Maharana Jitendra, Patra Mahesh Chandra, De Bidhan Chandra, Sahoo Bikash Ranjan, Behera Bijay Kumar, De Sachinandan, Pradhan Sukanta Kumar
Biotechnology Laboratory, Central Inland Fisheries Research Institute, Kolkata, 700120, West Bengal, India.
J Mol Recognit. 2014 May;27(5):260-75. doi: 10.1002/jmr.2357.
Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)-a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2-leucine rich repeat (LRR) domain activates NF-κB signaling through a protein-protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD-CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD-CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2-LRR confer MDP recognition by hydrophobic and hydrogen bond (H-bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2-CARDa and the electronegative surface on zRIP2-CARD reinforced mostly by H-bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2-CARDa and Asp494 of zRIP2-CARD. Arg11 and Lys53 of zNOD2-CARDa and Ser498 and Glu508 of zRIP2-CARD are critical residues for CARD-CARD interaction and NOD2 signaling. The 2.7 Å H-bond between Lys104 of the linker and Glu508 of zRIP2-CARD suggests a possible role of the linker for shaping CARD-CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD-CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective.
核苷酸结合寡聚化结构域（NOD2）是天然免疫的关键组成部分，对胞壁酰二肽（MDP）具有高度特异性，MDP是细菌细胞壁的一种肽聚糖成分。NOD2富含亮氨酸重复序列（LRR）结构域对MDP的识别通过NOD2的半胱天冬酶激活和募集结构域（CARD）与下游受体相互作用激活蛋白激酶2（RIP2）之间的蛋白质-蛋白质相互作用来激活NF-κB信号通路。由于缺乏晶体/NMR结构，对MDP识别和CARD-CARD相互作用了解甚少。在此，我们使用各种计算机模拟方法预测了斑马鱼NOD2（zNOD2）中可能的MDP和CARD-CARD结合表面。结果表明，位于zNOD2-LRR凹面的保守残基Phe819、Phe871、Trp875、Trp929、Trp899和Arg845通过疏水和氢键（H键）相互作用实现MDP识别。分子动力学模拟揭示了zNOD2-CARDa上的正电表面与zRIP2-CARD上的负电表面之间的稳定结合，这种结合主要由氢键和静电相互作用加强。重要的是，在zNOD2-CARDa的Arg60和zRIP2-CARD的Asp494之间观察到一个3.5埃的盐桥。zNOD2-CARDa的Arg11和Lys53以及zRIP2-CARD的Ser498和Glu508是CARD-CARD相互作用和NOD2信号传导的关键残基。连接子的Lys104与zRIP2-CARD的Glu508之间2.7埃的氢键表明连接子在塑造CARD-CARD相互作用中可能发挥的作用。这些发现与现有的诱变数据一致。我们首次深入了解了斑马鱼中的MDP识别和CARD-CARD相互作用，这将有助于从更广泛的角度理解NOD信号传导的分子基础。
