In silico functional and structural characterization of Streptococcus pneumoniae atypical Rib domain-containing hypothetical protein unravels conserved immunogenic epitopes

In silico functional and structural characterization of Streptococcus pneumoniae atypical Rib domain-containing hypothetical protein unravels conserved immunogenic epitopes | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 21 July 2025 V1 Latest version Share on In silico functional and structural characterization of Streptococcus pneumoniae atypical Rib domain-containing hypothetical protein unravels conserved immunogenic epitopes Authors : Stephen Kyle C. Arcan 0009-0004-1174-6198 [email protected] , Gyraldin Marietony D. Gan , Debrah Jannsen N. Almazan , Zairus N. Duquilla , Azenith Vincent D. Barbosa , and Rainier Ulrich D. Velasco Authors Info & Affiliations https://doi.org/10.22541/au.175313166.61464735/v1 Published Proteins: Structure, Function, and Bioinformatics Version of record Peer review timeline 341 views 207 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Streptococcus pneumoniae is a high-mortality pathogen with broad-spectrum antibiotic resistance, necessitating the development of alternative therapies, such as antigenic protein-based vaccines, which have recently gained interest due to their novelty. Here, we characterized antigenic hypothetical proteins (HPs) of S. pneumoniae and determined their potential as vaccine construct targets. Subcellular localization reported 10 extracellular proteins, six of which were antigenic and non-allergenic, thus making them ideal vaccine construct targets. Functional annotation through conserved protein domain and motif prediction identified a unique, atypical Rib (aRib) domain from WP_001166178.1, widely distributed on bacterial cell surface proteins. A comparison with a native Rib domain showed domain atrophy, highlighting the lack of structural core elements. Further analysis revealed non-covalent interactions of Thr47, Ala48, Val41, and Phe38 interacting with an alpha-D-mannopyranose ligand, triggering S. pneumoniae colonization and capsule synthesis mechanism, with highly dynamic and flexible residues present on the ligand binding site. A robust immune response was observed from an immune response simulation, likely attributed to the presence of predicted 4 cytotoxic T lymphocyte (CTL), 10 helper T lymphocyte (HTL), and 5 B-cell lymphocyte (BCL) epitopes. Therefore, the study presents a novel protein for designing a vaccine construct against S. pneumoniae , thus offering a new target for future vaccinology studies In silico functional and structural characterization of Streptococcus pneumoniae atypical Rib domain-containing hypothetical protein unravels conserved immunogenic epitopes Running title: Immunogenic epitopes in S. pneumoniae aRib protein Stephen Kyle C. Arcan*, Gyraldin Marietony D. Gan, Debrah Jannsen N. Almazan, Zairus N. Duquilla, Azenith Vincent D. Barbosa, Rainier Ulrich D. Velasco Department of Biological Sciences, College of Science, Pamantasan ng Lungsod ng Maynila (University of the City of Manila), General Luna, corner Muralla St, Intramuros, Manila, 1002 Metro Manila, Philippines *Corresponding author: [email protected] Co-authors email address: [email protected] ; [email protected] ; [email protected] ; [email protected] ; [email protected] Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of Interest Statement No potential conflict of interest relevant to this article was reported. Funding information This study was not supported by any sponsor or funder. Ethics Statement No experiments involving animals were conducted in this study. Author’s Contributions SKCA - Conceptualization, Writing (Original Draft and Review/Editing), Project Administration, Methodology, Data Curation, Formal Analysis, Visualization, GMDG - Conceptualization, Writing (Original Draft and Review/Editing), Data Curation, Formal Analysis, Visualization, DJNA - Data Curation, Formal Analysis, Writing (Original Draft), ZND - Data Curation, Formal Analysis, Writing (Original Draft), AVDB - Data Curation, Formal Analysis, RUDV - Supervision, Writing (Review/Editing) ABSTRACT Streptococcus pneumoniae is a high-mortality pathogen with broad-spectrum antibiotic resistance, necessitating the development of alternative therapies, such as antigenic protein-based vaccines, which have recently gained interest due to their novelty. Here, we characterized antigenic hypothetical proteins (HPs) of S. pneumoniae and determined their potential as vaccine construct targets. Subcellular localization reported 10 extracellular proteins, six of which were antigenic and non-allergenic, thus making them ideal vaccine construct targets. Functional annotation through conserved protein domain and motif prediction identified a unique, atypical Rib (aRib) domain from WP_001166178.1, widely distributed on bacterial cell surface proteins. A comparison with a native Rib domain showed domain atrophy, highlighting the lack of structural core elements. Further analysis revealed non-covalent interactions of Thr47, Ala48, Val41, and Phe38 