Figure 1.
Solutions of the docking studies between the human TLR4-MD-2 complex and glucosamine molecules performed with GRID protocol.
The human TLR4-MD-2 complex is shown with its points of interaction with glucosamine molecules. Blue – human TLR4-MD-2 complex structure represented as ribbons; Red – interacting protein residues and glucosamine molecules with the latter shown as CPK, scaled to the number of molecules at each spot (0.9 for small spots, 1.7 for medium spots and 2 for large spots). The black ellipse highlights MD-2.
Figure 2.
Interaction studies between the dendrimers and human MD-2.
(A) Solvent accessible surface area (SASA) was determined for each human MD-2 residue; (B) Docking studies performed with Patchdock; (C) Docking studies performed with Hex. Black columns – dendrimer modified with 8 glucosamines. Grey columns – unmodified dendrimer.
Figure 3.
Difference between the number of interactions of human MD-2 with the unmodified dendrimer and the partially glycosylated dendrimer.
Black columns – docking study performed with Hex; Grey columns – docking study performed with Patchdock. The Patchdock results showed a larger number of interactions with human MD-2 along its whole sequence. The Hex results showed a clear preference for interactions with residues lining the entrance to human MD-2's hydrophobic pocket (i.e., residues 84 to 127).
Figure 4.
Number of interactions between human MD-2 and ligands normalised to the energy values for each interaction.
The graph shows the difference between the number of interactions with human MD-2 of the unmodified dendrimer and the partially glycosylated dendrimer from the docking study performed with Hex after it was normalised for the energy value for each interaction. The graph also shows the residues that line the opening of human MD-2's hydrophobic pocket. The secondary structure of human MD-2 is shown with the solid grey arrows representing the ß6 (Pro88 to Ile94) and ß7 strands (Val113 to Ser120). The grey box represents the α-helix (Phe104 to Lys109). The lines represent the loops.
Figure 5.
Crystal structure of human MD-2 with PDB entry 2E56 displayed as its electrostatic potential surface.
(A – left hand side figure):- Residues Arg90, Lys91, Ser118 and Lys122 on human MD-2 form electrostatic interactions with lipid A. The interaction of Lys122 and Arg90 with the hydrophilic moiety of lipid A tethers LPS to human MD-2's cavity [15]. The 4′ phosphate on lipid A's first glucosamine binds to Ser118 and the 1′ phosphate on lipid A's second glucosamine binds to Lys122. In addition, Tyr102 is crucial for the subsequent hydrogen bond interaction of the human MD-2-LPS complex with human TLR4 [55]. (B – middle figure):- The residues Lys91, Arg96, Tyr102 and Ser118 form electrostatic interactions with the partially glycosylated dendrimer. (C – right hand side figure):- Human MD-2 is shown in its “top right” orientation. The residues Ser98, Tyr102, Arg106, Lys109, Thr112, Asn114 and Thr116 form electrostatic interactions with the partially glycosylated dendrimer. These residues, which border the entrance of human MD-2's hydrophobic pocket, are labelled in the figures.
Figure 6.
Snapshots of a molecular dynamics simulation of the partially glycosylated dendrimer with human MD-2 obtained with Hex.
These figures show the molecule blocking the entrance to human MD-2's hydrophobic pocket. Top left and across the page to top right:- 0 ns; 0.8 ns and 1.6 ns. Bottom left and across the page to bottom right:- 2.4 ns; 3.2 ns and 4 ns. Human MD-2 is displayed as ribbons, the partially glycosylated dendrimer as tube, and the surface glucosamines as CPK.
Figure 7.
Surface contact area between human MD-2 and the partially glycosylated dendrimer.
(A) 0 ns, (B) 1.6 ns, (C) 2.4 ns. Left – frontal view of human MD-2. Right – side view of human MD-2. The human MD-2 surface is shown in grey, the partially glycosylated dendrimer's surface is shown in pink with 80% transparency, and their contact surface area is shown in green.
Figure 8.
Interactions observed during a 4.8 ns molecular dynamics simulation of the partially glycosylated dendrimer – human MD-2 complex.
Red – number of contacts of 1.3 Å between the dendrimer's surface glucosamine molecules and human MD-2. Blue – number of contacts of 1.3 Å between the partially glycosylated dendrimer (taking the molecule in its entirety) and human MD-2. Green – number of intermolecular hydrogen bond contacts between the partially glycosylated dendrimer (taking the molecule in its entirety) and human MD-2. Taken together, these results show that the affinity of the partially glycosylated dendrimer for human MD-2 involved both the dendrimer's surface glucosamine molecules and several of its peripheral carboxylic acid branches. Notably, these hydrogen bond interactions increased with time. This enabled the partially glycosylated dendrimer to block the entrance to human MD-2's hydrophobic pocket and thereby prevent access of the lipid chains of LPS.
Figure 9.
Interaction of the partially glycosylated dendrimer's surface with human MD-2's surface.
Residues shown in red are <1.3 Å away from the surface of human MD-2. Glucosamine residues are shown in ball and stick configuration. (A) Side view of human MD-2's pocket; (B) frontal view of human MD-2's pocket.
Figure 10.
Schematic representation of the proposed mechanism of action of partially glycosylated dendrimers on human MD-2.
Green – hydrophobic residues. Red and blue – hydrophilic regions.