X-Ray Crystal Structure Analyses of Inante Immunological TLR-Related Receptor Proteins for Sepsis Treatments

点击翻译按钮获取中文摘要

Innate immunity is the first line of defense against microbial infections. Among microbial components recognized by pathogen sensors, lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is a potent stimulant of immune responses. Excessive responses to the endotoxic LPS frequently result in severe sepsis, a rapidly progressing inflammatory disease with very high mortality.MD-2 forms a stable complex with Toll-like receptor 4 (TLR4) on the cell surface. Lipid A, the primary immunostimulatory core of LPS, is diverse in several species, and these variations are discriminated by the TLR4/MD-2 complex as endotoxic or as anti-endotoxic. Its precursor lipid IVa, the tetra-acylated form of lipid A, acts as an antagonist in human cells but as an agonist in mouse cells.Human MD-2, encoded as a 160 amino-acid glycoprotein with a 16 amino-acid secretion signal, was expressed in methyltropic yeast Pichia pastoris. Polysaccharide moieties of MD-2 were trimmed off with endoglycosidase treatment which leaves a single N-acetyl-glucosamine at each glycosylation site. Thus obtained MD-2 with residues 17-160 was successfully crystallized, but showed crystal twinning. Twinned crystals were transformed into single crystals through optimization of cryoprotectant. The structure of the native MD-2 crystal was determined at 2.0 A resolution. Cocrystals with the lipid IVa complex were obtained from a mixture of MD-2 and lipid IVa. The structure of the complex was refined at 2.2 A resolution.MD-2 shows a deep hydrophobic cavity sandwiched by two (3-sheets, in which four acyl chains of the lipid IVa are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. In the native structure, unexpected electron-densities were observed in the cavity, and were attributed to bound lipidic molecules. These structures suggest that MD-2 plays a principal role in endotoxin recognition, and provide a basis for antiseptic drug development.