interacting with an alpha-D-mannopyranose ligand, triggering S. pneumoniae colonization and capsule synthesis mechanism, with highly dynamic and flexible residues present on the ligand binding site. A robust immune response was observed from an immune response simulation, likely attributed to the presence of predicted 4 cytotoxic T lymphocyte (CTL), 10 helper T lymphocyte (HTL), and 5 B-cell lymphocyte (BCL) epitopes. Therefore, the study presents a novel protein for designing a vaccine construct against S. pneumoniae , thus offering a new target for future vaccinology studies Keywords: Streptococcus pneumoniae , epitope, vaccinology, protein domain, Atypical Rib domain INTRODUCTION Streptococcus pneumoniae is a Gram-negative pathogen capable of causing diseases such as sinusitis, otitis media, pneumonia, sepsis, and meningitis when it invades beyond its commensal niche 1 . The 2016 Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study identified it as the leading cause of pneumonia infections and deaths worldwide, responsible for 197.05 million episodes and over 1.1 million deaths across all age groups 2 . The study also revealed that morbidity and mortality rates were highest among children under 5 years (44.69 million episodes; 340,000 deaths) and the elderly (29.43 million episodes; 490,000 deaths) 2 . Beyond pneumonia, S. pneumoniae is the primary cause of meningitis and sepsis in children, with most fatalities occurring in developing countries across Africa and Asia 3 . While pneumococcal disease was traditionally treated with antimicrobials, the rise of antibiotic- and multidrug-resistant strains has shifted the focus to vaccination as the most effective preventive strategy, particularly for high-risk groups like children and the elderly. Currently, two pneumococcal conjugate vaccines (PCVs)—Prevnar 13 and Synflorix (10-valent)—are licensed for use 4 . Although these vaccines are effective, their protection is limited to the specific serotypes they contain, leaving no defense against the 90+ non-vaccine serotypes now emerging. This poses a significant challenge, not only in high-income countries but especially in developing regions, where a wider diversity of serotypes contributes to disease burden. Hypothetical proteins (HPs) are sequences predicted to be present within an organism’s genetic material but with little to no evidence of their function. These proteins are often determined by the presence of open reading frames (ORFs), but such a protein product lacks homologous structure/function in the databases. Characterizing HPs can be done using modified in silico pipelines utilizing both structural and functional annotation 5,6 . Recent studies are exploring the potential of HPs for alternative therapeutic approaches, therefore expanding the knowledge on their infection mechanisms and identifying potential vaccine candidates and drug targets. A previous study identified a novel drug target from Klebsiella pneumoniae hypothetical proteins, alleviating the drug evasion mechanism. Another study extensively annotated the functions of several hypothetical proteins from Mycobacterium tuberculosis , identifying twelve proteins playing a significant role in its pathogenicity, with ten of which as promising vaccine candidates 7,8 . We employed an in silico pipeline to functionally and structurally characterize hypothetical proteins (HPs) from S. pneumoniae SMRU183 and identify potential vaccine targets by predicting conserved immunogenic epitopes. Extracellular HPs that were antigenic, non-allergenic, and non-toxic were selected from the database as promising candidates. Additionally, multiple conserved epitopes capable of inducing immune responses—including those from cytotoxic T-lymphocytes (CTLs), helper T-lymphocytes (HTLs), and B-lymphocytes—were identified in HP with known conserved domains and motifs. This study addresses the current knowledge gap regarding uncharacterized pathogenic proteins, suggesting their potential metabolic roles and thereby proposing novel therapeutic targets relevant to pneumococcal disease. MATERIALS & METHODS 2.1. Sequence retrieval and subcellular localization Hypothetical proteins from S. pneumoniae strain SMRU183 (Accession Number: NZ_CKMT01000004.1) were manually curated from the National Center for Biotechnology Information (NCBI) Genome browser. Protein sequences were thereafter submitted to the PSORTb v3.0.3 (https://psort.org/psortb/) web server to predict their respective subcellular localization 9 . 2.2. Antigenicity and allergenicity evaluation Extracellular HPs were then subjected to antigenicity screening using the VaxiJen v2.0 web server (with a default threshold value of 0.4 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) 10 . HPs reported as antigenic were further screened based on their allergenicity using the AllerTOP v2.0 web server (http://www.ddg-pharmfac.net/AllerTOP/) 11 . Antigenic and non-allergenic HPs were utilized in subsequent analyses. 