{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"Research Square"}],"indexed":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T00:16:28Z","timestamp":1773879388795,"version":"3.50.1"},"posted":{"date-parts":[[2026,2,25]]},"group-title":"In Review","reference-count":66,"publisher":"Springer Science and Business Media LLC","license":[{"start":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T00:00:00Z","timestamp":1771977600000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,14]]},"abstract":"<title>Abstract<\/title>\n                <p>Carbohydrate-binding modules (CBMs) are widespread auxiliary domains in carbohydrate-active enzymes, yet their contribution to enzyme function and evolvability remains unclear, particularly in redox enzymes. Here, we report the first biochemically and structurally characterized bacterial galactose oxidase. Unlike its fungal counterparts, this enzyme is produced in an active form containing a catalytic Cu(II)\u2013tyrosyl radical cofactor and two N-terminal CBM32 domains. Biochemical, structural, and computational analyses reveal functional asymmetry between the duplicated CBMs, which differentially modulate soluble expression, stability and polysaccharide binding. Deletion of the first CBM markedly enhances solubility while preserving catalytic competence, and binding assays identify this module as the dominant contributor to galactan affinity. Directed evolution promotes CBM truncation and recombination, yielding functional chimeric variants and uncovering a residue influencing polysaccharide recognition. These findings expand the diversity of copper radical oxidases and highlight domain architecture as an evolvable determinant of redox enzyme behavior.<\/p>","DOI":"10.21203\/rs.3.rs-8880588\/v1","type":"posted-content","created":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T07:05:11Z","timestamp":1772003111000},"source":"Crossref","is-referenced-by-count":0,"title":["Evolvable Carbohydrate-Binding Modules Shape Function and Engineering of a Bacterial Galactose Oxidase"],"prefix":"10.21203","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0082-9591","authenticated-orcid":false,"given":"Ligia","family":"Martins","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3003-0580","authenticated-orcid":false,"given":"Andr\u00e9","family":"Taborda","sequence":"additional","affiliation":[{"name":"nstituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5522-4085","authenticated-orcid":false,"given":"Tom\u00e1s","family":"Fraz\u00e3o","sequence":"additional","affiliation":[{"name":"nstituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]},{"given":"Tiago","family":"Lopes","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]},{"given":"Carolina","family":"Dias","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]},{"given":"Felipe","family":"Folgosa","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]},{"given":"Marcia","family":"Renio","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6854-7278","authenticated-orcid":false,"given":"M. Rita","family":"Ventura","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2917-2100","authenticated-orcid":false,"given":"Ferran","family":"Sancho","sequence":"additional","affiliation":[{"name":"Zymvol Biomodeling"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0602-819X","authenticated-orcid":false,"given":"Patricia","family":"Borges","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Quimica e Biologica - Universidade Nova de Lisboa"}]}],"member":"297","reference":[{"key":"ref1","doi-asserted-by":"crossref","unstructured":"1. Boraston, A.B., Bolam, D.N., Gilbert, H.J. & Davies, G.J. