Critical residues involved in Toll-like receptor 4 activation by cationic lipid nanocarriers are not located at the lipopolysaccharide-binding interface

Abstract

DiC14-amidine is a cationic lipid that was originally designed as a lipid nanocarrier for nucleic acid transport, and turned out to be a Toll-like receptor 4 (TLR4) agonist as well. We found that while E. coli lipopolysaccharide (LPS) is a TLR4 agonist in all species, diC14-amidine nanoliposomes are full agonists for human, mouse and cat receptors but weak horse agonists. Taking advantage of this unusual species specificity, we used chimeric constructs based on the human and horse sequences and identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine. Interestingly, these regions lie outside the known LPS-binding domain. Competition experiments also support our hypothesis that diC14-amidine interacts primarily with TLR4 hydrophobic crevices located at the edges of the TLR4/TLR4* dimerization interface. We have characterized potential binding modes using molecular docking analysis and suggest that diC14-amidine nanoliposomes activate TLR4 by facilitating its dimerization in a process that is myeloid differentiation 2 (MD-2)-dependent and cluster of differentiation 14 (CD14)-independent. Our data suggest that TLR4 may be activated through binding at different anchoring points, expanding the repertoire of TLR4 ligands to non-MD-2-binding lipids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1915-1) contains supplementary material, which is available to authorized users.

An immunoinformatic approach driven by experimental proteomics: in silico design of a subunit candidate vaccine targeting secretory proteins of Leishmania donovani amastigotes

“…Notably, this rearrangement also involved participation of other critical histidine (His431, His555) residues at the TLR4-TLR4* interface [80] unlike the unbound structure (Additional file 8: Figure S5). Overall, these events are congruent with non-canonical TLR4 activation model mediated by microbial peptides, metals and cationic lipid nano-carriers, which are suggested to not confer canonical interaction with other monomers but to induce bond rearrangement among receptor monomers upon interaction [74][75][76][77]. Although the exact mechanism remains to be elucidated, our observations suggest that the vaccine construct may possess a characteristic peptide feature of a non-canonical TLR4 ligand [81,82], which may facilitate TLR4-TLR4* dimerization for downstream activation of immune cells.…”

“…Vaccine-specific, but not parasite protein-specific humoral response was predicted, and this can be used as a biomarker of vaccine efficacy [46,73] without eliciting a parasite-specific B cell response. Moreover, the construct structure showed a good binding affinity in previously reported binding cavity of TLR4 [74][75][76][77].…”

“…Additionally, reduction in electrostatic surface potential at the vaccine-bound TLR4 interface was observed after docking, which was consistent in post-simulation structural interface. Simultaneously, it was observed that a homo-dimer destabilizing His458-His458* repulsion [76] at pre-dock TLR4 was nullified and superseded post-dock by a solvent stable pihydrophobic interaction. It is thus possible that a change in the interpolated charge difference between pre-dock and post-dock TLR4 interface could have contributed to the bonding rearrangement between TLR4 ECDs.…”

An immunoinformatic approach driven by experimental proteomics: in silico design of a subunit candidate vaccine targeting secretory proteins of Leishmania donovani amastigotes

“…Vaccine specific, but not parasite protein specific humoral response was predicted, and this can be used as biomarker of vaccine efficacy [46,111] without eliciting parasite specific B cell response. Moreover, the construct structure showed good binding affinity in previously reported binding cavity of TLR4 [112][113][114][115].…”

“…Besides, reduction in electrostatic surface potential at the vaccine bound TLR4 interface was observed after docking, which was consistent in post-simulation structural interface. Simultaneously, it was observed that a homo-dimer destabilizing His458-His458* repulsion [114] at pre-dock TLR4 was nullified and superseded post-dock by a solvent stable pi-hydrophobic interaction. It is thus possible that change in interpolated charge difference between pre-dock and post-dock TLR4 interface could have contributed to the bonding rearrangement between TLR4 ECDs.…”