2.3. Functional annotation 2.3.1. Conserved protein domain and motif prediction The HPs were functionally annotated according to their structural and architectural motifs, which consequently describe their roles in bacterial growth, metabolism, and/or pathogenicity, using InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/). Conserved domains and motifs were predicted using NCBI Conserved Domain Search (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) 8,12 and the Motif Search web server (https://www.genome.jp/tools/motif/MOTIF.html), respectively. 2.3.2. Physicochemical characterization Physicochemical properties of the shortlisted HPs, their physicochemical properties, such as predicted molecular weight (MW), theoretical isoelectric point (pI), estimated half-life, aliphatic and instability index (II), and the grand average of hydropathicity (GRAVY), among others, were determined using the Expasy ProtParam tool (https://web.expasy.org/protparam/) 13 . 2.3.3. Binding pocket prediction Prediction of amino acid residues potentially involved in the protein-ligand interaction of the HPs was performed using the Computed Atlas of Surface Topography of the Universe of Protein Fold (CASTpFold) server (https://cfold.bme.uic.edu/castpfold/compute) and COACH (https://zhanggroup.org/COACH/) 14 . A molecular docking simulation was also performed using AutoDock Tools version 1.5.7 software developed by the Molecular Graphics Laboratory (MGL) of The Scripps Research Institute to determine residue-atom interactions 15 . 2.4. Structural characterization 2.4.1. Protein structure modeling and refinement The 3D models of the HPs were predicted using ColabFold v1.5.5 from AlphaFold 2.0. Protein structure quality was subsequently assessed using ERRAT and PROCHECK from the SAVES tool (https://saves.mbi.ucla.edu/). The structure refinement of the models was performed using the Galaxy Refine server (https://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE) 2.4.2. Normal mode analysis-based molecular dynamics simulations A molecular dynamics simulation based on normal-mode analysis (NMA) was performed to map significant conformational changes of the HPs under simulated physiological conditions, which gives insights into its predicted stability and flexibility. Here, the iMods server (https://imods.iqf.csic.es/) was used to analyze protein dynamics based on NMA mobility, deformability, B-factor, eigenvalue, variance, covariance map, and elastic network 16 . 2.5. Epitope prediction and immune response simulation 2.5.1. Cytotoxic T lymphocyte (CTL) epitope prediction NetCTL 1.2 web server was employed to predict CTL epitopes (https://services.healthtech.dtu.dk/services/NetCTL-1.2/) for supertypes A2, A3, and B7 which cover >88% of the global population. The parameters for prediction on the web server were set to default, with a weight on C-terminal cleavage as 0.15, a weight on TAP transport efficiency as 0.05, and an epitope identification threshold of 0.75 17 . Predicted epitopes were further evaluated based on their antigenicity using VaxiJen v2.0, allergenicity using AllerTOP v2.0, and toxicity using ToxinPred (https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php). Final epitopes were denoted as antigenic, non-allergenic, and non-toxic. 2.5.2. Helper T lymphocyte (HTL) epitope prediction NetMHCII 2.3 web server was utilized in predicting HTL epitopes (https://services.healthtech.dtu.dk/services/NetMHCII-2.3/) using the 30 reference alleles. Default settings were observed, such as the threshold value of -99.9, a stronger binder % rank threshold of 2, and a weaker binder % rank threshold of 10. Predicted strong binding (“SB”) epitopes entail high binders and were further shortlisted according to their antigenicity, allergenicity, and toxicity 18 . 2.5.3. B cell lymphocyte (BCL) epitope prediction BepiPred-2.0 web server (https://services.healthtech.dtu.dk/services/BepiPred-2.0/) was used for linear BCL epitope prediction. The predicted epitopes above the 50% threshold value were selected, and their structural conformation (coiled or helix) and proximity to the surface (buried or exposed) were documented. The number of predicted linear B-cell epitopes should be adequate and enough to stimulate the humoral immune responses of the host 19,20 . Similarly, predicted epitopes were further characterized based on their antigenicity, allergenicity, and toxicity. 2.5.4. Epitope conservancy analysis Antigenic, non-allergenic, and non-toxic epitopes were further analyzed for their conservancy across different strains of S. pneumoniae . Here, the Epitope Conservancy Tool from Immune Epitope Database (IEDB) Analysis Resource (http://tools.iedb.org/conservancy/). We obtained 45 S. pneumoniae sequences containing the reported conserved domain from NCBI as the reference. 