling and energy minimization techniques. Validation of the final model demonstrated high structural quality: 96.4% of residues were positioned within the favored regions of the Ramachandran plot, and no outliers were observed. Additional quality metrics further supported the model's reliability, including a low root mean square deviation (RMSD) of 0.24 Å, a Z-score of –6.10, a MolProbity score of 0.91, and a Verify3D score of 93.81%. Taken together, these results indicate that the refined mutant structure is both stable and accurately folded, making it well-suited for subsequent structural and dynamic analysis. Validation Metrics for TP53 Structure Metric Value Ramachandran Favored (%) 96.4 MolProbity Score 0.91 Ramachandran Outliers (%) 0 RMSD (Å) 0.24 Z-Score -6.0993 Verify3D Score (%) 93.81 3.2 Analysis of Binding Sites Showed Pocket Formation on Mutation The binding site predictions were from seven independent tools and compiled them to compare the wild-type (WT) and mutant TP53 structures. The wild-type protein showed randomly arranged and isolated pockets within this protein, with critical residues like MET 246 and THR 140 distant (Pockets 6 and 1). The C238Y binding mode was not widely distributed among multiple clusters, as it showed one single consensus binding location (Pocket 0), centered at GLU 221 and flanked by PRO 219, TYR 220 and PRO 222. All tools identified this site, indicating consensus confidence and potential druggability. Structural differences: Panel a shows side-by-side pockets (Figure 2) comparing pocket organization between both structures. A consensus binding site was defined as a region supported by predictions from ≥3 independent servers Table 1. Binding Sites Predicted in Wild-Type TP53 by Each Server Server Predicted Binding Residues (Wild-Type TP53) FTMap MET 246, ARG 174, SER 240, VAL 172 CryptoSite ARG 158, ARG 174, VAL 172, MET 246 DREAMM A174, A209, A212, A225, A243, A248 (membrane-associated loop) PocketMiner VAL 147, ARG 174, GLU 180, MET 246 PockDrug ARG 174, SER 240, MET 246, ARG 248 DoGSiteScorer VAL 172, MET 246, THR 140 P2Rank MET 246, ARG 174, ARG 248, PHE 134 Table 2. Binding Sites Predicted in C238Y Mutant TP53 by Each Server Server Predicted Binding Residues (Mutant C238Y TP53) FTMap GLU 221, PRO 219, TYR 220, MET 160 CryptoSite GLU 221, TYR 220, PRO 219, MET 160 DREAMM A208, A209, A221, A225, A248 (loop flexibility/membrane residues) PocketMiner GLU 221, TYR 220, MET 160, PRO 222 PockDrug GLU 221, TYR 220, PRO 219, TYR 126 DoGSiteScorer GLU 221, PRO 219, TYR 220 P2Rank GLU 221, TYR 220, PRO 222, MET 160 Table 3. Consensus Binding Site – Wild-Type TP53 Residue Predicted By Servers Included in Consensus? MET 246 FTMap, CryptoSite, PocketMiner, PockDrug, P2Rank ✅ Yes ARG 174 FTMap, CryptoSite, PockDrug, P2Rank ✅ Yes VAL 172 FTMap, CryptoSite, DoGSiteScorer ✅ Yes SER 240 FTMap, PockDrug ✅ Yes (2 servers only) PHE 134 P2Rank ❌ No THR 140 DoGSiteScorer ❌ No Table 4. Consensus Binding Site – C238Y Mutant TP53 Residue Predicted By Servers Included in Consensus? GLU 221 FTMap, CryptoSite, PocketMiner, PockDrug, DoGSiteScorer, P2Rank ✅ Yes TYR 220 CryptoSite, PocketMiner, PockDrug, P2Rank, DoGSiteScorer ✅ Yes PRO 219 FTMap, CryptoSite, PockDrug, DoGSiteScorer ✅ Yes MET 160 FTMap, CryptoSite, PocketMiner, P2Rank ✅ Yes PRO 222 PocketMiner, P2Rank ✅ Yes (borderline) TYR 126 PockDrug ❌ No How the Consensus binding sites Were Reached How the Consensus Was Reached A residue was considered to be part of the consensus if it was predicted by ≥3 out of the 7 tools. The pocket is predominantly occupied by MET 246, ARG 174, and VAL 172, which sit in the core DNA‐binding domain for wild type TP53[16] For the mutant, the consensus rearranged toward a remodeled, flexible loop centering on GLU 221 with support of dynamic analysis Results and Structural Analysis: Insights into the C238Y Mutation 3.1 A Reliable mutant model I wanted to make sure that my mutant model was as accurate as possible. Therefore, I carefully developed and validated a 3D structure model of the TP53 protein with C238Y point mutation. The model passed all my quality checks : 96.4% of its amino acids sat in the most energetically favorable regions of the Ramachandran plot, indicative of proper geometry. One of the most important validation step is the find how close my model was to the original model by comparing the RMSD of my model compared to original structure. A very low RMSD = 0.24 Å for the model was obtained using matchmaker module alignmemt in UCSF Chimera molecular software. The Z-score of the model and MolProbity clash score were perfect .This confirmed a model that is stable and energetically sound so that the further analysis can be pursued. 3.2 Structural Remodeling of the Binding Pocket When I looked at the predicted drug-binding sites for the normal (wild-type) TP53 and the mutated TP53 there was a dramatic structural shift.. In the normal (wild-type) TP53 protein, potential binding sites were spread out across the surface as separate, unconnected areas. But in the C238Y mutant, this pattern shifted. These scattered regions came together to form a single, continuous pocket centered around the GLU 221 residue. This change seems to be caused by the mutation, which reshaped the surface of the protein and created a new space where a drug might be able to bind. The pocket also includes nearby residues like PRO 219, TYR 220, and PRO 222—consistently highlighted by all the prediction tools used in this study. This points to a clear structural change brought about by the mutation. 3.3 Increased Flexibility: Creating Space for Drug Binding Proteins are not rigid—they constantly shift and move. molecular dynamics simulations showed that the area around GLU 221 in the C238Y mutant became much more flexible compared to the wild-type protein. This was noticeable in the loop between residues A208 and A209, which showed greater movement. Such flexibility may allow the newly formed pocket to briefly open, creating an opportunity for drug molecules to enter. In simpler terms, the mutation may act like a door that open just long enough for a potential drug to pass through—something that wouldn’t be possible in the more stable, less flexible wild-type structure. Pocket Accessibility In the wild-type TP53 protein, hydrogen bonds help stabilize the region around GLU 221, keeping it tightly packed. However, in the C238Y mutant, those stabilizing bonds are no longer present. One clear example is the increased distance of 27.19A between TYR 238 (the mutated residue) and GLU 221, which shows that the mutation disrupts the original structural arrangement. Without these hydrogen bonds, the local space becomes more open. The mutation removes the original support holding the structure together, making room for a new pocket to form—one that wasn’t previously accessible in the wild type tp53. 3.5 Increased Surface Exposure: A Pocket Ready for Drugs To understand whether this new pocket might actually be reachable by ligands , I analyzed its solvent-accessible surface area (SASA). This measurement reveals how much of a protein’s surface is exposed to its surrounding environment. When comparing the wild-type and mutant structures, I found that GLU 221—and its neighboring residues like PRO 219, TYR 220, and PRO 222—were much more exposed in the mutant. This increased exposure suggests that the mutation made the surface more open, which could allow small molecules o to access and bind the pocket more easily. 