Deciphering the ATP-binding mechanism(s) in NLRP-NACHT 3D models using structural bioinformatics approaches

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), the first line of defense, are the cytosolic pattern recognition receptors (PRRs) that regulate the inflammatory activity in response to invading pathogens. NLRs are the members of AAA+ ATPase superfamily that comprises of N-terminal EBD(s), a centrally positioned NOD/NACHT and varying range of LRRs towards the C-terminal end. Due to the lack of structural data, the functional aspects of NLRP-signaling mechanism, which includes pathogen recognition, nucleotide-binding, and sensor-adaptor-effector interactions, are not fully understood. In this study, we implemented structural bioinformatics approaches including protein modeling, docking, and molecular dynamics simulations to explore the structural-dynamic features of ADP-/ATP-Mg2+ binding in NLRPNACHT models. Our results indicate a similar mode of ATP-Mg2+ binding in all NLRPNACHT models and the interacting residues are found consistent with reported mutagenesis data. Accompanied by the key amino acids (proposed to be crucial for ATP-Mg2+ coordination), we further have noticed that some additional conserved residues (including ‘Trp’ of the PhhCW motif, and ‘Phe’ and ‘Tyr’ of the GFxxxxRxxYF motif) are potentially interacting with ATP during dynamics; which require further experimentation for legitimacy. Overall, this study will help in understanding the ADP-/ATP-Mg2+ binding mechanisms in NLRPs in a broader perspective and the proposed ATP-binding pocket will aid in designing novel inhibitors for the regulation of inflammasome activity.

核苷酸结合寡聚化结构域（NOD）样受体（NLRs）作为机体防御的第一道防线，是一类胞质模式识别受体（pattern recognition receptors, PRRs），可调控针对入侵病原体的炎症应答活性。NLRs属于AAA+ ATP酶超家族成员，其结构包含N端效应结合结构域（effector binding domain, EBDs）、中央位置的NOD/NACHT结构域，以及C端区域的多种富亮氨酸重复序列（leucine-rich repeats, LRRs）。由于结构数据的匮乏，包括病原体识别、核苷酸结合以及传感器-接头蛋白-效应蛋白相互作用在内的NLRP信号通路机制的功能层面尚未被完全阐明。本研究采用结构生物信息学方法，包括蛋白质建模、分子对接以及分子动力学模拟，以探究NLRP NACHT结构域模型中ADP/ATP-Mg²+结合的结构动态特征。研究结果显示，所有NLRP NACHT结构域模型的ATP-Mg²+结合模式均相似，且其相互作用残基与已报道的诱变数据一致。除了被认为对ATP-Mg²+配位至关重要的关键氨基酸残基外，我们还进一步观察到一些额外的保守残基——包括PhhCW基序中的色氨酸（tryptophan, Trp），以及GFxxxxRxxYF基序中的苯丙氨酸（phenylalanine, Phe）和酪氨酸（tyrosine, Tyr）——在分子动力学过程中可能与ATP发生相互作用；这一点仍需进一步实验验证其合理性。总体而言，本研究将有助于从更广阔的视角理解NLRPs家族中ADP/ATP-Mg²+的结合机制，而本次提出的ATP结合口袋将为设计调控炎症小体活性的新型抑制剂提供参考。