Development of an in silico Pipeline Supporting Interaction Characterization of a Novel Anti-TF Glycan Binding Protein The Thomsen-Friedenreich (TF) antigen is a disaccharide (Gal 1-3GalNAc⍺-) that is overexpressed on the cell surface of many cancers, including 90% of lung, breast, prostate, and colon carcinomas. Prior work evolved a novel Sso7d-based glycan binding protein (GBP) against the TF antigen, herein termed “2.4.i”. The evolved ssoGBP 2.4.i demonstrated selective recognition to the TF antigen and structurally similar Lewis C (Lec) antigen (Gal 1-3GlcNAc-), and further recognized aberrant glycan epitopes on TF-expressing cancer cells in vitro. Preliminary predictive models of 2.4.i-TF and 2.4.i-Lec interactions were generated with the web-based High Ambiguity Driven biomolecular DOCKing (HADDOCK) platform. However, recent structural investigations into the 2.4.i-TF interaction have provided experimental evidence not aligning with the preliminary model. Thus, a user-defined docking pipeline with accurate carbohydrate modeling and incorporation of recent structural data for further refinement of a putative binding pocket was critically needed to augment understanding (and ultimate refinement) of anti-TF ssoGBP as functional reagents for cancer diagnostics and therapeutics. In this study, a localized, user-defined HADDOCK pipeline was developed and optimized for protein-glycan docking and used in conjunction with complementary computational tools to interrogate receptor-ligand interactions. Using 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) data of chemical shift perturbations, amino acid residues implicated in binding were established in restraint parameters to guide docking computations. The in silico results align with NMR data gathered from experimental methodology. Notably, simulations preliminarily suggest the possibility of multiple binding modalities of the TF antigen to 2.4.i. Null mutant variants of interest were studied similarly; in particular, the Y28A variant of 2.4.i was investigated to determine whether the mutation induces structural rearrangements that enable neighboring residues to participate in π–π stacking interactions, thereby stabilizing the protein and enhancing ligand binding. In addition to computational work, microscale thermophoresis was performed on Y28A-2.4.i to assess binding affinity to a stereochemically locked, multivalent presentation of the TF antigen. Further work will corroborate HADDOCK docking models, utilizing tools such as CORCEMA-ST and/or PLUMED to directly compare simulated NMR data of models to empirical data. Future directions include deeper investigation of alternative 2.4.i-TF binding configurations. Optimized computational modeling of structural changes introduced by null mutations of interest (including Y28A-2.4.i) and subsequent study of these binding interactions will further characterize binding pocket structure/function. Concurrently, in vitro methods will include probing point mutations to elucidate the roles of individual residues in recognition and binding. Joseph J. Park Author Keean Kawamoto Author Miguel Martinez Author Megan Kizer Author Chemistry, Physical and theoretical Computer simulation Modeling Biophysics--Research Computational Biology Molecular dynamics Proteins Computational chemistry Biochemistry Glycans Glycan-binding proteins GBPs 2.4.i HADDOCK GROMACS GaMD All rights reserved In Copyright text_resources instructional posters riu Providence, Rhode Island Brown University Library 2026 2026-08-04 1 document born digital 10.26300/gcet-dv43