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382, 769\u2009\u2212\u200981 (2004).","DOI":"10.1042\/BJ20040892"},{"key":"ref2","doi-asserted-by":"crossref","unstructured":"2. You, Y. et al. Carbohydrate binding modules: Compact yet potent accessories in the specific substrate binding and performance evolution of carbohydrate-active enzymes. Biotechnol Adv 73, 108365 (2024).","DOI":"10.1016\/j.biotechadv.2024.108365"},{"key":"ref3","doi-asserted-by":"crossref","unstructured":"3. Sidar, A. et al. Carbohydrate Binding Modules: Diversity of Domain Architecture in Amylases and Cellulases From Filamentous Microorganisms. Front Bioeng Biotechnol 8, 871 (2020).","DOI":"10.3389\/fbioe.2020.00871"},{"key":"ref4","doi-asserted-by":"crossref","unstructured":"4. Forsberg, Z. et al. Discovery of a Copper-Binding Carbohydrate-Binding Module Regulating the Activity of Lytic Polysaccharide Monooxygenases. J Am Chem Soc 147, 45104\u201345118 (2025).","DOI":"10.1021\/jacs.5c14016"},{"key":"ref5","doi-asserted-by":"crossref","unstructured":"5. Fong, J.K. & Brumer, H. Copper radical oxidases: galactose oxidase, glyoxal oxidase, and beyond! Essays Biochem 67, 597\u2013613 (2023).","DOI":"10.1042\/EBC20220124"},{"key":"ref6","doi-asserted-by":"crossref","unstructured":"6. Savino, S. & Fraaije, M.W. The vast repertoire of carbohydrate oxidases: An overview. Biotechnol Adv 51, 107634 (2021).","DOI":"10.1016\/j.biotechadv.2020.107634"},{"key":"ref7","doi-asserted-by":"crossref","unstructured":"7. Levasseur, A., Drula, E., Lombard, V., Coutinho, P.M. & Henrissat, B. Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol Biofuels 6, 41 (2013).","DOI":"10.1186\/1754-6834-6-41"},{"key":"ref8","doi-asserted-by":"crossref","unstructured":"8. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42, 490\u2013495 (2014).","DOI":"10.1093\/nar\/gkt1178"},{"key":"ref9","doi-asserted-by":"crossref","unstructured":"9. Bissaro, B. et al. Tandem metalloenzymes gate plant cell entry by pathogenic fungi. Sci Adv 8, eade9982 (2022).","DOI":"10.1126\/sciadv.ade9982"},{"key":"ref10","doi-asserted-by":"crossref","unstructured":"10. Kersten, P.J. & Kirk, T.K. Involvement of a new enzyme, glyoxal oxidase, in extracellular H2O2 production by Phanerochaete chrysosporium. J Bacteriol 169, 2195\u2009\u2212\u2009201 (1987).","DOI":"10.1128\/jb.169.5.2195-2201.1987"},{"key":"ref11","doi-asserted-by":"crossref","unstructured":"11. Cleveland, M.E. et al. A survey of substrate specificity among Auxiliary Activity Family 5 copper radical oxidases Cell Mol Life Sci 78, 8187\u20138208 (2021).","DOI":"10.1007\/s00018-021-03981-w"},{"key":"ref12","doi-asserted-by":"crossref","unstructured":"12. Koschorreck, K., Alpdagtas, S. & Urlacher, V.B. Copper-radical oxidases: A diverse group of biocatalysts with distinct properties and a broad range of biotechnological applications. Eng Microb 2, 100037 (2022).","DOI":"10.1016\/j.engmic.2022.100037"},{"key":"ref13","doi-asserted-by":"crossref","unstructured":"13. Yin, D. et al. Structure\u2013function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family. Nat Comm 6, 10197 (2015).","DOI":"10.1038\/ncomms10197"},{"key":"ref14","doi-asserted-by":"crossref","unstructured":"14. Ito, N. et al. Novel thioether bond revealed by a 1.7 \u00c5 crystal structure of galactose oxidase. Nature 350, 87\u201390 (1991).","DOI":"10.1038\/350087a0"},{"key":"ref15","doi-asserted-by":"crossref","unstructured":"15. Mathieu, Y. et al. Discovery of a Fungal Copper Radical Oxidase with High Catalytic Efficiency toward 5-Hydroxymethylfurfural and Benzyl Alcohols for Bioprocessing. ACS Catal 10, 3042\u20133058 (2020).","DOI":"10.1021\/acscatal.9b04727"},{"key":"ref16","doi-asserted-by":"crossref","unstructured":"16. Chaplin, Amanda K. et al. GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of Streptomyces lividans. Biochem. J. 469, 433\u2013444 (2015).","DOI":"10.1042\/BJ20150190"},{"key":"ref17","doi-asserted-by":"crossref","unstructured":"17. Chaplin, A.K. et al. Active-site