Structural insights into TLR4 activation by SARS-CoV-2 spike protein: implications for inflammatory response modulation.

Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD2) plays a central role in innate immune sensing and inflammatory responses during viral infections. Emerging evidence suggests that viral glycoproteins, including the SARS-CoV-2 spike (S) protein, can aberrantly activate TLR4, contributing to cytokine storms; however, the molecular basis remains unclear. In this study, we investigated the recognition of the SARS-CoV-2 spike protein, in both its monomeric and trimeric forms, by the TLR4/MD2 receptor complex using a comprehensive in silico framework. Protein-protein docking, extended molecular dynamics simulations (500 ns), interaction profiling, principal component analysis, free energy landscape mapping, and binding-affinity calculations were employed. The S1 subunit, particularly the receptor-binding domain (RBD) and N-terminal domain (NTD), emerged as the principal interface for TLR4 and MD2-a novel finding. The spike monomer exhibited stronger and more stable interactions than the trimer, supported by a greater number of hydrogen bonds and salt bridges, lower binding energies, and distinct PCA/energy landscape features. Two N-linked glycosylation sites in the monomer were positioned proximal to the MD2 binding pocket, compared to one in the trimer, suggesting a possible role in modulating receptor activation. Several hotspot residues were also identified as potential therapeutic targets. Collectively, these findings support a model in which the SARS-CoV-2 spike protein engages TLR4/MD2 through domain-specific interactions that may modulate innate immune signalling.