Identification of Key Residues That Confer Rhodobacter sphaeroides LPS Activity at Horse TLR4/MD-2
Identification of Key Residues That Confer Rhodobacter sphaeroides LPS Activity at Horse TLR4/MD-2
复制标题
DOI：
10.1371/journal.pone.0098776
复制
发表时间：
2014-05-30
期刊：
影响因子：
3.7
通讯作者：
Bryant, Clare E.
中科院分区：
文献类型：
--
作者：
Irvine, Katherine L.;Gangloff, Monique;Bryant, Clare E.
支持六酰化脂质A和四酰化前体脂质IVa如何激活Toll样受体4（TLR 4）的分子决定因素已被充分理解，但其他脂质A物质如何诱导激活尚不清楚。物种特异性研究已经阐明了TLR 4/MD-2如何识别不同的脂质A结构，例如四酰化脂质IVa需要直接的静电相互作用来激动。在这项研究中，我们研究如何pentacylated脂多糖从球形红细菌（RSLPS）拮抗人类TLR 4/MD-2和激活马受体复合物使用的计算方法和跨物种诱变。在功能水平上，我们表明RSLPS是马TLR 4/MD-2的部分激动剂，具有比脂质IVa更大的功效。这些数据表明RSLPS信号传导中额外酰基链的重要性。基于对接分析，我们提出了一个模型，用于定位的RSLPS脂质A部分（RSLA）内的MD-2腔在TLR 4二聚体接口，它允许在马受体复合物的活动。至于脂质IVa，RSLPS激动作用需要与MD-2和TLR 4的物种特异性接触，但RSLA的R2链从MD-2口袋突出以接触脯氨酸442附近的TLR 4二聚体。我们的模型解释了为什么RSLPS仅部分依赖于马TLR 4残基R385，不像脂质IVa。如在人TLR 4中发现的，脯氨酸442突变成丝氨酸残基，揭示了该位点在RSLPS信号传导中的重要性;马TLR 4 R385 G/P442 S双突变完全消除RSLPS活性，而其对应物人TLR 4 G384 R/S441 P不能恢复RSLPS活性。我们的数据突出了配体定位中细微变化的重要性，并表明可能不直接参与配体结合的TLR 4和MD-2残基可以决定给定配体的信号传导结果。这表明受体复合物内的合作结合机制，这在TLR信号传导中变得越来越重要。
The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.