2.5.5 Immune response simulation An immune response simulation was carried out to model the predicted cellular and humoral response against the vaccine candidate. We used the C-Imm-Sim web server (https://150.146.2.1/C-IMMSIM/index.php?page=1), setting a two-step injection with 30 simulation days in between. Other parameters were left as default. RESULTS AND DISCUSSION 3.1. Several antigenic hypothetical proteins were identified from the Streptococcus pneumoniae proteome Proteome mining of S. pneumoniae SMRU183 revealed 1,965 proteins, 228 of which are denoted as hypothetical. Ten (4.4%) out of 228 hypothetical proteins (Supplementary Table 1) were reported as extracellular and were used for subsequent analyses. Extracellular and cell surface proteins are favored as vaccine design targets due to their direct participation in host-pathogen interaction and contact with immune cell receptors, inducing robust responses 21 . Extracellular hypothetical proteins were characterized using web servers VaxiJen v2.0, and Allertop 2.0 to predict their antigenicity and allergenicity, respectively. This ensures their ability to induce potent immunological pathways and functionality while avoiding autoimmune responses and allergic reactions within their hosts 22,23 . The screening reported 6 out of 10 highly immunogenic hypothetical proteins (Table 1). The reported antigenicity and non-allergenicity of the hypothetical proteins were predicted to stimulate viable adaptive immune responses attributed to the presence of epitopes, a region of interaction between the antigen and the antibodies and other immune cells 24,25 . 3.2. Functional annotation of the antigenic hypothetical proteins revealed a unique protein domain Structural domain prediction through the NCBI Conserved Domain Search Tool (NCBI-CDD) and InterPro reported no significant results on hypothetical proteins WP_000521619.1, WP_001808548.1, WP_044812623.1, WP_224757147.1, and WP_224757285.1. Larger proteins tend to exhibit more complex conformation, allowing them to fold into functional domains. Generally, relatively short amino acids would still yield a functional domain since the size ranges from 50 to 200 residues; however, given the length of their secondary structure, the complexity of their conformation would be insufficient, and their function as a conserved protein domain would be limited 26 . Su and colleagues (2013) reported that small proteins of less than 100 residues in length would still function and participate in biological processes, including but not limited to cellular metabolism, regulation, interaction, and signalling 27 . Moreover, further analysis revealed that the 199-residue hypothetical protein WP_001166178.1 yields an atypical Rib (aRib) domain on positions 125-198 (Figure 1). An aRib domain (Pfam family: PF18938) is a unique subdomain whose topology is similar to the Rib domain and is characterized by the small three antiparallel beta-sheets at the C-terminus end of the protein and a long alpha helix, replacing the short helical turn of the Rib domain, with its extended loop covering the missing space that results from the smaller β-sheet in aRib at the N-terminus end 28 . Additionally, this domain frequently occurs in tandem repeats and is often linked with other cell surface domains like DUF1542, FIVAR, and GA domains. The Rib (resistance to protease, immunity, group B) protein domain is commonly expressed on group B Streptococcus (GBS) cell surfaces. This protein is a member of the alpha-like proteins of GBS family, which also includes alpha C and has a similar overall structure, including related N termini with a series of identical Rib domains 29 . Whelan et al. (2019) described the Rib protein structure with two distinct antiparallel beta-sheets on its C-terminus end and central structures composed of four parallel beta-sheets. They also mentioned that Rib proteins are a good example of domain atrophy, where a significant number of core structural elements in their protein structure are missing. Missing beta-sheets forming an immunoglobulin (Ig) fold were reported from a structural comparison of Rib proteins from the He_PIG family (Pfam famility: PF05345) 30 . Further characterization of WP_001166178.1 and its probable catalytic activities, its active site, putative ligands, and amino acid residues were predicted using CASTpFold and COACH web servers. Other HPs were not subjected to such analyses due to their lack of a functional domain. The Computed Atlas of Surface Topography of the Universe of Protein Fold (CASTpFold) server identifies topographic features, area, and volume, and computes imprints using the alpha shape method developed in computational geometry 31 . CASTpFold predicted a binding cleft with 304.471 solvent-accessible surface area and 168.119 solvent-accessible surface volume from the non-aRib domain of WP_001166178.1, with the reported amino acid residues summarized in Supplementary Table 2. COACH server also reported that WP_001166178.1 can recognize alpha-D-mannopyranose as a ligand. To validate this, we performed a molecular docking simulation with a 100-Lamarckian Genetic Algorithm using AutoDock4. Even though we had prior information on the probable binding cleft of the protein, a blind docking simulation was still carried out to confirm our previous findings. Our simulation suggests high-affinity interactions between WP_001166178.1 and alpha-D-mannopyranose, with the lowest Gibbs free energy reported being -9.14 kcal/mol. The top ligand cluster is