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

先天免疫是抵抗微生物感染的第一道防线。在病原体传感器识别的微生物组分中，革兰氏阴性菌外膜中的脂多糖（LPS）是免疫应答的有效刺激物。对内毒素LPS的过度反应经常导致严重的脓毒症，这是一种进展迅速的炎性疾病，具有非常高的死亡率。脂质A，LPS的主要免疫刺激核心，在几个物种中是不同的，并且这些变化通过TLR 4/MD-2复合物作为内毒素或作为抗内毒素来区分。人MD-2是一种由160个氨基酸组成的糖蛋白，具有16个氨基酸的分泌信号。MD-2的多糖部分用内切糖苷酶处理修剪掉，这在每个糖基化位点留下单个N-乙酰基-葡糖胺。由此获得的具有残基17-160的MD-2成功地结晶，但显示出晶体孪晶。通过对冷冻保护剂的优化，将双晶转化为单晶。天然MD-2晶体的结构在2.0 A分辨率下测定。从MD-2和脂质IVa的混合物中获得具有脂质IVa复合物的共晶体。复合物的结构在2.2 A分辨率下得到了精细化。MD-2显示了夹在两个β-片层之间的深疏水空腔，其中脂质IVa的四个酰基链被完全限制。磷酸化葡糖胺部分位于腔的入口处。在天然结构中，在腔中观察到意外的电子密度，并归因于结合的双金属分子。这些结构表明MD-2在内毒素识别中起主要作用，并为抗菌药物的开发提供了基础。

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.