The Biophysical Characterisation and SAXS Analysis of Human NLRP1 Uncover a New Level of Complexity of NLR Proteins

NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs.

核苷酸结合寡聚化结构域样受体（NOD-like receptors, NLRs）是一类重要的种系编码模式识别受体，在炎症信号通路的调控中发挥关键作用。它们作为危险感知传感器，启动炎症应答与细胞因子的产生。由于NLR功能异常会引发慢性炎症与自身免疫疾病，学界对阐明其分子层面的工作机制有着极高的研究热情。迄今为止，学界已对NLRs的生物学功能积累了大量研究认知，但由于难以获得可溶性且稳定的重组NLR蛋白，相关生物物理与结构生物学研究始终受到掣肘。NLRP1是一类可形成炎性小体的NLR，据信可通过结合胞壁酰二肽（muramyl dipeptide, MDP）被激活，并诱导半胱天冬氨酸蛋白酶1（caspase 1）的活化。本研究成功鉴定出NLRP1的一段可溶性片段，该片段包含NACHT寡聚化结构域（NACHT oligomerization domain）与推定的MDP感知亮氨酸富集重复序列（Leucine-Rich Repeat, LRR）结构域。我们对该蛋白构建体开展了生物物理与生化表征，并通过尺寸排阻色谱-小角X射线散射（SEC-SAXS）分析，计算得到了其低分辨率分子轮廓。研究数据显示，该蛋白可与三磷酸腺苷（ATP）组成型结合，且水解该三磷酸核苷酸的能力可忽略不计；同时该蛋白呈现单体延伸构象，这与炎性小体复合物中NLRC4所采取的构象高度相似。此外，我们的实验结果表明，仅存在MDP并不足以促进该NACHT-LRR片段发生自身寡聚化，这提示MDP要么结合于NACHT-LRR模块之外的区域，要么并非NLRP1的天然配体。综上，本研究数据表明，NLRP1的作用机制与近期报道的其他NLR家族成员存在显著差异。