【作者】 陈朋；

【导师】 滕脉坤； 牛立文；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 磷酸核糖焦磷酸合成酶PRS(负责催化ATP和核糖5磷酸R5P生成AMP与磷酸核糖焦磷酸PRPP)对于核苷酸,脱氧核苷酸以及它们的衍生物的生物合成都是必不可少的。人类的PRS1的单碱基突变与诸多的疾病,诸如高尿酸血症和痛风,CMTX5综合征,Arts综合征,X染色体连锁的非综合的感神经性耳聋DFN2乃至癌症等直接关联。但是PRS1的催化机制和可能的致病机制目前尚不清楚。我们利用X射线晶体学方法解析了人源野生型PRS1、疾病相关突变体的晶体结构,并利用冷冻电镜方法重构了它们的四级结构。结合PRS1或者其与不同底物或者抑制剂分子复合物(PRS1, PRS1-AMPNPP, PRS1-AMPNPP-R5P, PRS1-ADP)的不同构象的负染电镜结构,证实了前人提出的可能PRS1六聚体为其发挥生物学功能单位的推论,提出了四级结构的相互作用界面。综合基因突变、生化分析和序列比对的信息,阐明了其与底物和抑制剂相互作用的区域,提出了一种可能的PRS1催化及产物PRPP释放的机制。该研究结果对于理解由单点突变导致疾病的发生机制具有重要意义并对由PRS1突变引起的疾病治疗和药物设计提供了参考。PRS1D52H突变将会导致病人红细胞中PRPP浓度显著提高并最终引起高尿酸血症和严重的痛风疾病。D52H突变体的晶体结构显示PRS1第52位天冬氨酸被组氨酸替代的突变可能会完全破坏原先其周围稳定的相互作用网络。酶活实验表明D52H突变体和野生型的PRS1具有类似的催化活力。结合结构比对并通过酶活实验和等温滴定微量热实验证实这种相互作用网络的改变会进一步影响病人PRS1的ADP结合口袋的构象并降低其对抑制剂ADP的敏感性。这一结果很好的解释了PRS1D52H突变体的致病机制,同时也是对于上一篇提出模型的验证。与PRS1可能相互作用的蛋白包括PAP39, PAP41, PRS2, PRS3以及p300KIX结构域。PAP39一直都是作为PRS1可能的负调控蛋白被报道,而PRS1与p300KIX结构域相互作用对酶活的影响未见报道。我们通过PAP39pull down实验证实PAP39可以和PRS1在体外以1：1摩尔比相互作用,并分析了它们的酶动力学作用,结果表明,PAP39应该是PRS1的激活蛋白。我们还通过C端6his标签的PRS1pull down实验证实PRS1可以和KIX结构域在体外以1：1摩尔比相互作用。更多还原

【Abstract】 Phosphoribosyl pyrophosphate synthase is responsible for the synthesis of PRPP and AMP using ATP and R5P as substrates. Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated with multiple diseases such as hyperuricemia and gout, Charcot-Marie-Tooth disease-5(CMTX5), Arts syndrome, X-linked nonsyndromic sensorineural deafness (DFN2) and even cancers because of single base mutations. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism.Here, we solved the wild type structure of PRS1and6of its pathogenetic mutant structures using X-Ray crystallography. We also reconstructed the3D cryo-EM structure of the PRS1apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we proved the hexamer is the functional unit, which also provided the quaternary structure interaction interfaces. With our combined findings based on mutagenesis, biochemical assays and sequence analysis, we reveal conserved substrates and inhibitor binding motifs. Based on these findings, we propose a possible dynamic catalytic mechanism and PRPP release tunnel. The possible mechanism has broad implications for understanding all pathogenic mutants and provides a framework for future therapy or drug design investigations.The D52H missense mutation of PRS1will lead to a conspicuous PRPP content elevation in the erythrocyte of patients and finally induce hyperuricemia and serious gout. In this study, the enzyme activity analysis indicated that D52H-mutant possessed similar catalytic activity to the wild type PRS1, and the2.27A resolution D52H-Mutant crystal structure revealed that the stable interaction network surrounding the52position of PRS1would be completely destroyed by the substitution of histidine. Combined enzyme activity assays, ITC experiments, and structural analysis, we proved that these interaction variations would further influence the conformation of ADP binding pocket of D52H-mutant and reduced the inhibitor sensitivity of PRS1in patient’s body. It might be the D52H pathogenetic mechanism, which also confirmed the viewpoint in the last paper.Many proteins including PAP39, PAP41, PRS2, PRS3and KIX domain of p300are reported to interact with PRS1. It has been reported that PAP39might be a negative regulator of PRS1, however, the influence of the interaction between PRS1and p300KIX domain is unknown. Here, we first confirmed the in vitro interaction between PAP39and PRS1as1:1(molar ratio) by conducting an N-his6-tagged PAP39pull down experiment. Then, we assayed the enzyme activity. PAP39alone displayed no activity, however, when it was added to equal molar amounts of PRS1it displayed a2-fold increased kcat compared with PRS1alone. This result implied that PAP39is an activator of PRS1. Our result was contradictory to an earlier report from1994; however, we stand behind our more rigorous experiment. In addition, we also confirmed the in vitro interaction between p300KIX domain and PRS1as1:1(molar ratio) by conducting a C-his6-tagged PRS1pull down experiment.更多还原