3.6 A Larger Pocket: Improving Drug Binding When comparing the size and shape of the binding pockets, the difference between the wild-type and the C238Y mutant TP53 was obvious. In the wild-type protein, potential pockets appeared as several small and scattered regions. In contrast, the C238Y mutation gave rise to a single, more spacious and continuous cavity. This new pocket wasn’t just bigger in volume; it also acquired a higher druggability score, suggesting it is more likely to accommodate small-molecule . The mutation reshaped a fragmented and less accessible site into a well-defined target ideal for drug targeting. Table 5. Comparative Binding Pocket Properties in TP53 Wild-Type vs. C238Y Mutant Feature Wild-Type TP53 (2OCJ) Mutant TP53 (C238Y) Consensus Binding Residues PHE 134, THR 140, MET 246 GLU 221, TYR 220, PRO 219 Pocket ID(s) P_1 (THR 140, PHE 134), P_6 (MET 246) P_0 Pocket Volume (ų) P_1: 252.67; P_6: 133.25 P_0: 299.26 Surface Area (Ų) P_1: 470.66; P_6: 190.09 P_0: 456.03 Drug Score P_1: 0.48; P_6: 0.29 P_0: 0.53 Pocket Type Fragmented, shallow pockets Unified, deep, flexible pocket Druggability Implication Lower — scattered sites reduce ligand binding Higher — improves binding 3.7 Mechanistic Model of Mutation-Driven Pocket Formation By incorporating structural and dynamic analysis , I proposed the following mechanistic pathway: → C238Y Mutation → Differentiable Loop (RMD) → Loss of Hydrogen Bonds → Increased SASA and Spatial Disconnection → Unified Cryptic Pocket Formation This cascade driven by mutational events explains the birth of a novel druggable pocket that is focused specifically on GLU 221—a finding that has not been reported in the literature with potentially important implications for drug development. Analysis of Solvent Exposure and Secondary Structure 4.1 SASA (Solvent Accessibility) Analysis of the structures using the GetArea webserver revealed greater solvent exposure at the loop containing GLU 221 and the surrounding residues (C238Y mutant). This greater accessibility indicates that the pocket has switched from a buried site to a more solvent-accessible target for ligand binding 4.2 PlayMolecule MD Simulation Analysis Radius of Gyration (Rg): The Rg values were stable in the wild-type structure during the MD simulation suggesting the structural compactness. However the C238Y mutant showed fluctuations around global Rg reflecting overall flexibility and partial instability of the mutant. Secondary Structure Transitions: Secondary Structure Transitions: DSSP-based secondary structure monitoring revealed a shift from helical to coil regions near the GLU 221 loop in the mutant structure. This change aligns with observations of increased loop dynamics and pocket plasticity. Discussion 5.1 A Druggable Site In TP53 Unlocked by Mutation The comparison of the crystal structures between the wild-type and C238Y mutant TP53 (Table 6) reveals an intriguing structural shift. Binding cavities in the wild-type spread shallowly and randomly, lacking strong crystal clustering. In the C238Y mutant, a stronger crystal cluster also formed, with previously dispersed pockets merging into a clearly defined single cavity. This transformation, opened a new binding site for drugs. Where the wild-type lacked such a pocket, the C238Y mutation introduced it, as noted in structural analysis. Residues like PHE 134 THR 140, and MET 246 dD within these pockets (P_1 and P_6) are reorganized. Changes occurred in the adjacent loops, specifically near GLU 221, resulting from the cysteine-to-tyrosine change at position 238. It generates a new pocket (P_0), comprising GLU 221, TYR 220, and PRO 219. It's a cryptic pocket that wasn't visible under normal conditions but emerges prominently when structural changes take place. The generally accepted view that TP53 is undruggable is challenged by this observation. TP53's potential for drug interaction is not fixed. Mutation-driven remodeling can modify it. This has important implications for the developmentdrug process, offering new possibilities for therapeutic interventions. The traditional understanding of TP53's undruggable paradigm is longer valid, making these insights particularly transformative. Together with these visual checks, there were different quantitative shifts witnessed, and they were observed to coincide with drugs. The density of pockets P_1 and P_6 combined measured about 386 ų. The scores for druggability were low, 0.48 and 0.29. The mutant pocket P_0, smaller at 299 ų, had a higher score of 0.53. This means that pharmacological relevance, as well as structural organization, is improved by the C238Y mutation. A variety of quantitative shifts was seen, supporting this visual confirmation. Mechanistically, this shift is supported by several observations: The mobility of the loop between residues 200 and 220 was notably elevated based on RMSF data. Solvent exposure of GLU 221 was higher, making it more likely that it was broken, thus allowing greater loop mobility. The observation regarding the separation distance between TYR 238 and GLU 221 indicates an increase in this distance in C238Y, allowing the geometry of a newly formed pocket. This evidence, when considered collectively, shows the mechanistic sequence: the C238Y mutation leads GLU 221 to reposition and reveal a hidden site. That's important. Furthermore, this sequence explains how a point mutation can modify local topology, providing an opportunity that could be targeted with drugs. 6.1 Structuring the Changes The direct observation of conformational and dynamic variations, as well as access to binding sites, has not been performed before this study. This is new territory. Structural changes due to the mutation affect the microenvironment around GLU 221, resulting in loosening stabilizing contacts and increased flexibility in conformation. These changes were found to create a surface pocket suitable for drug targeting, absent in the original protein and associated with potential therapeutic targets. 6.2 Why Multiple Prediction Tools Were Used Relying on a number of software tools to generate the results makes the process robust and reliable; these involved five end-user applications, such as CryptoSite and PocketMiner, employed as the standard cryptographic applications there are. The identification process was carried out by these tools efficiently, taking advantage of their unique capabilities. Efficiency was maintained in the investigation of binding sites. Energy-favorable binding hotspots were identified using FTMap. Druggability scoring methodologies, which include those based on structural features like P2Rank and DoGSiteScorer, as well as machine learning approaches such as PockDrug, were used for clarity evaluation. Clarity was achieved with the use of DREAMM. The membrane-relevant exposure residues in TP53 were considered significant biologically. All tools, even with different methods, agreed. The result: the C238Y mutant exposes a new area. It's centered around GLU 221. The shared conclusion of various methodologies confirms its relevance, while the joint interpretation of a single method pointed to a singular insight: a ligand-accessible region, previously unexposed, has been revealed through mutant analysis. Despite different approaches, agreement was found. It showcases GLU 221 as a hub within the protein structure. How the C238Y Mutation Restructs the Binding Surface The C238Y mutation substitutes a small cysteine with a larger tyrosine, creating steric pressure that forces nearby loops to shift their positions. This shift may move GLU 229 to a position that exposes it systematically. The C238Y mutation acts by exposing GLU 221 more effectively. RMSF, distance mapping, and SASA calculations align with these observed structural changes. \"A structurally new pocket results, and it's not just new—it's also suitable for drug targeting,\" Koasiakoff said. What emerges from this is a biologically important and druggable pocket. 6.4 Drug Design Opportunities The cleft's location presents notable interest, and because it is not a static feature of the protein structure, this makes it even more intriguing; it opens only with allosteric targeting, where targeting takes place. The pocket's dynamic nature is due to the active shaping by protein movement. It offers ligands a chance for flexibility-dependent binding. 6.5 Changing the Ways We Discover Drugs for TP53 The traditional belief that TP53 is universally nondruggable is questioned by this study. A different perspective is introduced. The current studies are critical for these features. Consequently, it is demonstrated that a cryptic pocket is associated with the C238Y mutant, which is not present in normal cells. Various tumors, such as uterine leiomyosarcoma and osteosarcoma, exhibit the mutation, so researchers think this structural information might have significant implications for translation. Wide translational implications could result. Since oncogenic mutations were seen to remodel surface proteins, a cell surface pocket was expected. A cryptic pocket was identified. This is associated with the C238Y mutant and is absent in the wild-type, showing the value of considering tumor-specific mutations when exploring therapeutic options. Therefore, this study calls into question the long-held belief of TP53 being universally nondruggable. Structural changes could be exploited for therapy developments, given the discoveries. Outlook and Future Perspectives A new role for the TP53 C238Y mutation has been discovered, revealing a hidden binding pocket around residue L331. In this context, conformational data and measurement of spatial distances from various pocket prediction tools were used and combined. It has been proved that a series of conformational changes