Site-specific evaluation of mutation-based mimics of histone glycation in the nucleosome

Histone glycation is a non-enzymatic post-translational modification (PTM) associated with aging and metabolic stress, yet its residue-specific structural and functional effects remain poorly understood. Because selectively installing defined glycation adducts is experimentally challenging, amino acid substitution by site-directed mutagenesis is commonly used to mimic such PTMs; however, the validity of these substitutions as models of specific glycation adducts has not been systematically assessed.

Here, we performed atomistic molecular dynamics simulations of the nucleosome core particle to compare wild-type systems, advanced glycation end products (AGEs), and substitution-based mimics. Three sites were examined: H2BK43 and H4K31 modified as Nε-(carboxymethyl)lysine (CML), and H3R42 modified as methylglyoxal-derived hydroimidazolone (MG-H1), with glutamine used to mimic CML and tyrosine to mimic MG-H1.

Glutamine substitutions used to mimic CML reproduced the direction of local structural changes induced by CML at H2BK43 and H4K31, including increased protein contact, flexibility, and solvent exposure. In contrast, tyrosine substitution did not reproduce the effects of MG-H1 at H3R42, instead markedly reducing DNA engagement and electrostatic interactions. Microsecond-scale simulations further revealed a replicate-dependent propensity for asymmetric DNA entry/exit breathing in systems containing the H3R42Y mutation, a behavior not observed in wild-type or other modified systems.

These findings indicate that the reliability of mutation-based glycation mimics depends on both the specific glycation chemistry and the local structural context. Consequently, such models should be structurally or biochemically validated before being used to infer nucleosome dynamics or biological consequences.