Structural Modulation and Binding of HLA-DQ8 by Cysteine-to-Serine Mutated Insulin Peptide: Insights from Molecular Dynamics Simulations

Type 1 diabetes (T1D) is driven by autoreactive CD4⁺ T-cell responses to pancreatic beta cell antigens presented by disease-associated human leucocyte antigen (HLA) class II molecules. However, the molecular mechanisms by which subtle antigenic modifications promote pathogenic immunity remain incompletely defined. Recent immunopeptidomic studies have identified a cysteine-to-serine substitution at position 19 of the insulin B chain, referred to as InsC19S, as a microenvironment-driven neoepitope that can be presented by HLA class II molecules, including HLA-DQ8, and is recognized by diabetogenic CD4⁺ T cells. In this study we explore potential structural and thermodynamic mechanisms that may contribute to the enhanced immunogenicity associated with this single-amino-acid modification. Using molecular dynamics simulations combined with coarse-grained free-energy-perturbation analyses, we compared HLA DQ8 complexes bound to wild-type (WT) insulin and InsC19S peptides. The InsC19S variant is predicted in simulations to exhibit enhanced binding stability, characterized by increased hydrogen bond occupancy, reduced peptide conformational mobility, and a more favorable binding free energy. In addition, the modified peptide is predicted to induce peptide-dependent conformational adjustments within the HLA-DQ8 peptide-binding groove, resulting in expansion of the conformational landscape and stabilization of distinct low-energy states that are not accessed by the WT complex. Principal component analysis and free-energy landscape mapping suggest that this mutation may promote altered collective motions within HLA DQ8 that are consistent with enhanced peptide major histocompatibility complex (MHC) persistence and optimized antigen presentation geometry. Together, these computational observations suggest a structural framework that may help explain the preferential presentation and pathogenic recognition of InsC19S reported in experimental studies. These findings provide a molecular-level framework that may help link microenvironment-driven insulin neoepitope formation to altered peptide–MHC stability and conformational dynamics in HLA-DQ8.