Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins

Abstract

A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

Fig. 1. Schematics of: major structural and topological features of endotoxin

Left) Integration of LPS, via lipid A fatty acyl chains, into the outer leaflet of the outer membrane of GNB; Center) organization of large E aggregates after extraction and purification o of E from GNB; Right) structure of E. coli LPS.

Fig. 2. Model for LBP/CD14/MD2-dependent transformation of E promoting TLR4 dependent cell activation by E

Potency of E is amplified by interactions with E-binding proteins that modify the presentation of E to TLR4. See text for additional details. Note that TLR4-expressing cells include those that express TLR4 ± MD-2.

Fig. 3. Comparison of TLR4 activation (left) and cell surface TLR4 binding (right) by the monomeric complexes of hexaacylated meningococcal LOS with either wild-type (wt) or mutant (F126A or Y102A) human MD-2 or of tetraacylated eritoran.MD-2

TLR4 activation was measured by induced extracellular accumulation of IL-8; TLR4 binding was measured by inhibition of TLR4-dependent binding of LOS.MD-2[¹²⁵I] (60 pM) as described in (33).

Fig. 4. Ribbon and stick models of eritoran or LPS.MD-2 ± TLR4_ECD derived from published X-ray crystal structures (–38)

Note that the left two panels show only the ligand.MD-2 structures while the TLR4 structure is not displayed.

Fig. 5

Effect of change in surface TLR4 expression on potency of LOS.MD-2^wt, as expressed by concentration of LOS.MD-2^wt added (left) or amount of LOS.MD-2^wt bound (right

Fig. 6. Ribbon models of MD-2 bound with lipid IVA (36) or LPS (37) based on X-ray crystal structure

The structure of MD-2 bound to LPS is based on LPS.MD-2.TLR4_ECD with TLR4 not displayed “. The MD-2 is shown in gray while the ligand is shown in black. Note the different orientation of Phe¹²⁶ in the two complexes. Note also the position of the side chain of Tyr¹⁰² within the hydrophobic pocket of MD-2.

Fig. 7. NMR data for [¹³C]LOS.MD-2^wt and [¹³C]LOS.MD-2^F126A complexes

¹³C/¹H high resolution HSQC spectra (A–C) of ¹³CH₃ region of [¹³C]LOS.MD-2^wt (A) and [¹³C]LOS.MD-2^F126A (B) complexes; (C) represents the overlay of the two spectra. (D) Comparison of the paramagnetic relaxation effect (PRE) of Gd(DPTA-BMA) on the ¹³CH₃ peaks of [¹³C]LOS.MD-2 (wt and F126A) complexes. Data were collected on Avance II 800 MHz NMR spectrometer.