composed of 54 ligand conformations whose proximity was reported on the predicted binding cleft. The residue-atom interactions of the complex with the lowest reported Gibbs free energy, which was also part of the 54-membered ligand cluster, were further elucidated in Figure 2 where it shows that alpha-D-mannopyranose forms a conventional hydrogen bond, with Thr47, Ala48, Val41, and Phe38, which were also predicted to be present in binding cleft (Supplementary Table 2), through its oxygen atoms. These types of molecular interactions yield an enhanced protein-ligand stability, paving the way for activating antagonistic or agonistic effects on its downstream pathways 32 . Alpha-D-mannopyranose or alpha-D-mannose is an alpha configuration of D-mannopyranose, which has a crucial role as a product of cellular activity, pathways, and metabolism. Bacterial colonization and invasion can be aided by mannose-specific attachment. This prediction suggests the potential contribution of alpha-d-mannopyranose ligand in the virulence of S. pneumoniae through colonization. Zhi et al. (2018) reported that short hydrophobic peptides (Shp) induce Rgg144/Shp144 and Rgg939/Shp939 QS systems in response to sugars found in the respiratory tract, such as galactose and mannose 33 . 3.3. The HP, WP_001166178.1, exhibits ideal physicochemical and stereochemical properties for protein expression, purification, and in vivo validation Various physicochemical properties were predicted using the Expasy ProtParam web server to assess the suitability of WP_001166178.1 as a vaccine candidate. The analysis revealed that the 199-residue protein has a predicted molecular weight of 21.5 kDa and a GRAVY value of -0.627, indicating its hydrophilic nature. Dey et al. (2022) reported that proteins smaller than 110 kDa are suitable for vaccine development, as they can be easily purified using protein chromatography platforms 22 . This HP also contains 23 negatively and 21 positively charged amino acid residues, contributing to its theoretical isoelectric point (pI: 6.09). Its aliphatic index, a measure of thermostability based on aliphatic amino acid content, was predicted to be 73.52, suggesting good thermal stability. Additionally, it exhibits an ideal half-life in both in vivo and in vitro systems: 30 hours in mammalian reticulocytes, >20 hours in yeast, and Since ideal vaccine candidates require stable physicochemical properties and high solubility 34 , these findings indicate that WP_001166178.1 is likely to be stably expressed in both in vitro and in vivo expression systems and can be easily purified due to its favorable predicted properties. 3.4. Structural characterization reveals loss of core beta-sheets in atypical Rib protein and highly dynamic protein regions The 3D structure model of the hypothetical protein (HP) WP_001166178.1, was generated using AlphaFold and compared it with known Rib protein crystal structures. The pdb100 database served as the template reference, ranking models based on the highest average per-residue local distance difference test (pLDDT) scores, followed by relaxation to optimize local geometry. The pLDDT and predicted IDDT (pIDDT) analyses revealed high to very high confidence in the aRib domain, but low to very low confidence in regions lacking significant domain hits (Figures 3B-C). This discrepancy likely stems from the absence of high-resolution templates in databases, leading to suboptimal secondary structure folding 35 . ERRAT yielded a quality factor of 85.88% for WP_001166178.1, suggesting localized errors in protein folding (Figure 4A). ERRAT is a protein structure validation tool that assesses predicted models by comparing non-bonded atomic interactions against statistics from highly refined crystallographic structures, where scores >90% indicate a well-refined structure 36 . Moreover, PROCHECK evaluates the stereochemical quality of protein structures by analyzing residue geometry, dihedral angles (ψ and φ), chirality, and non-bonded interactions. The Ramachandran plot from PROCHECK revealed that 94.4% (167/177) of non-glycine/non-proline residues occupy the most favored regions, while 5.1% and 0.6% fall into additionally allowed and disallowed regions, respectively (Figure 4B). No residues were observed in generously allowed regions. Although structures with high-quality 37 , minor deviations may arise from factors such as topology errors, overinterpretation, or insufficient quaternary conformation 38 . Subjecting the model to GalaxyRefine significantly improved the structural quality where it achieved an ERRAT score of 99.459%, indicating excellent atomic-level accuracy. PROCHECK analysis also showed enhanced stereochemical properties, with 95.5% of non-glycine/non-proline residues in the most favored regions of the Ramachandran plot and 4.5% in additionally allowed regions. Notably, no residues were observed in generously allowed or disallowed regions. Given its high quality, this refined model was selected as the receptor for molecular docking simulations. A comparative analysis of the secondary structures in the Rib and atypical