is triggered by this mutation. Loops destabilize, native hydrogen bonds are lost, and increased local flexibility occurs, exposing a site that could become therapeutic. This study questions the assumption that TP53 cannot be targeted therapeutically. Rather, it indicates that specific cancer-related mutations might alter the protein's surface, creating potential for treatment development. The finding holds particular relevance for drug targeting. The cryptic pocket identified here provides a mutation-specific chance for targeting with fragment-based or allosteric drug design swiftly. In contrast to traditional beliefs, certain mutations may open up new vistas on the TP53 protein surface, suggesting innovative approaches for drug discovery. A fragment-based approach can exploit these new binding possibilities. This observation is particularly noteworthy in a system where the C238Y mutation alters TP53. Suddenly, the presence of a pocket offers a new direction. The mutation-specific nature of this pocket could see it targeted by novel therapeutic strategies, challenging established ideas regarding TP53's previously thought limitations.There are several important paths to build on the findings of this study: Virtual screening of FDA-approved drugs and natural compounds should be conducted to uncover molecules that specifically interact with the pocket revealed by the C238Y mutation. These candidate molecules should then be tested through molecular dynamics simulations to evaluate how stably they bind under conditions that mimic the cellular environment. Finally, experimental validation using structural techniques such as NMR spectroscopy or X-ray crystallography—alongside functional testing in cancer cell lines carrying the TP53 C238Y mutation—will be essential to confirm their biological relevance. These steps can help translate this structural finding into fruitful therapeutic strategies. 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Additional Declarations The authors declare no competing interests. Supplementary Files GraphicalAbstract.docx 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6370188\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":438011842,\"identity\":\"89767e09-19d7-49d6-aa97-bd80b4e2609c\",\"order_by\":0,\"name\":\"hoosdally shakeel\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDACCRDBxiDDx8B8gDQtPGwMbAkka+ExIE6H/Ozmgw9/lNnxsEnkfN3woWJbYgN77+MX+LQY3DmWbMxzLhmoJXfbzRlnbic28Bw3s8CrRSLHTJqxjRms5TZvG1CLRBobXifKz8gxk/zZVg9y2LPbvP+I0MJwI8dMgrftMEgL223eBrAW5gd4HXYjDeSX4zxsPM/Mbs44dtu4jecYG15L5Gckg0KsWo6fPfnZjQ81t2X72duYP+DVAwcCCRAaaAWbBHFa+A/AmcTaMgpGwSgYBSMEAABJcUeoZL+uEQAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"ministry of education and higher scientific research\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"hoosdally\",\"middleName\":\"\",\"lastName\":\"shakeel\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-04-03 14:26:37\",\"currentVersionCode\":1,\"declarations\":{\"humanSubjects\":false,\"vertebrateSubjects\":false,\"conflictsOfInterestStatement\":false,\"humanSubjectEthicalGuidelines\":false,\"humanSubjectConsent\":false,\"humanSubjectClinicalTrial\":false,\"humanSubjectCaseReport\":false,\"vertebrateSubjectEthicalGuidelines\":false},\"doi\":\"10.21203/rs.3.rs-6370188/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6370188/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":79880042,\"identity\":\"38f091b8-f516-431d-99ce-03d0a90a3968\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:50:35\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":90906,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSASA Comparison at GLU 221 Region\\u003c/p\\u003e\\n\\u003cp\\u003eBar chart comparing SASA values of pocket residues in wild-type and C238Y mutant TP53. Mutant shows increased surface exposure.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/f1d664354b7ba6ef11004666.png\"},{\"id\":79879654,\"identity\":\"b85fa0f6-877e-49ea-b380-3ec459affd55\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":112422,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eResidue Flexibility (RMSF) of Pocket Residues\\u003c/p\\u003e\\n\\u003cp\\u003eBar chart of RMSF values for residues 200–250. A208–A209 show the highest flexibility in the mutant TP53.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/86e9eebeeed489e550feba1e.png\"},{\"id\":79879649,\"identity\":\"0127ebd9-65ad-4b61-9071-0dd0255106bd\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":165633,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA. Radius of Gyration – Wild-Type TP53\\u003c/p\\u003e\\n\\u003cp\\u003eStable Rg values indicate compact structure of wild-type TP53.\\u003c/p\\u003e\\n\\u003cp\\u003eB. Radius of Gyration – C238Y Mutant TP53\\u003c/p\\u003e\\n\\u003cp\\u003eElevated Rg in mutant TP53 suggests unfolding or increased flexibility.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/b1880c55cbb2f6f2deec847d.png\"},{\"id\":79879659,\"identity\":\"e5f11fcb-0755-4738-b65e-4a7bdbccbbfd\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":221034,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA. RMSD – Wild-Type TP53\\u003c/p\\u003e\\n\\u003cp\\u003eWild-type TP53 shows low RMSD for both backbone and sidechains over time.\\u003c/p\\u003e\\n\\u003cp\\u003eB. RMSD – C238Y Mutant TP53\\u003c/p\\u003e\\n\\u003cp\\u003eMutant TP53 displays increasing RMSD, indicating loss of structural stability.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/e396ec13a4c3567a6573c848.png\"},{\"id\":79879661,\"identity\":\"4e3d475f-d9a6-4603-8ec1-daa31e268c41\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":98918,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRMSF Zoomed View (A200–A209)\\u003c/p\\u003e\\n\\u003cp\\u003eZoomed RMSF plot from CABS-flex showing highest flexibility at A208–A209.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/8f111fd24edde750df24c437.png\"},{\"id\":79879662,\"identity\":\"6384799d-b57c-437e-adae-ab6fda25b4e1\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":109072,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSecondary Structure Transitions – Mutant TP53\\u003c/p\\u003e\\n\\u003cp\\u003eTime-dependent shift from alpha-helix to coil/loop observed in the mutant structure.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/940c1741b961ca1ee3d0258f.png\"},{\"id\":79879657,\"identity\":\"6684ad16-6bec-4bd0-8ab7-b61e9c0fa40e\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":369345,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTYR 238 – GLU 221 Distance Measurement\\u003c/p\\u003e\\n\\u003cp\\u003eSnapshot from Chimera showing 27.81 Å distance between disconnected pocket-forming residues.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/e5b76648304f135288b9f42c.png\"},{\"id\":79879651,\"identity\":\"657a7e7c-b88e-4d01-95f6-7e00b02e0469\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":33547,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eLoop Flexibility Diagram for Ligand Access\\u003c/p\\u003e\\n\\u003cp\\u003eSchematic showing upward motion of A208–A209 providing transient access to GLU 221 pocket.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/7592ef818b53fe76c2363584.png\"},{\"id\":79879666,\"identity\":\"3c032949-5a89-47d9-a932-c0c9c770781d\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":95488,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSurface rendering of wild-type TP53 showing shallow, fragmented binding pockets (P_1 and P_6) identified via multiple prediction tools.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/64c27b355d75183757fa6971.png\"},{\"id\":79880045,\"identity\":\"4d48c963-533d-484e-bc36-1f661d5c32e2\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:50:35\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":33920,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSurface rendering of C238Y mutant TP53 showing a unified, deeper cryptic pocket (P_0) centered on GLU 221.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/69a7f57ee1a4bdfe45f746b1.png\"},{\"id\":79880043,\"identity\":\"1cfc1fc4-d655-463f-8b45-705672907f77\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:50:35\",\"extension\":\"png\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":180751,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eUnnumbered image in the Methods section.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"methossectionfigure.