{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T22:52:35Z","timestamp":1768344755349,"version":"3.49.0"},"reference-count":50,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T00:00:00Z","timestamp":1768262400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004054","name":"Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia","doi-asserted-by":"publisher","award":["DRP-19-130-2025"],"award-info":[{"award-number":["DRP-19-130-2025"]}],"id":[{"id":"10.13039\/501100004054","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>The zoonotic potential of bat coronaviruses, especially HKU5, is a significant issue because of their capacity to utilize human angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. This study offers structural insights into the binding kinetics of HKU5 (Bat Merbecovirus HKU5) receptor-binding domain (RBD) spike protein with human ACE2 through a multiscale computational method. This study employed structural modeling, 300-nanosecond (ns) molecular dynamics (MD) simulations, alanine-scanning mutagenesis, and computational peptide design to investigate ACE2 recognition by the HKU5 RBD and its interactions with peptides. The root mean square deviation (RMSD) investigation of HKU5\u2013ACE2 complexes indicated that HKU5 exhibited greater flexibility than SARS-CoV-2, with RMSD values reaching a maximum of 1.2 nm. Free energy analysis, Molecular Mechanics\/Generalized Born Surface Area (MM\/GBSA), indicated a more robust binding affinity of HKU5 to ACE2 (\u0394GTotal = \u221221.61 kcal\/mol) in contrast to SARS-CoV-2 (\u0394GTotal = \u22125.82 kcal\/mol), implying that HKU5 binding with ACE2 had higher efficiency. Additionally, a peptide was designed from the ACE2 interface, resulting in the development of 380 single-site mutants by mutational alterations. The four most promising mutant peptides were selected for 300-nanosecond (ns) MD simulations, subsequently undergoing quantum chemical calculations (DFT) to evaluate their electronic characteristics. MM\/GBSA of \u221237.83 kcal\/mol indicated that mutant-1 exhibits the most favorable binding with HKU5, hence potentially inhibiting ACE2 interaction. Mutant-1 formed hydrogen bonds involving Glu74, Ser202, Ser204, and Asn152 residues of HKU5. Finally, QM\/MM calculations on the peptide\u2013HKU5 complexes showed the most favorable \u0394E_interaction of \u2212170.47 (Hartree) for mutant-1 peptide. These findings offer a thorough comprehension of receptor-binding dynamics and are crucial for evaluating the zoonotic risk associated with HKU5-CoV and guiding the design of receptor-targeted antiviral treatments.<\/jats:p>","DOI":"10.3390\/ani16020237","type":"journal-article","created":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T15:52:57Z","timestamp":1768319577000},"page":"237","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structural Insights into the Receptor-Binding Domain of Bat Coronavirus HKU5-CoV-2: Implications for Zoonotic Transmission via ACE2"],"prefix":"10.3390","volume":"16","author":[{"given":"Manal A.","family":"Babaker","sequence":"first","affiliation":[{"name":"Department of Chemistry, Faculty of Science, Majmaah University, Al Majmaah 11952, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nariman","family":"Sindi","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Othman Yahya","family":"Alyahyawy","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Technology (MLT), Faculty of Applied Medical Sciences, King Abdulaziz University, Rabigh 80200, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1076-9740","authenticated-orcid":false,"given":"Ehssan","family":"Moglad","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics, College of Pharmacy, Prince Sattam bin Abdulaziz University, P.O. Box 173, Alkharj 11942, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohieldin","family":"Elsayid","sequence":"additional","affiliation":[{"name":"Clinical Laboratory Sciences-Jeddah, College of Applied Medical Sciences, King Saud bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Jeddah 22384, Saudi Arabia"},{"name":"King Abdullah International Medical Research Center, King Abdulaziz Medical City, Jeddah 22384, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9513-8202","authenticated-orcid":false,"given":"Thamir M.","family":"Eid","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia"},{"name":"Experimental Biochemistry Unit, King Fahad Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0546-6697","authenticated-orcid":false,"given":"Mohamed Eltaib","family":"Elmobark","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hisham N.","family":"Altayb","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1038\/nature12711","article-title":"Isolation and Characterization of a Bat SARS-like Coronavirus That Uses the ACE2 Receptor","volume":"503","author":"Ge","year":"2013","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1016\/j.cell.2020.03.045","article-title":"Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2","volume":"181","author":"Wang","year":"2020","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/j.ijid.2020.08.050","article-title":"Bats in Ecosystems and Their Wide Spectrum of Viral Infectious Potential Threats: SARS-CoV-2 and Other Emerging Viruses","volume":"102","author":"Pachar","year":"2021","journal-title":"Int. J. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1007\/s00018-020-03580-1","article-title":"Protein Structure Analysis of the Interactions between SARS-CoV-2 Spike Protein and the Human ACE2 Receptor: From Conformational Changes to Novel Neutralizing Antibodies","volume":"78","author":"Mercurio","year":"2021","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4932","DOI":"10.1038\/s41467-025-60286-3","article-title":"ACE2 from Pipistrellus Abramus Bats Is a Receptor for HKU5 Coronaviruses","volume":"16","author":"Catanzaro","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1729","DOI":"10.1016\/j.cell.2025.01.042","article-title":"Bat-Infecting Merbecovirus HKU5-CoV Lineage 2 Can Use Human ACE2 as a Cell Entry Receptor","volume":"188","author":"Chen","year":"2025","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"6822","DOI":"10.1038\/s41467-025-61583-7","article-title":"HKU5 Bat Merbecoviruses Engage Bat and Mink ACE2 as Entry Receptors","volume":"16","author":"Keeler","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"748","DOI":"10.1038\/s41586-022-05513-3","article-title":"Close Relatives of MERS-CoV in Bats Use ACE2 as Their Functional Receptors","volume":"612","author":"Xiong","year":"2022","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1126\/science.abd9909","article-title":"De Novo Design of Picomolar SARS-CoV-2 Miniprotein Inhibitors","volume":"370","author":"Cao","year":"2020","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5143","DOI":"10.1021\/acsnano.0c02857","article-title":"Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2","volume":"14","author":"Han","year":"2020","journal-title":"ACS Nano"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e02451-20","DOI":"10.1128\/mBio.02451-20","article-title":"Stapled Peptides Based on Human Angiotensin-Converting Enzyme 2 (ACE2) Potently Inhibit SARS-CoV-2 Infection In Vitro","volume":"11","author":"Curreli","year":"2020","journal-title":"mBio"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1711","DOI":"10.1016\/j.cell.2024.12.032","article-title":"Molecular Basis of Convergent Evolution of ACE2 Receptor Utilization among HKU5 Coronaviruses","volume":"188","author":"Park","year":"2025","journal-title":"Cell"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Tse, A.L., Acreman, C.M., Ricardo-Lax, I., Berrigan, J., Lasso, G., Balogun, T., Kearns, F.L., Casalino, L., McClain, G.L., and Chandran, A.M. (2024). Distinct Pathways for Evolution of Enhanced Receptor Binding and Cell Entry in SARS-like Bat Coronaviruses. PLoS Pathog., 20.","DOI":"10.1101\/2024.06.24.600393"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The Protein Data Bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology Modelling of Protein Structures and Complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"W365","DOI":"10.1093\/nar\/gkx407","article-title":"HDOCK: A Web Server for Protein\u2013Protein and Protein\u2013DNA\/RNA Docking Based on a Hybrid Strategy","volume":"45","author":"Yan","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"W338","DOI":"10.1093\/nar\/gkz383","article-title":"mCSM-PPI2: Predicting the Effects of Mutations on Protein\u2013Protein Interactions","volume":"47","author":"Rodrigues","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_18","unstructured":"Bauer, P., Hess, B., and Lindahl, E. (2026, January 07). GROMACS 2022 Source Sode. Zenodo. Available online: https:\/\/zenodo.org\/records\/8134358."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2135","DOI":"10.1002\/jcc.23354","article-title":"CHARMM36 All-Atom Additive Protein Force Field: Validation Based on Comparison to NMR Data","volume":"34","author":"Huang","year":"2013","journal-title":"J. Comput. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"10089","DOI":"10.1063\/1.464397","article-title":"Particle Mesh Ewald: An N\u22c5log(N) Method for Ewald Sums in Large Systems","volume":"98","author":"Darden","year":"1993","journal-title":"J. Chem. Phys."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"174501","DOI":"10.1063\/1.4872239","article-title":"Structure and Dynamics of TIP3P, TIP4P, and TIP5P Water near Smooth and Atomistic Walls of Different Hydroaffinity","volume":"140","author":"Harrach","year":"2014","journal-title":"J. Chem. Phys."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1002\/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H","article-title":"LINCS: A Linear Constraint Solver for Molecular Simulations","volume":"18","author":"Hess","year":"1997","journal-title":"J. Comput. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"014101","DOI":"10.1063\/1.2408420","article-title":"Canonical Sampling through Velocity Rescaling","volume":"126","author":"Bussi","year":"2007","journal-title":"J. Chem. Phys."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"075503","DOI":"10.1103\/PhysRevLett.90.075503","article-title":"Predicting Crystal Structures: The Parrinello-Rahman Method Revisited","volume":"90","author":"Laio","year":"2003","journal-title":"Phys. Rev. Lett."},{"key":"ref_25","unstructured":"(Sim(Ana) Analogue Release 2024, 2024). Sim(Ana) Analogue Release 2024, version 1.1."},{"key":"ref_26","unstructured":"(Trajecta(Ana) Analogue Release 2024, 2024). Trajecta(Ana) Analogue Release 2024, version 1.1."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1016\/j.jmb.2007.05.022","article-title":"Inference of Macromolecular Assemblies from Crystalline State","volume":"372","author":"Krissinel","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Berthold, R.M., Glen, R.C., Diederichs, K., Kohlbacher, O., and Fischer, I. (2005). Detection of Protein Assemblies in Crystals. Proceedings of the Computational Life Sciences, Springer.","DOI":"10.1007\/11560500"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"6473","DOI":"10.1038\/s41598-024-57247-z","article-title":"De Novo Antioxidant Peptide Design via Machine Learning and DFT Studies","volume":"14","author":"Hesamzadeh","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"9381","DOI":"10.1039\/D1CP00206F","article-title":"Protein\u2013Ligand Free Energies of Binding from Full-Protein DFT Calculations: Convergence and Choice of Exchange\u2013Correlation Functional","volume":"23","author":"Gundelach","year":"2021","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1672","DOI":"10.1002\/jcc.23317","article-title":"Corrected Small Basis Set Hartree-Fock Method for Large Systems","volume":"34","author":"Sure","year":"2013","journal-title":"J. Comput. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"054107","DOI":"10.1063\/1.4927476","article-title":"Consistent Structures and Interactions by Density Functional Theory with Small Atomic Orbital Basis Sets","volume":"143","author":"Grimme","year":"2015","journal-title":"J. Chem. Phys."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1652","DOI":"10.1021\/acs.jctc.8b01176","article-title":"GFN2-xTB\u2014An Accurate and Broadly Parametrized Self-Consistent Tight-Binding Quantum Chemical Method with Multipole Electrostatics and Density-Dependent Dispersion Contributions","volume":"15","author":"Bannwarth","year":"2019","journal-title":"J. Chem. Theory Comput."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1002\/wcms.30","article-title":"Density Functional Theory with London Dispersion Corrections","volume":"1","author":"Grimme","year":"2011","journal-title":"WIREs Comput. Mol. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Chelikowsky, J.R. (2005). Nanostructures, Electronic Structure Of. Encyclopedia of Condensed Matter Physics, Elsevier.","DOI":"10.1016\/B978-0-323-90800-9.00290-0"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1198","DOI":"10.1002\/anie.200802019","article-title":"QM\/MM Methods for Biomolecular Systems","volume":"48","author":"Senn","year":"2009","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"25009","DOI":"10.1021\/acsami.3c01430","article-title":"An Electrostatically Embedded QM\/MM Scheme for Electrified Interfaces","volume":"15","author":"Abidi","year":"2023","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3098","DOI":"10.1103\/PhysRevA.38.3098","article-title":"Density-Functional Exchange-Energy Approximation with Correct Asymptotic Behavior","volume":"38","author":"Becke","year":"1988","journal-title":"Phys. Rev. A"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1002\/wcms.81","article-title":"The ORCA Program System","volume":"2","author":"Neese","year":"2012","journal-title":"WIREs Comput. Mol. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e1327","DOI":"10.1002\/wcms.1327","article-title":"Software Update: The ORCA Program System, Version 4.0","volume":"8","author":"Neese","year":"2018","journal-title":"WIREs Comput. Mol. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"669","DOI":"10.1002\/jcc.10189","article-title":"Energies, Structures, and Electronic Properties of Molecules in Solution with the C-PCM Solvation Model","volume":"24","author":"Cossi","year":"2003","journal-title":"J. Comput. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"064104","DOI":"10.1063\/1.5012601","article-title":"B97-3c: A Revised Low-Cost Variant of the B97-D Density Functional Method","volume":"148","author":"Brandenburg","year":"2018","journal-title":"J. Chem. Phys."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1634","DOI":"10.1038\/sj.emboj.7600640","article-title":"Receptor and Viral Determinants of SARS-Coronavirus Adaptation to Human ACE2","volume":"24","author":"Li","year":"2005","journal-title":"EMBO J."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"8904","DOI":"10.1074\/jbc.M111.325803","article-title":"Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus","volume":"287","author":"Wu","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1038\/s41579-023-00878-2","article-title":"The Evolution of SARS-CoV-2","volume":"21","author":"Markov","year":"2023","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1146\/annurev-virology-100114-055029","article-title":"Coronavirus Host Range Expansion and Middle East Respiratory Syndrome Coronavirus Emergence: Biochemical Mechanisms and Evolutionary Perspectives","volume":"2","author":"Peck","year":"2015","journal-title":"Annu. Rev. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/j.cell.2020.02.058","article-title":"Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein","volume":"181","author":"Walls","year":"2020","journal-title":"Cell"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"bbab243","DOI":"10.1093\/bib\/bbab243","article-title":"Computational Design of Ultrashort Peptide Inhibitors of the Receptor-Binding Domain of the SARS-CoV-2 S Protein","volume":"22","author":"Pei","year":"2021","journal-title":"Brief. Bioinform."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e2413465122","DOI":"10.1073\/pnas.2413465122","article-title":"Structure-Guided Engineering of a Mutation-Tolerant Inhibitor Peptide against Variable SARS-CoV-2 Spikes","volume":"122","author":"Nakamura","year":"2025","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1016\/0006-291X(75)90482-9","article-title":"Delineation of the Intimate Details of the Backbone Conformation of Pyridine Nucleotide Coenzymes in Aqueous Solution","volume":"66","author":"Bose","year":"1975","journal-title":"Biochem. Biophys. Res. Commun."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/2\/237\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T16:05:07Z","timestamp":1768320307000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/2\/237"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,13]]},"references-count":50,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["ani16020237"],"URL":"https:\/\/doi.org\/10.3390\/ani16020237","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,13]]}}}