Glycosylation stabilizes TNFα and receptor complexes: structural and biophysical implications

Glycosylation is a ubiquitous post-translational modification that can reshape protein stability and protein-protein recognition, yet its mechanistic impact on cytokine-receptor interactions remains incompletely understood. Here, the influence of glycosylation of tumor necrosis factor alpha (TNFα) on tumor necrosis factor receptor I (TNFR1) and II (TNFR2) binding was investigated. For this purpose, biophysical measurements with molecular modeling and molecular dynamics (MD) simulations were conducted. The thermal stability of glycosylated and non-glycosylated TNFα, glycosylated receptor ectodomains, and their complexes was assessed by nano differential scanning fluorescence (nanoDSF). Glycosylated TNFα exhibited higher thermal stability and formed receptor complexes with increased apparent thermal stability compared with the non-glycosylated preparation. Binding affinity was quantified using microscale thermophoresis (MST), revealing that glycosylated TNFα binds TNFR2 more tightly than non-glycosylated TNFα corresponding to a four-fold affinity increase. For TNFR1, binding was detected for glycosylated TNFα, whereas no detectable binding of the non-glycosylated preparation was observed under the MST conditions used. Complementary MD simulations of TNFα/TNFR1 and TNFα/TNFR2 complexes, indicated that TNFα glycosylation are associated with increased complex stability, with effects dependent on glycan structure and receptor type. However, linear interaction energy analysis showed consistently favorable contributions of glycans only to complex stability, with stronger stabilization for longer glycans and a more pronounced effect for TNFR1 than for TNFR2. Together, our results are consistent with an ensemble-based qualitative model in which glycosylated TNFα forms more stable receptor complexes and may display enhanced receptor binding affinity through glycan-associated stabilization effects.