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/46296bf2d9b616bd3beab64a.png\"},{\"id\":79880953,\"identity\":\"d8efbaea-9aa6-4c5b-8943-338574f3de21\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 04:06:36\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2615886,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/a1f24233-99c2-41be-9351-588a4a5357e9.pdf\"},{\"id\":79879655,\"identity\":\"245a2abc-b26e-4d8c-8a95-ece51699aa7d\",\"added_by\":\"auto\",\"created_at\":\"2025-04-04 03:42:35\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1158921,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"GraphicalAbstract.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6370188/v1/b4bf04c44ddbafab94b1f4e7.docx\"}],\"financialInterests\":\"The authors declare no competing interests.\",\"formattedTitle\":\"\\u003cp\\u003eStructure-Based Discovery of a Cryptic Druggable Pocket in TP53 C238Y: Implications for Targeted Therapy\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"SIMPLE SUMMARY\",\"content\":\"\\u003cp\\u003eTP53 gene is really important for keeping our cells healthy. It does this by fixing damaged DNA, managing cell growth, and telling cells to die if they\\u0026apos;re too damaged to repair. But when TP53 has mutations,these defenses stop working, and cells start growing out of control. One of the big problems in trying to create drugs that target TP53 is that it\\u0026apos;s very flexible and doesn\\u0026apos;t have obvious places where drugs can easily attach. This has led to it being called \\u0026quot;undruggable\\u0026rdquo;. However, some recent findings suggest that certain mutations can change the protein\\u0026apos;s shape, revealing hidden areas where drugs might be able to bind. In this research, I used computer-based methods to study the C238Y mutation in TP53 and see how it changes the protein\\u0026apos;s structure. The results showed that this mutation causes some reshaping of the protein\\u0026apos;s surface, exposing a previously hidden pocket that could be a target for drugs. These results suggest that we should take another look at TP53 as a potential target for cancer drugs, which could lead to the development of drugs that work specifically against certain TP53 mutations .\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe TP53 gene\\u0026ensp;plays a key role in maintaining genetic material stability and is one of the most frequently mutated genes in human cancers [1]. The protein it encodes, p53, plays a key\\u0026ensp;role as a tumor suppressor by coordinating multiple protective responses in cells. This includes pathogens of DNA repair, apoptosis, and\\u0026ensp;cell cycle regulation under stress or damage [2, 3]. However,\\u0026ensp;TP53 missense mutations can significantly impair these protective mechanisms [4]. These types of mutations are often linked to more aggressive\\u0026ensp;tumor growth, lower response to treatment and worse outcomes for patients. The C238Y mutation\\u0026ensp;in the DNA binding core domain is the most structurally and functionally relevant of these mutations. In my previous investigation of uterine leiomyosarcoma (ULMS) [5],\\u0026ensp;I described \\u0026nbsp;a case of a 48-year-old female diagnosed with uterine leiomyosarcoma that contained this particular C238Y alteration in TP53. In addition, this mutation was also identified in other malignancies, such as osteosarcoma\\u0026ensp;[6], further supporting the clinical importance of this mutation.\\u003c/p\\u003e\\n\\u003cp\\u003eHere, I present a detailed structural and rigid-body perspective\\u0026ensp;comparison of the wild-type TP53 and the C238Y mutant, which allows us to characterize a mutation-induced remodeling of the binding pockets[7,8], solvent accessibility, and flexibility[9]. Combining structure-based predictions from multiple sources with molecular dynamics simulations [10] and analysis of solvent exposure[11,12], the study reveals a cryptic, druggable pocket that is specifically exposed by the C238Y\\u0026ensp;mutant, which challenges the long-held view of TP53 as an \\u0026lsquo;undruggable\\u0026rsquo; target [13].\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e2.1 Construction of the\\u0026ensp;Molecular Model Used: Wild-Type and Mutant TP53\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn order to\\u0026ensp;begin my \\u0026nbsp; investigation, I \\u0026nbsp;needed an \\u0026nbsp;accurate 3D representations of the TP53 protein in both its normal (wild-type) and C238Y mutant forms. I started with a very high-resolution (1.8\\u0026ensp;\\u0026Aring;) crystal structure from the PDB (PDB ID: 2OCJ) a sort of \\u0026lsquo;blueprint\\u0026rsquo; of the wild-type (wt) protein. Next, I launched the PyMOL[14]\\u0026ensp;molecular visualization program to \\u0026nbsp; remove the cysteine at position 238, replacing it with tyrosine. This generation leads\\u0026ensp;to a C238Y mutant model. Since mutations can sometimes cause structural\\u0026ensp;distortions,and \\u0026nbsp; I minimized the structure \\u0026nbsp;using UCSF Chimera[15], which means I \\u0026nbsp;\\u0026lsquo;relaxed\\u0026rsquo; the structure to relieve any possible steric clashes among residues. To improve my model further and verify its \\u0026nbsp;physical plausibility, I used the CHIRON web server[16] which refined protein structures \\u0026nbsp;Lastly, to validate the \\u0026nbsp;model, I used the SAVES 6.0 suite[17], multiple \\u0026nbsp;tests including topology checks such as proper amino acid geometries Ramachandran plots, atomic clashes, MolProbity scores, and background checks such as root-mean-square deviation and Z-scores to assess\\u0026ensp;overall structural \\u0026nbsp; quality model. This was an essential step to ensure that further \\u0026ensp;analysis were based on reliable \\u0026nbsp;model structure.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.2 \\u0026nbsp;Binding\\u0026ensp;Sites identification:using different software tools based on different identication methods\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo \\u0026nbsp;identify potential ligand-binding pockets, both \\u0026nbsp;wild type WT and mutant TP53 structures were analyzed using seven web-based prediction tools: FTMap[18], CryptoSite[19], PocketMiner[20], DoGSiteScorer[21], P2Rank[22], PockDrug[23], and DREAMM[24]. Each software used \\u0026nbsp; different methodologies\\u0026mdash;ranging from fragment-based mapping , cryptic site prediction , machine learning algorithms and membrane-targeted scoring\\u0026mdash;to detect hotspots and druggable regions. Binding site residues predicted by each tool were identified \\u0026nbsp;and aligned to highlight overlapping or common sites. These shared regions were identified \\u0026nbsp;to define consensus binding sites that would be \\u0026nbsp;relevant for ligand binding.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.3 \\u0026nbsp;Molecular Dynamics \\u0026nbsp;Simulations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProteins are dynamic and \\u0026ensp;they have a conformation. To study the influence of the C238Y mutation on the TP53\\u0026ensp;flexibility and dynamics, I carried out molecular dynamics (MD) simulations. My initial approach applied coarse-grained simulations using CABS-flex 2.0 to analyse \\u0026nbsp;\\u0026ensp; residue-level dynamics and contact maps. Furthermore I then performed \\u0026nbsp;simulations with the SimpleRun module in the PlayMolecule suite with the AMBER force field. Those were\\u0026ensp;fine-tuned to track structural changes over time, both pocket motion and radius of gyration. That\\u0026ensp;gave \\u0026nbsp;some insight into how the mutation might affect how flexible the protein is, and therefore how it would affect drug binding\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.4 \\u0026nbsp;Hydrogen Bonds\\u0026ensp;and Distance Measurements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eI wanted to know the \\u0026nbsp;local effects \\u0026nbsp;of the C238Y mutation, in particular in the vicinity of GLU 221, which seemed to be significant for the formation of a\\u0026ensp;possible binding pocket. The hydrogen bond \\u0026nbsp;in this region were analyzed by Visual Molecular Dynamics\\u0026ensp;(VMD). UCSF Chimera was also used to measure the distances for important residues, specifically TYR\\u0026ensp;238 and GLU 221, to measure \\u0026nbsp;this structural change \\u0026nbsp;caused by the mutation. These measurements yielded actual numbers on how the mutation changes \\u0026ensp;the local environment\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.5 Measuring \\u0026ensp;solvent exposure using solvent accessibility surface area (SASA)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe Solvent Accessibility Surface Area (SASA) was calculated by \\u0026nbsp;using \\u0026nbsp; GetArea server to assess the accessibility of the potential drug-binding pocket of the C238Y mutation to solvents\\u0026ensp;. \\u0026nbsp; I studied GLU 221\\u0026ensp;and the residues surrounding it, such as PRO 219, TYR 220, and PRO 222 from the wild-type and mutant structures. By checking the SASA numbers, I\\u0026ensp;could find out if the mutation made the pocket more hidden or visible.