Fig 1: The IFL of Ebola glycoprotein GP1/2 docks in the LPS binding site of the TLR4/MD2 dimer(A and B) The best docking pose for IFL (A) overlaps perfectly with LPS binding site of MD2 (B). Both are depicted in yellow spheres.(C) Trimeric prefusion GP1,2 as observed in crystal structure 3CSY (Lee et al., 2008).(D) Monomeric sGP1, 2 in the same orientation as observed in the trimer.(E) IFL deletion mutant (marked with asterisk) maintains the overall fold of GP1/2 but is unable to crosslink TLR4/MD2 into a signaling competent complex.(F) TLR4-MD2 can be crosslinked by one or two sGP1,2 molecules (second binding site not represented).(G) Close-up view of the IFL inside MD2 hydrophobic cavity. Structural representations are generated with The PyMOL Molecular Graphics System, v.2.5.2, Schrödinger.
Fig 2: WT and deglycosylated GP interacts directly with TLR4 and MD2 except for the fusion loop mutant GP?FuLoImmunoprecipitation conducted with purified TLR4, MD2, and GP with different states of glycosylation via specific antibodies for the human proteins. Here, 2 µg TLR4 and MD2 and 5 µg GP were incubated and immunoprecipitated with either anti-TLR4 or anti-MD2 antibodies immobilized to protein A/G beads.(A) Input for co-immunoprecipitation (coIP) with TLR4.(B) All GP species were co-precipitated with TLR4 except GP?FuLo.(C) Input for coIP with MD2.(D) All GP species were co-precipitated with MD2 except GP?FuLo. All deglycosylated GP species were capable of binding TLR4. The “+” indicates the presence of TLR4 or MD2 whereas the “–“ indicates the absence in the coIP sample.Black arrows indicate TLR4 (A and B) and MD2 (C and D). GP, WT glycoprotein; OG, GP protein after treatment with O-glycosidase; PF, GP after treatment with PNGase F; EH, GP after treatment with Endo H; FL, GP with deleted internal fusion loop GP?FuLo; G-, GP expressed in Expi293 GnTI- cells.
Fig 3: sGP1,2 binds TLR4/MD2 beyond the LPS binding pocket(A) Protein-protein contacts include residues from both TLR4 chains as well as MD2.(B) Two copies of Ebola GP1/2 glycoproteins crosslink TLR4/MD2 into a signaling competent complex.(C) GP1/2 displays additional contacts outside the hydrophobic cavity of MD2, to which the IFL binds, increasing the interface to also potentially include glycan-mediated contacts.
Fig 4: The internal fusion loop of GP is critical for TLR4 activation(A) The pre-treatment of stable hTLR4/MD2/CD14 HEK293 cells with RSLA reduces TLR4 activation by GP and LPS equally, suggesting a similar mechanism. The cells were pre-incubated with RSLA at concentrations of 100 ng/µL, 300 ng/µL, and 1 µg/µL for 1 h pre-induction, followed by LPS concentrations of 10 and 100 ng/µL and 100 and 500 ng/µL of GP for 6 h, and NF-?B activity was assessed.(B) Sequence analysis of GP according to the Kyte and Doolittle scale for hydrophobicity reveals five regions of high hydrophobicity.(C) Deletion of the internal fusion loop sequence, hit no. 4, in fully glycosylated GP diminished TLR4 activation as evident from NF-?B signaling assays. The cells were incubated with 100 ng/µL LPS and 500 ng/µL GP for 6 h, and activity was assessed. The negative control consisted of purified GP, treated with proteases for 1 h, and was followed by boiling for 20 min.The negative control was set to 1-fold change and the values normalized to it. Statistical significance was assessed using a one-way ANOVA test. Data presented as means of triplicates (A and C).
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