Rib (aRib) proteins was performed (Figure 3D). The Streptococcus pyogenes Rib domain (PDB: 6S5Z) adopts a classic Ig-like fold, characterized by four beta-sheets arranged in a β-sandwich configuration. In contrast, the aRib domain retains only three beta-sheets and features a prominent long alpha helix—a defining structural hallmark of its atypical conformation. This divergence suggests evolutionary loss of core beta-sheets from the ancestral Ig-like fold, enabling structural diversification into the observed aRib architecture 30 . Normal mode analysis (NMA) revealed key dynamic properties of WP_001166178.1, with distinct patterns of flexibility and stability across its structure. The N-terminal region exhibited high deformability, corresponding to its uncharacterized domain and likely representing an intrinsically disordered region (Figure 5A). Such structural deformation often facilitates ligand binding through induced-fit conformational changes, consistent with our earlier predictions of binding clefts and docking simulations. In contrast, the C-terminal domain showed limited deformation, reflecting its stable structural core 39 . Thermodynamic analysis of Cα B-factors confirmed the overall stability of the protein fold. Notably, the first principal mode (λ₁ = 3.71×10⁻⁵) dominated the observed dynamics, accounting for 80% of the variance and representing large-scale functional motions. The rapid eigenvalue decay (modes 2-20 contributing <20% combined variance) indicated that higher-frequency modes corresponded primarily to localized fluctuations rather than biologically relevant motions (Figure 5B) 40 . Cross-correlation matrices revealed coordinated residue motions, suggesting potential allosteric networks, while deformability mapping aligned precisely with regions of low confidence (pLDDT <70) in the predicted structure (Figures 5C-D). These dynamic features highlight mechanistically important regions for functional interactions, explain the structural basis for observed binding properties, identify key targets for experimental validation of the proposed dynamic model, and integrate our previous structural and docking analyses, providing a comprehensive framework for understanding WP_001166178.1’s functional architecture and its potential as a therapeutic target 41 . 3.5. WP_001166178.1 elicits a robust immune response attributed to conserved immunogenic epitopes, implying its potential as a vaccine construct target Epitopes are immunogenic oligopeptides or antigenic regions capable of binding directly to immune cell receptors. Recently, epitope-based vaccines have gained significant attention in vaccine research due to their potential to elicit targeted immune responses. Ahmad et al. (2016) demonstrated that epitope-based vaccines can effectively induce both prophylactic and therapeutic effects by triggering pathogen-specific immunity. This approach offers multiple advantages, including precise immune targeting, resource efficiency, and the ability to induce long-lasting immunity while minimizing unwanted immune reactions 42 . Using immunoinformatic tools, we predicted potential epitopes for cytotoxic T lymphocytes (CTL), helper T lymphocytes (HTL), and B lymphocytes (BCL). Initial screening with NetCTL 1.2 identified 39 CTL epitopes from six antigenic hypothetical proteins, while NetMHCII 2.3 predicted 183 HTL epitopes. BepiPred-2.0 analysis revealed 18 BCL epitopes. These candidate epitopes were subsequently evaluated for antigenicity and allergenicity to ensure optimal immunogenicity and safety. Following rigorous screening, we identified 12 high-confidence CTL epitopes, 51 HTL epitopes, and 11 BCL epitopes. Notably, WP_001166178.1 yielded four CTL, 10 HTL, and five BCL epitopes (Tables 2-4). This protein was particularly significant as it contained a conserved domain—a crucial feature for computational vaccine design 43 . Among all hypothetical proteins analyzed, only WP_001166178.1 possessed this conserved domain, underscoring its potential as a prime vaccine candidate. Furthermore, our analysis of epitope conservation across 45 Streptococcus pneumoniae strains (Supplementary Table 3) revealed variable conservation rates, with some immune targets remaining stable across strains—making them ideal candidates for broad-spectrum protection—while others exhibited frequent mutations, suggesting immune evasion mechanisms. For vaccine development, this dichotomy presents two strategic approaches: (1) targeting conserved epitopes to induce cross-reactive immunity or (2) incorporating variable epitopes in updated formulations to address strain-specific variations. A balanced integration of both strategies could enhance durable and adaptable protection 44,45 . Future studies may employ either a multi-epitope vaccine approach, given the wide range of immunogenic and conserved epitopes with varying rates of conservation, or a subunit vaccine design. The former depends on the rational construction of the authors and will be highly dependent on the permutation of selected epitopes. The latter, however, is a more direct application since the