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.6 Structures Features Comparison\\u0026ensp;using Pocket Volume and Structural Overlays\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLastly, to visualize the\\u0026ensp;differences in TP53 structural changes between wild-type and mutant, consensus binding site residues were mapped onto the protein surface in PyMOL. I performed pocket\\u0026ensp;size estimations using DoGSiteScorer to quantify the volume available for drug binding. I constructed structural overlays to compare pocket formation and conformational states directly and illustrate how the C238Y mutation could reveal cryptic binding\\u0026ensp;pockets\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e.1 \\u003cstrong\\u003eValidation of the TP53 C238Y Mutant Model\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;The C238Y mutant structure was successfully generated and optimized through a combination of structural modeling and energy minimization techniques. Validation of the final model demonstrated high structural quality: 96.4% of residues were positioned within the favored regions of the Ramachandran plot, and no outliers were observed. Additional quality metrics further supported the model\\u0026apos;s reliability, including a low root mean square deviation (RMSD) of 0.24 \\u0026Aring;, a Z-score of \\u0026ndash;6.10, a MolProbity score of 0.91, and a Verify3D score of 93.81%. Taken together, these results indicate that the refined mutant structure is both stable and accurately folded, making it well-suited for subsequent structural and dynamic analysis.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eValidation Metrics for TP53 Structure\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eMetric\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eValue\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eRamachandran Favored (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003e96.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eMolProbity Score\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003e0.91\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eRamachandran Outliers (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003e0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eRMSD (\\u0026Aring;)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003e0.24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eZ-Score\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003e-6.0993\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eVerify3D Score (%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003e93.81\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.2 Analysis of Binding Sites Showed Pocket\\u0026ensp;Formation on Mutation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe \\u0026nbsp;binding site predictions were \\u0026nbsp;from seven independent tools and compiled them\\u0026ensp;to compare the wild-type (WT) and mutant TP53 structures. The wild-type protein showed randomly arranged and isolated pockets within this protein, with critical\\u0026ensp;residues like MET 246 and THR 140 distant (Pockets 6 and 1). The C238Y binding mode was not widely distributed among multiple clusters, as it showed one single consensus binding location (Pocket 0), centered at GLU 221 and flanked\\u0026ensp;by PRO 219, TYR 220 and PRO 222. All tools identified this\\u0026ensp;site, indicating consensus confidence and potential druggability. Structural differences: Panel a shows side-by-side pockets\\u0026ensp;(Figure 2) comparing pocket organization between both structures.\\u003c/p\\u003e\\n\\u003cp\\u003eA consensus binding site was defined as a region supported by predictions from \\u0026ge;3 independent servers\\u003c/p\\u003e\\n\\u003cp\\u003eTable 1. Binding Sites Predicted in Wild-Type TP53 by Each Server\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eServer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003ePredicted Binding Residues (Wild-Type TP53)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eFTMap\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eMET 246, ARG 174, SER 240, VAL 172\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eCryptoSite\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eARG 158, ARG 174, VAL 172, MET 246\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eDREAMM\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eA174, A209, A212, A225, A243, A248 (membrane-associated loop)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003ePocketMiner\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eVAL 147, ARG 174, GLU 180, MET 246\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003ePockDrug\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eARG 174, SER 240, MET 246, ARG 248\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eDoGSiteScorer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eVAL 172, MET 246, THR 140\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eP2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eMET 246, ARG 174, ARG 248, PHE 134\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTable 2. Binding Sites Predicted in C238Y Mutant TP53 by Each Server\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eServer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003ePredicted Binding Residues (Mutant C238Y TP53)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eFTMap\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221, PRO 219, TYR 220, MET 160\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eCryptoSite\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221, TYR 220, PRO 219, MET 160\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eDREAMM\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eA208, A209, A221, A225, A248 (loop flexibility/membrane residues)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003ePocketMiner\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221, TYR 220, MET 160, PRO 222\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003ePockDrug\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221, TYR 220, PRO 219, TYR 126\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eDoGSiteScorer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221, PRO 219, TYR 220\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eP2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 50%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221, TYR 220, PRO 222, MET 160\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTable 3. Consensus Binding Site \\u0026ndash; Wild-Type TP53\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eResidue\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePredicted By Servers\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eIncluded in Consensus?\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eMET 246\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFTMap, CryptoSite, PocketMiner, PockDrug, P2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eARG 174\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFTMap, CryptoSite, PockDrug, P2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eVAL 172\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFTMap, CryptoSite, DoGSiteScorer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eSER 240\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFTMap, PockDrug\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes (2 servers only)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePHE 134\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e❌\\u0026nbsp;No\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eTHR 140\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eDoGSiteScorer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e❌\\u0026nbsp;No\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTable 4. Consensus Binding Site \\u0026ndash; C238Y Mutant TP53\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eResidue\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePredicted By Servers\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eIncluded in Consensus?