Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2.
Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2.
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DOI：
10.4049/jimmunol.1502074
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发表时间：
2016-03-01
期刊：
影响因子：
--
通讯作者：
Jerala R
中科院分区：
文献类型：
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作者：
Vašl J;Oblak A;Peternelj TT;Klett J;Martín-Santamaría S;Gioannini TL;Weiss JP;Jerala R
骨髓分化因子 (MD-2) 是一种细胞外蛋白，与 Toll 样受体 4 (TLR4) 的胞外域相关，在细菌脂多糖 (LPS) 的识别中发挥着关键作用。尽管总体结构和功能高度相似，人类 (h) 和小鼠 (m) MD-2 仍表现出一些与物种相关的差异。 hMD-2 在没有 TLR4 的情况下能够结合 LPS，而 mMD-2 仅当 mMD-2 和 mTLR4 在同一细胞中共表达时才支持 LPS 响应。此前，LPS 结合袋边缘的带电残基已被归因于这种差异。在本研究中，定点诱变用于探索 MD-2 结合口袋内的疏水残基，作为 hMD-2 和 mMD-2 之间功能差异的来源。虽然hMD-2结合袋中残基61和63的疏水性降低保留了野生型hMD-2的特征，但135位缬氨酸相对较小的丙氨酸变化完全消除了LPS与hMD-2突变体的结合。然而，突变体保留了 LPS 与 TLR4 的复合物结合以及细胞活化，从而产生了类似鼠的表型。这些结果得到了分子动力学模拟的支持。我们认为 MD-2 135 位的残基控制着结合袋的动力学及其容纳脂质 A 的能力，脂质 A 受到结合的 TLR4 的变构影响。
Myeloid differentiation factor (MD-2) is an extracellular protein, associated with the ectodomain of Toll-like receptor 4 (TLR4), that plays a critical role in the recognition of bacterial lipopolysaccharide (LPS). Despite high overall structural and functional similarity, human (h) and murine (m) MD-2 exhibit several species-related differences. hMD-2 is capable of binding LPS in the absence of TLR4, whereas mMD-2 supports LPS responsiveness only when mMD-2 and mTLR4 are coexpressed in the same cell. Previously, charged residues at the edge of LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within MD-2 binding pocket, as the source of functional differences between hMD-2 and mMD-2. While decreased hydrophobicity of residues 61 and 63 in hMD-2 binding pocket retained the characteristics of wild type hMD-2, a relatively minor change of valine to alanine at position 135 completely abolished the binding of LPS to the hMD-2 mutant. The mutant, however, retained the LPS binding in complex with TLR4 and also cell activation, resulting in a murine-like phenotype. These results were supported by the molecular dynamics simulation. We propose that the residue at position 135 of MD-2 governs the dynamics of the binding pocket and its ability to accommodate lipid A, which is allosterically affected by bound TLR4.