maturation and activity of the copper-radical oxidase GlxA are governed by a tryptophan residue. Biochem J 474, 809\u2013825 (2017).","DOI":"10.1042\/BCJ20160968"},{"key":"ref18","doi-asserted-by":"crossref","unstructured":"18. Whittaker, M.M. & Whittaker, J.W. Ligand interactions with galactose oxidase: mechanistic insights. Biophys J 64, 762\u2013772 (1993).","DOI":"10.1016\/S0006-3495(93)81437-1"},{"key":"ref19","doi-asserted-by":"crossref","unstructured":"19. Whittaker, M.M., Ballou, D.P. & Whittaker, J.W. Kinetic Isotope Effects as Probes of the Mechanism of Galactose Oxidase. Biochem 37, 8426\u20138436 (1998).","DOI":"10.1021\/bi980328t"},{"key":"ref20","doi-asserted-by":"crossref","unstructured":"20. Baron, A.J. et al. Structure and mechanism of galactose oxidase. The free radical site. J Biol Chem 269, 25095-105 (1994).","DOI":"10.1016\/S0021-9258(17)31504-1"},{"key":"ref21","doi-asserted-by":"crossref","unstructured":"21. Knowles, P.F. et al. Spectroscopic Studies of the Active Site of Galactose Oxidase. Inorg Chem 34, 3895\u20133902 (1995).","DOI":"10.1021\/ic00119a010"},{"key":"ref22","doi-asserted-by":"crossref","unstructured":"22. Lee, Y.K., Whittaker, M.M. & Whittaker, J.W. The electronic structure of the Cys-Tyr(*) free radical in galactose oxidase determined by EPR spectroscopy. Biochem 47, 6637-49 (2008).","DOI":"10.1021\/bi800305d"},{"key":"ref23","doi-asserted-by":"crossref","unstructured":"23. Whittaker & Whittaker. A tyrosine-derived free radical in apogalactose oxidase. J Biol Chem 265, 9610\u20139613 (1990).","DOI":"10.1016\/S0021-9258(19)38711-3"},{"key":"ref24","doi-asserted-by":"crossref","unstructured":"24. Faria, C.B. et al. Production of Galactose Oxidase Inside the Fusarium fujikuroi Species Complex and Recombinant Expression and Characterization of the Galactose Oxidase GaoA Protein from Fusarium subglutinans. Mol Biotechnol 61, 633\u2013649 (2019).","DOI":"10.1007\/s12033-019-00190-6"},{"key":"ref25","doi-asserted-by":"crossref","unstructured":"25. Paukner, R. et al. Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization. PLoS One 9, e100116 (2014).","DOI":"10.1371\/journal.pone.0100116"},{"key":"ref26","doi-asserted-by":"crossref","unstructured":"26. Paukner, R., Staudigl, P., Choosri, W., Haltrich, D. & Leitner, C. Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli. Protein Expr Purif 108, 73\u201379 (2015).","DOI":"10.1016\/j.pep.2014.12.010"},{"key":"ref27","doi-asserted-by":"crossref","unstructured":"27. Humphreys, K.J., Mirica, L.M., Wang, Y. & Klinman, J.P. Galactose oxidase as a model for reactivity at a copper superoxide center. J Am Chem Soc 131, 4657-63 (2009).","DOI":"10.1021\/ja807963e"},{"key":"ref28","doi-asserted-by":"crossref","unstructured":"28. McPherson, M.J. et al. Galactose oxidase: Molecular analysis and mutagenesis studies. Biochem Soc Trans 21, 752\u2013756 (1993).","DOI":"10.1042\/bst0210752"},{"key":"ref29","doi-asserted-by":"crossref","unstructured":"29. Tressel, P. & Kosman, D.J. A simplified purification procedure for galactose oxidase. Anal Biochem 105, 150-3 (1980).","DOI":"10.1016\/0003-2697(80)90437-6"},{"key":"ref30","doi-asserted-by":"crossref","unstructured":"30. Jun, H., Bing, Y., Keying, Z., Xuemei, D. & Daiwen, C. Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2. New Biotechnol 26, 53\u201359 (2009).","DOI":"10.1016\/j.nbt.2009.04.002"},{"key":"ref31","doi-asserted-by":"crossref","unstructured":"31. DeChellis, A. et al. Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass. ACS Sus Chem Eng 12, 3500\u20133516 (2024).","DOI":"10.1021\/acssuschemeng.3c06266"},{"key":"ref32","doi-asserted-by":"crossref","unstructured":"32. Passaro, S. et al. Boltz-2: Towards Accurate and Efficient Binding Affinity Prediction. bioRxiv, 2025.06.14.659707 (2025).","DOI":"10.1101\/2025.06.14.659707"},{"key":"ref33","doi-asserted-by":"crossref","unstructured":"33. Reynolds, M.P. et al. Structure and mechanism of galactose oxidase: catalytic role of tyrosine 495. J Biol Inorg Chem 2, 327\u2013335 (1997).","DOI":"10.1007\/s007750050139"},{"key":"ref34","doi-asserted-by":"crossref","unstructured":"34. Rogers, M.S. et al. The stacking tryptophan of galactose oxidase: a second-coordination sphere residue that has profound effects on tyrosyl radical behavior and enzyme catalysis. Biochem 46, 4606-18 (2007).","DOI":"10.1021\/bi062139d"},{"key":"ref35","doi-asserted-by":"crossref","unstructured":"35. Miton, C.M. & Tokuriki, N. Insertions and Deletions (Indels): A Missing Piece of the Protein Engineering Jigsaw. Biochemistry 62, 148\u2013157 (2023).","DOI":"10.1021\/acs.biochem.2c00188"},{"key":"ref36","doi-asserted-by":"crossref","unstructured":"36. Wittmund, M., Cadet, F. & Davari, M.D. Learning Epistasis and Residue Coevolution Patterns: Current Trends and Future Perspectives for Advancing Enzyme Engineering. ACS Catal 12, 14243\u201314263 (2022).","DOI":"10.1021\/acscatal.2c01426"},{"key":"ref37","doi-asserted-by":"crossref","unstructured":"37. Abbott, D.W., Eirin-Lopez, J.M. & Boraston, A.B. Insight into ligand diversity and novel biological roles for family 32 carbohydrate-binding modules. Mol Biol Evol 25, 155\u2009\u2212\u200967 (2008).","DOI":"10.1093\/molbev\/msm243"},{"key":"ref38","doi-asserted-by":"crossref","unstructured":"38. Oberg, N., Zallot, R. & Gerlt, J.A. EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for Genomic Enzymology Tools. J Mol Biol 435, 168018 (2023).","DOI":"10.1016\/j.jmb.2023.168018"},{"key":"ref39","doi-asserted-by":"crossref","unstructured":"39. Dur\u00e3o, P. et al. Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies. J Biol Inorg Chem 11, 514\u2013526 (2006).","DOI":"10.1007\/s00775-006-0102-0"},{"key":"ref40","doi-asserted-by":"crossref","unstructured":"40. Petasis, D.T. & Hendrich, M.P. Quantitative Interpretation of Multifrequency Multimode EPR Spectra of Metal Containing Proteins, Enzymes, and Biomimetic Complexes. Methods Enzymol 563, 171\u2013208 (2015).","DOI":"10.1016\/bs.mie.2015.06.025"},{"key":"ref41","doi-asserted-by":"crossref","unstructured":"41. Juanhuix, J. et al. Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron. J. Synchrotron Rad. 21, 679\u00e2\u0080-689 (2014).","DOI":"10.1107\/S160057751400825X"},{"key":"ref42","doi-asserted-by":"crossref","unstructured":"42. von Stetten, D. et al. D30A-3 (MASSIF-3)\u0080\u0093 a beamline for macromolecular crystallography at the ESRF with a small intense beam. J Synchrotron Rad 27, 844-\u0080\u0093851 (2020).","DOI":"10.1107\/S1600577520004002"},{"key":"ref43","doi-asserted-by":"crossref","unstructured":"43. Kabsch, W. XDS. Acta Crystalogr D 66, 125\u2013132 (2010).","DOI":"10.1107\/S0907444909047337"},{"key":"ref44","doi-asserted-by":"crossref","unstructured":"44. Vonrhein, C. et al. Advances in automated data analysis and processing within autoPROC, combined with improved characterisation, mitigation and visualisation of the anisotropy of diffraction limits using STARANISO. Acta Crystallogr A 74, 360 (2018).","DOI":"10.1107\/S010876731809640X"},{"key":"ref45","doi-asserted-by":"crossref","unstructured":"45. Evans, P.R. & Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr D 69, 1204-14 (2013).","DOI":"10.1107\/S0907444913000061"},{"key":"ref46","doi-asserted-by":"crossref","unstructured":"46. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D 67, 293\u2013302 (2011).","DOI":"10.1107\/S0907444911007773"},{"key":"ref47","doi-asserted-by":"crossref","unstructured":"47. Kantardjieff, K.A. & Rupp, B. Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals. Protein Sci 12, 1865-71 (2003).","DOI":"10.1110\/ps.0350503"},{"key":"ref48","doi-asserted-by":"crossref","unstructured":"48. Matthews, B.W. Solvent content of protein crystals. J Mol Biol 33, 491\u2013497 (1968).","DOI":"10.1016\/0022-2836(68)90205-2"},{"key":"ref49","doi-asserted-by":"crossref","unstructured":"49. McCoy, A.J. et al. Phaser crystallographic software. J Appl Crystallogr 40, 658\u2013674 (2007).","DOI":"10.1107\/S0021889807021206"},{"key":"ref50","doi-asserted-by":"crossref","unstructured":"50. McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Crystallogr D 61, 458\u2009\u2212\u200964 (2005).","DOI":"10.1107\/S0907444905001617"},{"key":"ref51","doi-asserted-by":"crossref","unstructured":"51. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D 67, 235\u2009\u2212\u200942 (2011).","DOI":"10.1107\/S0907444910045749"},{"key":"ref52","doi-asserted-by":"crossref","unstructured":"52. Perrakis, A., Harkiolaki, M., Wilson, K.S. & Lamzin, V.S. ARP\/wARP and molecular replacement. Acta Crystallogr D 57, 1445-50 (2001).","DOI":"10.1107\/S0907444901014007"},{"key":"ref53","doi-asserted-by":"crossref","unstructured":"53. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D 60, 2126-32 (2004).","DOI":"10.1107\/S0907444904019158"},{"key":"ref54","doi-asserted-by":"crossref","unstructured":"54. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D 67, 355\u2009\u2212\u200967 (2011).","DOI":"10.1107\/S0907444911001314"},{"key":"ref55","doi-asserted-by":"crossref","unstructured":"55. Painter, J. & Merritt, E.A. TLSMD web server for the generation of multi-group TLS models. J App Crystallogr 39, 109\u2013111 (2006).","DOI":"10.1107\/S0021889805038987"},{"key":"ref56","doi-asserted-by":"crossref","unstructured":"56. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Cryst. D 66, 12\u201321 (2010).","DOI":"10.1107\/S0907444909042073"},{"key":"ref57","unstructured":"57. Schrodinger, L.L.C. The PyMOL Molecular Graphics System Version 2.0. (New York USA, 2015)."},{"key":"ref58","doi-asserted-by":"crossref","unstructured":"58. Krieger, E. & Vriend, G. New ways to boost molecular dynamics simulations. Comp Chem 36, 996\u20131007 (2015).","DOI":"10.1002\/jcc.23899"},{"key":"ref59","doi-asserted-by":"crossref","unstructured":"59. Hornak, V. et al. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65, 712\u2009\u2212\u200925 (2006).","DOI":"10.1002\/prot.21123"},{"key":"ref60","doi-asserted-by":"crossref","unstructured":"60. Krieger, E., Nielsen, J.E., Spronk, C.A. & Vriend, G. Fast empirical pKa prediction by Ewald summation. J Mol Graph Model 25, 481-6 (2006).","DOI":"10.1016\/j.jmgm.2006.02.009"},{"key":"ref61","doi-asserted-by":"crossref","unstructured":"61. Mark, P. & Nilsson, L. Structure and Dynamics of the TIP3P, SPC, and SPC\/E Water Models at 298 K. Phys Chem A 105, 9954\u20139960 (2001).","DOI":"10.1021\/jp003020w"},{"key":"ref62","doi-asserted-by":"crossref","unstructured":"62. Jo, S., Kim, T., Iyer, V.G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J Comput Chem 29, 1859-65 (2008).","DOI":"10.1002\/jcc.20945"},{"key":"ref63","doi-asserted-by":"crossref","unstructured":"63. Lee, J. et al. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM\/OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory Comput. 12, 405\u2013413 (2016).","DOI":"10.1021\/acs.jctc.5b00935"},{"key":"ref64","doi-asserted-by":"crossref","unstructured":"64. Abraham, M.J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1\u20132, 19\u201325 (2015).","DOI":"10.1016\/j.softx.2015.06.001"},{"key":"ref65","doi-asserted-by":"crossref","unstructured":"65. Trott, O. & Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31, 455\u2009\u2212\u200961 (2010).","DOI":"10.1002\/jcc.21334"},{"key":"ref66","doi-asserted-by":"crossref","unstructured":"66. Eberhardt, J., Santos-Martins, D., Tillack, A.F. & Forli, S. AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. 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