\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFTMap, CryptoSite, PocketMiner, PockDrug, DoGSiteScorer, P2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eTYR 220\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eCryptoSite, PocketMiner, PockDrug, P2Rank, DoGSiteScorer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePRO 219\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFTMap, CryptoSite, PockDrug, DoGSiteScorer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eMET 160\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFTMap, CryptoSite, PocketMiner, P2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePRO 222\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePocketMiner, P2Rank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e✅\\u0026nbsp;Yes (borderline)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eTYR 126\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePockDrug\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e❌\\u0026nbsp;No\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eHow the Consensus binding sites\\u0026ensp;Were \\u0026nbsp; Reached\\u003c/p\\u003e\\n\\u003cp\\u003eHow the Consensus\\u0026ensp;Was Reached\\u003c/p\\u003e\\n\\u003cp\\u003eA residue was considered to be part of the consensus if it was predicted by \\u0026ge;3 out\\u0026ensp;of the 7 tools.\\u003c/p\\u003e\\n\\u003cp\\u003eThe pocket is predominantly occupied by MET 246, ARG 174, and VAL 172, which sit\\u0026ensp;in the core DNA‐binding domain for wild type TP53[16]\\u003c/p\\u003e\\n\\u003cp\\u003eFor the mutant, the consensus rearranged toward a\\u0026ensp;remodeled, flexible loop centering on GLU 221 with support of dynamic analysis\\u003c/p\\u003e\"},{\"header\":\"Results and Structural Analysis: Insights into the C238Y Mutation\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e3.1 A Reliable mutant model\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;I wanted to make sure that my mutant model was as accurate as possible. Therefore, I carefully developed and validated a 3D structure\\u0026ensp;model of the TP53 protein with C238Y point mutation. The model passed all my quality checks : 96.4% of its amino acids sat in the most energetically favorable regions of the \\u0026ensp;Ramachandran plot, indicative of proper geometry. One of the most important validation step is the find how close my model was \\u0026nbsp;to the original model by comparing the RMSD of my model compared to original structure. \\u0026nbsp;A very low RMSD = 0.24 \\u0026Aring; for the \\u0026nbsp;model was obtained using matchmaker module alignmemt in UCSF Chimera molecular software. The Z-score of the model and MolProbity clash score were perfect .This confirmed a model that is stable and energetically sound so that \\u0026nbsp;the further analysis can be pursued.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.2 \\u0026ensp; Structural Remodeling of the Binding Pocket\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWhen I looked at the predicted drug-binding sites for the normal (wild-type) TP53 and the mutated TP53 there was a dramatic structural shift.. In \\u0026nbsp;the normal (wild-type) TP53 protein, potential binding sites were spread out across the surface as separate, unconnected areas. But in the C238Y mutant, this pattern shifted. These scattered regions came together to form a single, continuous pocket centered around the GLU 221 residue. This change seems to be caused by the mutation, which reshaped the surface of the protein and created a new space where a drug might be able to bind. \\u0026nbsp;The pocket also includes nearby residues like PRO 219, TYR 220, and PRO 222\\u0026mdash;consistently highlighted by all the prediction tools used in this study. This points to a clear \\u0026nbsp;structural change brought about by the mutation.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.3 Increased Flexibility: Creating Space for Drug Binding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProteins are not rigid\\u0026mdash;they constantly shift and move. \\u0026nbsp;molecular dynamics simulations showed that the area around GLU 221 in the C238Y mutant became much more flexible compared to the wild-type protein. This was \\u0026nbsp;noticeable in the loop between residues A208 and A209, which showed greater movement. Such flexibility may allow the newly formed pocket to briefly open, creating an opportunity for drug molecules to enter. In simpler terms, the mutation may act like a door that \\u0026nbsp;open just long enough for a potential drug to pass through\\u0026mdash;something that wouldn\\u0026rsquo;t be possible in the more stable, less flexible wild-type structure.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePocket Accessibility\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn the wild-type TP53 protein, hydrogen bonds help stabilize the region around GLU 221, keeping it tightly packed. However, in the C238Y mutant, those stabilizing bonds are no longer present. One clear example is the increased distance of 27.19A between TYR 238 (the mutated residue) and GLU 221, which shows that the mutation disrupts the original structural arrangement. Without these hydrogen \\u0026nbsp;bonds, the local space \\u0026nbsp;becomes \\u0026nbsp; more open. The mutation removes the original support holding the structure together, making room for a new pocket to form\\u0026mdash;one that wasn\\u0026rsquo;t previously accessible in the wild type tp53.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.5 Increased \\u0026nbsp;Surface Exposure: A Pocket Ready for Drugs\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo understand whether this new pocket might actually be reachable by ligands , I analyzed its solvent-accessible surface area (SASA). This measurement reveals how much of a protein\\u0026rsquo;s surface is exposed to its surrounding environment. When comparing the wild-type and mutant structures, I found that GLU 221\\u0026mdash;and its neighboring residues like PRO 219, TYR 220, and PRO 222\\u0026mdash;were much more exposed in the mutant. This increased exposure suggests that the mutation made the surface more open, which could allow small molecules o to access and bind the pocket more easily.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.6 A Larger \\u0026nbsp;Pocket: Improving Drug Binding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWhen comparing the size and shape of the binding pockets, the difference between the wild-type and the C238Y mutant TP53 was obvious. In the wild-type protein, potential pockets appeared as several small and scattered regions. In contrast, the C238Y mutation gave rise to a single, more spacious and continuous cavity. This new pocket wasn\\u0026rsquo;t just bigger in volume; it also acquired \\u0026nbsp;a higher druggability score, suggesting it is more likely to accommodate small-molecule . \\u0026nbsp;The mutation reshaped a fragmented and less accessible site into a well-defined target ideal for drug targeting.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTable 5. Comparative Binding Pocket Properties in TP53 Wild-Type vs. C238Y Mutant\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eFeature\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eWild-Type TP53 (2OCJ)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMutant TP53 (C238Y)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eConsensus Binding Residues\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePHE 134, THR 140, MET 246\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eGLU 221, TYR 220, PRO 219\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePocket ID(s)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_1 (THR 140, PHE 134), P_6 (MET 246)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePocket Volume (\\u0026Aring;\\u0026sup3;)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_1: 252.67; P_6: 133.25\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_0: 299.26\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eSurface Area (\\u0026Aring;\\u0026sup2;)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_1: 470.66; P_6: 190.09\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_0: 456.