Protein co-scientist aided simulations of the structural and functional impact of O-GlcNAc Transferase TPR domain deletions and amino acid substitutions associated with X-Linked Intellectual Disability
O-GlcNAc transferase (OGT) is an essential glycosyltransferase that regulates cellular processes through the addition of O-linked {beta}-N-acetyglucosamine (O-GlcNAc) to target proteins. Substrate recognition and protein-protein interactions are mediated by the tetratricopeptide repeat (TPR) domain, yet the contributions of individual TPR repeats to structural stability and function remain poorly understood. Here, we employed an integrated computational framework combining an AI protein co-scientist, Speak to a Protein, which incorporates Boltz-2 for structure prediction and rDock for ligand binding evaluation, with external tools including AlphaFold3 for more advanced structure prediction and the HDOCK server for protein-protein docking. This framework was used to investigate how deletions of TPR domains and disease-associated amino acid substitutions within the TPR domain affect OGT structure and function. Five synthetic truncation OGT isoforms, thirteen single-TPR deletion variants, and five X-linked intellectual disability (XLID)-associated amino acid substitutions were modeled and aligned to wild-type OGT for comparison. Structural perturbations were quantified using backbone root mean square deviation (RMSD), while functional effects were assessed through docking analyses with UDP-GlcNAc, Host Cell Factor 1 (HCF1), and Tet methylcytosine dioxygenase 1 (TET1). Variation in TPR length altered global protein structure and induced long-range conformational changes within the catalytic domain despite no sequence modifications in that region. Single TPR deletions showed that individual repeats contribute unequally to structural stability, with deletions in mid-to-late TPR domains producing the largest structural disruptions. Disease-associated amino acid substitutions also generated conformational changes and reduced predicted ligand binding, consistent with known reductions in enzymatic activity in XLID. Protein-protein docking analyses demonstrated substrate-specific effects, as domain deletion and amino acid substitution variants of OGT had different interactions with TET1 and HCF1. Collectively, these findings identify the TPR domain as a regulatory domain that governs structure, ligand recognition, and protein interactions. More broadly, this work demonstrates how the integration of a protein co-scientist with protein structure modeling tools can be used to predict the structural and functional consequences of domain deletions or natural amino acid variants.