Abstract:
BACKGROUND: PRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While human PRPS1 (hPRPS1) and human PRPS2 (hPRPS2) are expressed in most tissues, human PRPS3 (hPRPS3) is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming filaments and cytoophidia is considered as a distinctive mechanism involving the polymerization of the protein. Previously we solved the filament structures of Escherichia coli PRPS (ecPRPS) using cryo-electron microscopy (cryo-EM) 1.
RESULTS: Order to investigate the function and molecular mechanism of hPRPS2 polymerization, here we solve the polymer structure of hPRPS2 at 3.08 Å resolution. hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity.
CONCLUSIONS: Findings suggest that the regulation of hPRPS2 polymer is distinct from ecPRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis.
摘要:
背景:PRPP合酶(PRPS)将焦磷酸基团从ATP转移到核糖-5-磷酸,以产生5-磷酸核糖-1-焦磷酸(PRPP),包括核苷酸在内的几种代谢物生物合成的关键中间体,二核苷酸和一些氨基酸。在人类基因组中编码有三种PRPS同种型。虽然人PRPS1(hPRPS1)和人PRPS2(hPRPS2)在大多数组织中表达,人PRPS3(hPRPS3)仅在睾丸中表达。尽管hPRPS1和hPRPS2共有95%的序列同一性,已经显示hPRPS2对变构抑制较不敏感,并且在某些癌症中在翻译水平上特异性上调。最近的研究表明,PRPS可以在原核生物和真核生物的多种生物中形成称为胞质的亚细胞区室。形成细丝和胞质被认为是涉及蛋白质聚合的独特机制。以前,我们使用低温电子显微镜(cryo-EM)1解决了大肠杆菌PRPS(ecPRPS)的细丝结构。
结果:为了研究hPRPS2聚合的功能和分子机理,在这里,我们以3.08µ分辨率求解hPRPS2的聚合物结构。hPRPS2六聚体在具有变构/竞争性抑制剂ADP的条件下堆叠到聚合物中。清楚地确定了ADP在经典变构位点和催化活性位点的结合模式。破坏六聚体间相互作用的点突变阻止了hPRPS2聚合并导致催化活性显著降低。
结论:研究结果表明,hPRPS2聚合物的调节与ecPRPS聚合物不同,并为具有结构基础的hPRPS2的调节提供了新的见解。