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eDrug Score\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_1: 0.48; P_6: 0.29\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eP_0: 0.53\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003ePocket Type\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eFragmented, shallow pockets\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eUnified, deep, flexible pocket\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eDruggability Implication\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eLower \\u0026mdash; scattered sites reduce ligand binding\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 33.3333%;\\\"\\u003e\\n \\u003cp\\u003eHigher \\u0026mdash; improves binding\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.7 Mechanistic Model\\u0026ensp;of Mutation-Driven Pocket Formation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBy incorporating structural and dynamic analysis , I proposed the following mechanistic\\u0026ensp;pathway:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026rarr; C238Y Mutation\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026rarr;\\u0026ensp;Differentiable Loop (RMD)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026rarr; Loss of Hydrogen Bonds\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026rarr; Increased SASA and Spatial Disconnection\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026rarr; \\u003cstrong\\u003eUnified Cryptic Pocket Formation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis cascade driven by mutational events explains the birth of a \\u0026nbsp;novel druggable pocket \\u0026nbsp;that is focused specifically on GLU\\u0026ensp;221\\u0026mdash;a finding that has not been reported in the literature with potentially important implications for drug development.\\u003c/p\\u003e\"},{\"header\":\"Analysis of Solvent Exposure and Secondary Structure\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e4.1 SASA (Solvent\\u0026ensp;Accessibility)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAnalysis of the structures using the GetArea webserver revealed greater solvent exposure at\\u0026ensp;the loop containing GLU 221 and the surrounding residues (C238Y mutant). This greater accessibility indicates that\\u0026ensp;the pocket has switched from a buried site \\u0026nbsp;to a more solvent-accessible target for ligand binding\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e4.2 PlayMolecule MD Simulation\\u0026ensp;Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRadius of Gyration (Rg):\\u003c/p\\u003e\\n\\u003cp\\u003eThe Rg values were stable in the\\u0026ensp;wild-type structure during the MD simulation suggesting the structural compactness. However the C238Y mutant showed fluctuations around global Rg reflecting overall flexibility and partial instability\\u0026ensp;of the mutant.\\u003c/p\\u003e\\n\\u003cp\\u003eSecondary\\u0026ensp;Structure Transitions:\\u003c/p\\u003e\\n\\u003cul type=\\\"disc\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eSecondary Structure Transitions:\\u003c/strong\\u003e\\u003cbr\\u003e\\u0026nbsp;DSSP-based secondary structure monitoring revealed a shift from helical to coil regions near the GLU 221 loop in the mutant structure. This change aligns with observations of increased loop dynamics and pocket plasticity.\\u003c/li\\u003e\\n\\u003c/ul\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e5.1 A Druggable Site In\\u0026ensp;TP53 Unlocked by Mutation\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe comparison of the crystal structures between the wild-type and C238Y mutant TP53 (Table 6) reveals an intriguing structural shift. Binding cavities in the wild-type spread shallowly and randomly, lacking strong crystal clustering. In the C238Y mutant, a stronger crystal cluster also formed, with previously dispersed pockets merging into a clearly defined single cavity. This transformation, \\u0026nbsp;opened \\u0026nbsp;a new binding site for drugs. Where the wild-type lacked such a pocket, the C238Y mutation introduced it, as noted in structural analysis. Residues like PHE 134 THR 140, and MET 246 dD within these pockets (P_1 and P_6) are reorganized. Changes occurred in the adjacent loops, specifically near GLU 221, resulting from the cysteine-to-tyrosine change at position 238. It generates a new pocket (P_0), comprising GLU 221, TYR 220, and PRO 219. It\\u0026apos;s a cryptic pocket that wasn\\u0026apos;t visible under normal conditions but emerges prominently when structural changes take place.\\u003c/p\\u003e\\n\\u003cp\\u003eThe generally accepted view that TP53 is undruggable is challenged by this observation. TP53\\u0026apos;s potential for drug interaction is not fixed. Mutation-driven remodeling can modify it. This has important implications for the developmentdrug \\u0026nbsp; process, offering new possibilities for therapeutic interventions. The traditional understanding of TP53\\u0026apos;s undruggable paradigm is longer valid, making these insights particularly transformative.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTogether with these visual checks, there were different quantitative shifts witnessed, and they were observed to coincide with drugs. The density of pockets P_1 and P_6 combined measured about 386 \\u0026Aring;\\u0026sup3;. The scores for druggability were low, 0.48 and 0.29. The mutant pocket P_0, smaller at 299 \\u0026Aring;\\u0026sup3;, had a higher score of 0.53. This means that pharmacological relevance, as well as structural organization, is improved by the C238Y mutation. A variety of quantitative shifts was seen, supporting this visual confirmation.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eMechanistically, this shift is\\u0026ensp;supported by several observations:\\u003c/p\\u003e\\n\\u003cp\\u003eThe mobility of the loop between residues 200 and 220 was notably elevated based on RMSF data. Solvent exposure of GLU 221 was higher, making it more likely that it was broken, thus allowing greater loop mobility. The observation regarding the separation distance between TYR 238 and GLU 221 indicates an increase in this distance in C238Y, allowing the geometry of a newly formed pocket. This evidence, when considered collectively, shows the mechanistic sequence: the C238Y mutation leads GLU 221 to reposition and reveal a hidden site. That\\u0026apos;s important. Furthermore, this sequence explains how a point mutation can modify local topology, providing an opportunity that could be targeted with drugs.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.1 Structuring\\u0026ensp;the Changes\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe direct observation of conformational and dynamic variations, as well as access to binding sites, has not been performed before this study. This is new territory. Structural changes due to the mutation affect the microenvironment around GLU 221, resulting in loosening stabilizing contacts and increased flexibility in conformation. These changes were found to create a surface pocket suitable for drug targeting, absent in the original protein and associated with potential therapeutic targets.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.2 Why Multiple Prediction Tools Were Used\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eRelying on a number of software tools to generate the results makes the process robust and reliable; these involved five end-user applications, such as CryptoSite and PocketMiner, employed as the standard cryptographic applications there are. The identification process was carried out by these tools efficiently, taking advantage of their unique capabilities. Efficiency was maintained in the investigation of binding sites.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEnergy-favorable binding hotspots were identified using\\u0026ensp;FTMap.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eDruggability scoring methodologies, which include those based on structural features like P2Rank and DoGSiteScorer, as well as machine learning approaches such as PockDrug, were used for clarity evaluation. Clarity was achieved with the use of DREAMM. The membrane-relevant exposure residues in TP53 were considered significant biologically. All tools, even with different methods, agreed. The result: the C238Y mutant exposes a new area. It\\u0026apos;s centered around GLU 221. The shared conclusion of various methodologies confirms its relevance, while the joint interpretation of a single method pointed to a singular insight: a ligand-accessible region, previously unexposed, has been revealed through mutant analysis. Despite different approaches, agreement was found. It showcases GLU 221 as a hub within the protein structure.