entire protein construct will be administered. To simulate and model the potential immune response of such an approach, we utilized the C-Imm-Sim server. We employed a two-step injection with 30 simulation days apart. The first-step injection elicited an antibody response peaking at 50,000 (arbitrary titer units) 12-days post-injection, while the secondary dose induced a robust recall response, with titers reaching 1.6 million 10 days post-injection and significant antigen clearance mediated by IgM and IgG (Figure 6A). IgM is the antibody primarily involved in the primary immune response and can trigger antigen clearance mechanisms through the complement pathway. IgG, on the other hand, is the major antibody involved in the secondary immune response and accounts for the majority (70-75%) of the antibody profiled in a human serum 46 . Moreover, we observed a similar robust secondary response with helper T-cell and B-cell populations 5- and 3-days post-injection during the second step, respectively (Figures 6B-C). An increased cytokine response was also reported during the simulation (Figure 6D). B cells and helper T (Th) cells play synergistic roles in protective immunity against bacterial infections. B cells mediate humoral immunity through antibody production, which opsonizes pathogens for phagocytosis or neutralizes bacterial toxins 46 . In our vaccine simulation, the rapid B-cell expansion (peaking 3 days post-injection) correlates with the observed surge in antibody titers (Figure 6A–C), mirroring real-world responses where B-cell activation is critical for long-term antibacterial defense 47 . Concurrently, Th cells (peaking at 5 days) provide essential cytokine signals, such as interleukins and IFN-γ, to direct B-cell class switching and optimize antibody efficacy against bacterial antigens 48 . The robust cytokine elevation (Figure 6D) further underscores Th-cell involvement in coordinating both humoral and cellular arms of immunity. In vaccine design, this interplay ensures durable memory responses, with Th cells enhancing B-cell affinity maturation—a key feature for combating extracellular bacteria like S. pneumoniae 49 . CONCLUSION This study employed an in silico pipeline to identify and characterize antigenic hypothetical proteins from S. pneumoniae with vaccine potential. Proteome mining revealed extracellular hypothetical proteins exhibiting antigenicity, non-allergenicity, and non-toxicity, which were reported to be key traits for vaccine candidates. Notably, functional annotation uncovered an atypical Rib domain from the hypothetical protein, WP_001166178.1, an example of Rib domain atrophy, characterized by the loss of core beta-sheets, indicative of domain atrophy. Biophysical analyses confirmed favorable physicochemical and stereochemical properties, including high flexibility in key residues, suggesting suitability for recombinant expression and purification. Epitope prediction further identified conserved, immunogenic regions capable of eliciting robust responses, as corroborated by immune simulation showing potent humoral and cellular activation. Our findings propose the atypical Rib domain as a promising subunit vaccine candidate against S. pneumoniae . Future work should prioritize in vitro and in vivo validation, including immunological assays to confirm protective efficacy. References 1. Whiley RA, Hardie JM. Streptococcus . In: Whitman WB, Rainey F, Kämpfer P, et al., eds. Bergey’s Manual of Systematics of Archaea and Bacteria . 1st ed. Wiley; 2015:1-86. Accessed March 9, 2023. https://onlinelibrary.wiley.com/doi/10.1002/9781118960608.gbm00612 2. Troeger C, Blacker B, Khalil IA, et al. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. 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Antigenicity and allergenicity of the extracellular hypothetical proteins Accession Number Length Antigenicity Score Allergenicity WP_224757285.1 99 1.0739 Antigen Non-allergen WP_000521619.1 46 0.8567 Antigen Non-allergen WP_001166178.1 199 0.815 Antigen Non-allergen WP_224757147.1 76 0.5756 Antigen Non-allergen WP_000807141.1 45 0.5686 Antigen Allergen WP_044812623.1 62 0.4787 Antigen Non-allergen WP_000807140.1 45 0.473 Antigen Allergen WP_001808548.1 52 0.4385 Antigen Non-allergen WP_000410533.1 49 0.3087 Non-antigen - WP_219300018.1 20 -0.3745 Non-antigen - Table 2. Predicted antigenic, non-allergenic, and non-toxic CTL Epitopes from WP_001166178.1 Alleles Peptide sequence MHC binding affinity Antigenicity score Conservancy HLA-A*0201, HLA A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0214, HLA-A*02:17, HLA-A*6802, HLA-A*69:01 ILCYFTITV 0.7174 1.3763 53.33% HLA-A*0201, HLA A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0214, HLA-A*02:17, HLA-A*6802, HLA-A*69:01 MQILCYFTI 0.616 1.4437 53.33% HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*3301, HLA-A*3303, HLA-A*6601, HLA-A*6801, HLA-A*7401 VSSSSQTAK 0.5677 1.6845 62.22% HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*3301, HLA-A*3303, HLA-A*6601, HLA-A*6801, HLA-A*7401 KIVSAKPTY 0.295 1.1221 53.33% Value for MHC binding affinity is 1-log 50K (aff); affinity (aff) in nM units not provided by NetCTL 1.2, Table 3. Predicted antigenic, non-allergenic, and non-toxic HTL epitopes of WP_001166178.1 Alleles Peptide sequence Prediction score Antigenicity score Conservancy DRB1*0401; DRB1*0405 SGHNFSSVSSSSQTA 0.6937 1.0314 26.67% DRB1*0401; DRB1*0405 GSGHNFSSVSSSSQT 0.6612 1.3438 26.67% DRB1*0802 CYFTITVVAKPNNSG 0.6064 0.5194 48.89% DRB1*0802 YFTITVVAKPNNSGE 0.6001 0.609 48.89% DRB1*0802 VKIVSAKPTYPDKIL 0.5943 0.5702 53.33% DRB1*0802 FTITVVAKPNNSGEV 0.5806 0.6244 48.89% DRB1*1201 DATALLKLKLNNVDN 0.6124 0.5906 46.67% DRB3*0202 EETGYNNNSSLYTT 0.7203 0.5277 4.44% Table 4. Antigenic, non-allergenic, and non-toxic linear BCL epitopes of WP_001166178.1 Peptide sequence Structural conformation Surface proximity Antigenicity score Conservancy KTSDATA CCCCHHH EEEEBEB 2.0211 51.11% PNNSGE CCCCCE EEEEEE 1.2826 53.33% NAKDNAGNWTSASNKKE ECCCCCCCCCCCCCCCE EBEEEEEEBEEEEEEEE 0.8015 44.44% LKEANKNHPAGAPTFAKGEGEHAN HHHHCCCCCCCCCCCCCCCCCCCC BEEBEEEBEEEBEEBEEEEEEEBE 0.7883 28.89% LNNVDNQPATEVPSS HCCCCCCCCCCCCCC BEEBEEEEBEEBEEE 0.6058 42.22% Structural conformation - C, coils; H, helices; E, sheets; Surface proximity - B, buried; E, exposed. Figure 1. Predicted conserved domains and motifs. An atypical Rib protein domain, spanning the C-terminal region (residue 126-198) of WP_001166178.1, was predicted to be conserved by the consensus of NCBI CDD, InterPro, and MotifFinder. MotifFinder also reported additional conserved motifs, which were not further analyzed due to higher e-values. Figure 2. Molecular interaction between alpha-D-mannopyranose and the residues of the predicted binding cleft of WP_001166178.1. Conventional hydrogen bonds were reported between the oxygen atoms of alpha-D-mannopyranose and the reported residues found on the WP_001166178.1 binding cleft, with relatively high solvent accessible surface, confirming the significance of the said region. Figure 3. Structural features of WP_001166178.1. (A) AlphaFold Tool was used to generate the 3D model of WP_001166178.1 with the aRib domain highlighted in blue, (B-C) Distribution of the pLDDT and pIDDT scores on the residues of WP_001166178.1, which entails the quality of the assigned secondary structure and folding, (D) Comparison of the aRib domain (left) and the Rib domain (right) from Streptococcus pyogenes (PDB: 6S5Z), highlighting the core beta-sheets in gray. Figure 4. Validation of pre- and post-refinement structural model of WP_001166178.1 using ERRAT and PROCHECK. (A) ERRAT scores showed significant improvement from 85.88% to 99.459%, resolving high-error-value-residues, (B) Ramachandran plots from PROCHECK reporting an overall improvement of WP_001166178.1 stereochemistry by refining residues from the disallowed regions. Figure 5. Normal-mode analysis-based molecular dynamics simulation of WP_001166178.1. (A) Deformability and B-factor of each residue, (B) eigenvalue and variance of each mode index, (C) covariance map of recorded motion during the simulation (red=correlated, white=no uncorrelated, blue=anticorrelated), (D) elastic network indicating the stiff regions (gray) Figure 6. Immune response simulation of WP_001166178.1 as a protein subunit vaccine. (A) antigen clearance and antibody titers during the simulation, (B-C) helper T-cells and B-cell population, and (D) concentration of cytokines Supporting Information Supplementary Table 1 . Subcellular localization of hypothetical proteins Supplementary Table 2. Amino acid residues and atoms involved in the predicted binding cleft of WP_001166178.1 Supplementary Table 3. Streptococcus pneumoniae strains used for epitope conservancy analysis Information & Authors Information Version history V1 Version 1 21 July 2025 Peer review timeline Published Proteins: Structure, Function, and Bioinformatics Version of Record 22 Jan 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords streptococcus pneumoniae atypical rib domain epitope protein domain vaccinology Authors Affiliations Stephen Kyle C. Arcan 0009-0004-1174-6198 [email protected] University of the City of Manila View all articles by this author Gyraldin Marietony D. Gan University of the City of Manila View all articles by this author Debrah Jannsen N. Almazan University of the City of Manila View all articles by this author Zairus N. Duquilla University of the City of Manila View all articles by this author Azenith Vincent D. Barbosa University of the City of Manila View all articles by this author Rainier Ulrich D. Velasco University of the City of Manila View all articles by this author Metrics & Citations Metrics Article Usage 341 views 207 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Stephen Kyle C. Arcan, Gyraldin Marietony D. Gan, Debrah Jannsen N. Almazan, et al. In silico functional and structural characterization of Streptococcus pneumoniae atypical Rib domain-containing hypothetical protein unravels conserved immunogenic epitopes. Authorea . 21 July 2025. DOI: https://doi.org/10.22541/au.175313166.61464735/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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