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHow the\\u0026ensp;C238Y Mutation Restructs the Binding Surface\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe C238Y mutation substitutes a small cysteine with a larger tyrosine, creating steric pressure that forces nearby loops to shift their positions. This shift may move GLU 229 to a position that exposes it systematically. The C238Y mutation acts by exposing GLU 221 more effectively. RMSF, distance mapping, and SASA calculations align with these observed structural changes. \\u0026quot;A structurally new pocket results, and it\\u0026apos;s not just new\\u0026mdash;it\\u0026apos;s also suitable for drug targeting,\\u0026quot; Koasiakoff said. What emerges from this is a biologically important and druggable pocket.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.4 Drug Design Opportunities \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe cleft\\u0026apos;s location presents notable interest, and because it is not a static feature of the protein structure, this makes it even more intriguing; it opens only with allosteric targeting, where targeting takes place. The pocket\\u0026apos;s dynamic nature is due to the active shaping by protein movement. It offers ligands a chance for flexibility-dependent binding.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.5 Changing the Ways We Discover Drugs for\\u0026ensp;TP53\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe traditional belief that TP53 is universally nondruggable is questioned by this study. A different perspective is introduced. The current studies are critical for these features. Consequently, it is demonstrated that a cryptic pocket is associated with the C238Y mutant, which is not present in normal cells. Various tumors, such as uterine leiomyosarcoma and osteosarcoma, exhibit the mutation, so researchers think this structural information might have significant implications for translation. Wide translational implications could result. Since oncogenic mutations were seen to remodel surface proteins, a cell surface pocket was expected. A cryptic pocket was identified. This is associated with the C238Y mutant and is absent in the wild-type, showing the value of considering tumor-specific mutations when exploring therapeutic options. Therefore, this study calls into question the long-held belief of TP53 being universally nondruggable. Structural changes could be exploited for therapy developments, given the discoveries.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eOutlook and Future\\u0026ensp;Perspectives\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA new role for the TP53 C238Y mutation has been discovered, revealing a hidden binding pocket around residue L331. In this context, conformational data and measurement of spatial distances from various pocket prediction tools were used and combined. It has been proved that a series of conformational changes is triggered by this mutation. Loops destabilize, native hydrogen bonds are lost, and increased local flexibility occurs, exposing a site that could become therapeutic. This study questions the assumption that TP53 cannot be targeted therapeutically. Rather, it indicates that specific cancer-related mutations might alter the protein\\u0026apos;s surface, creating potential for treatment development. The finding holds particular relevance for drug targeting. The cryptic pocket identified here provides a mutation-specific chance for targeting with fragment-based or allosteric drug design swiftly. In contrast to traditional beliefs, certain mutations may open up new vistas on the TP53 protein surface, suggesting innovative approaches for drug discovery. A fragment-based approach can exploit these new binding possibilities. This observation is particularly noteworthy in a system where the C238Y mutation alters TP53. Suddenly, the presence of a pocket offers a new direction. The mutation-specific nature of this pocket could see it targeted by novel therapeutic strategies, challenging established ideas regarding TP53\\u0026apos;s previously thought limitations.There are several important paths to build on the findings of this study:\\u003c/p\\u003e\\n\\u003col start=\\\"1\\\" type=\\\"1\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eVirtual screening\\u003c/strong\\u003e of FDA-approved drugs and natural compounds should be conducted to uncover molecules that specifically interact with the pocket revealed by the C238Y mutation.\\u003c/li\\u003e\\n \\u003cli\\u003eThese candidate molecules should then be tested through \\u003cstrong\\u003emolecular dynamics simulations\\u003c/strong\\u003e to evaluate how stably they bind under conditions that mimic the cellular environment.\\u003c/li\\u003e\\n \\u003cli\\u003eFinally, \\u003cstrong\\u003eexperimental validation\\u003c/strong\\u003e using structural techniques such as NMR spectroscopy or X-ray crystallography\\u0026mdash;alongside functional testing in cancer cell lines carrying the TP53 C238Y mutation\\u0026mdash;will be essential to confirm their biological relevance.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003cp\\u003eThese steps can help translate this structural finding into fruitful \\u0026nbsp;therapeutic strategies. The findings reinforce a growing realization in the field: that druggability in proteins like TP53, often labelled \\u0026nbsp;as too disordered or flexible to target, may in fact be highly dependent on the specific mutations they carry. This flexibility \\u0026nbsp;may offer a unique opportunity for designing mutation-specific treatments\\u0026mdash;bringing us closer to personalized cancer therapies.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eI acknowledged \\u0026nbsp;the use of public bioinformatics platforms including CABS-flex, PlayMolecule, and the SAVES 6.0 server. No external funding was received for this study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of Interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe author declares no conflicts of interest.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eKandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, et al. 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Fragment-based identification of druggable \\u0026apos;hot spots\\u0026apos; of proteins using Fourier domain correlation techniques. \\u003cem\\u003eBioinformatics\\u003c/em\\u003e. 2009 Jan 15;25(5):621\\u0026ndash;7. doi:10.1093/bioinformatics/btn023.\\u003c/li\\u003e\\n \\u003cli\\u003eCimermancic P, Weinkam P, Rettenmaier TJ, Bichmann L, Keedy DA, Woldeyes RA, Schneidman-Duhovny D, Demerdash ON, Mitchell JC, Wells JA, Fraser JS, Sali A. CryptoSite: Expanding the druggable proteome by characterization and prediction of cryptic binding sites. \\u003cem\\u003eJ Mol Biol\\u003c/em\\u003e. 2016 Apr 3;428(4):709\\u0026ndash;19. doi:10.1016/j.jmb.2016.01.029.\\u003c/li\\u003e\\n \\u003cli\\u003eAldeghi M, Gapsys V, de Groot BL. 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When TP53 can't do its job, cells start multiplying without control, and the cell's genetic material becomes unstable. Even though TP53 has long been known to be a key player in cancer it's been very difficult to develop drugs that target it. This is largely because of its flexible structure and the lack of clear binding sites for drugs. But, recent studies indicate that specific mutations can cause structural changes in TP53, creating new potential binding sites that could be useful for drug development. In this study, I used computer modeling and structural biological analysis to examine the c238y tp53 mutation . The results \\u0026nbsp;showed \\u0026nbsp;that this mutation dramatically reshapes the protein in the vicinity — it exposes a hidden pocket that could be a promising target for drugs. These results pave the way to conceptualising and designing therapies that are mutationally specific with the end goal being to disrupt or restore the default function of malfunctioning TP53 in cancer.\\u003c/p\\u003e\\n\\u003cp\\u003eThis structural study lays the foundation for a follow-up phase involving virtual screening and drug-binding validation targeting the revealed cryptic pocket.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Structure-Based Discovery of a Cryptic Druggable Pocket in TP53 C238Y: Implications for Targeted Therapy\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-04 03:42:30\",\"doi\":\"10.21203/rs.3.rs-6370188/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"2b2a2334-4646-4539-8043-e5062e240bd3\",\"owner\":[],\"postedDate\":\"April 4th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":46644683,\"name\":\"Computational Biology\"}],\"tags\":[],\"updatedAt\":\"2025-04-04T03:42:30+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-04-04 03:42:30\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6370188\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6370188\",\"identity\":\"rs-6370188\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}