Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA
Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA
复制标题
DOI：
10.1110/ps.0234503
复制
发表时间：
2003-04-01
期刊：
影响因子：
8
通讯作者：
Morikawa, K
中科院分区：
文献类型：
--
作者：
Matsumiya, S;Ishino, S;Morikawa, K
采用定点突变的方法，从极端嗜热古菌Pyrococcusfuriosus中制备了两个突变的增殖细胞核抗原PfuPCNA（D143 A）和PfuPCNA（D143 A/D147 A）。凝胶过滤的结果表明，D143和D147突变显著影响PfuPCNA三聚体结构的稳定性。PfuPCNA（D143 A/D147 A）仍具有刺激DNA聚合酶反应的活性，但PfuPCNA（D143 A/D147 A）失去了活性。测定了突变体PfuPCNAs的晶体结构。虽然野生型PCNA形成一个环形的三聚体与分子间氢键之间的N-和C-末端结构域，突变PfuPCNAs存在作为V形二聚体通过分子间氢键之间的两个C-末端结构域的晶体。由于突变的残基通过它们在野生型PfuPCNA中的侧链参与分子间离子对，这些离子对似乎在维持PfuPCNA三聚体的环形结构中起关键作用。晶体结构的比较揭示了有趣的构象。PfuPCNA亚基中每个结构域的柔性。PCNA的这种结构多样性可能参与了环的打开和关闭机制。
Two mutant proliferating cell nuclear antigens from the hyperthermophilic archaeon Pyrococcus furiosus, PfuPCNA(D143A) and PfuPCNA(D143A/D147A), were prepared by site-specific mutagenesis. The results from gel filtration showed that mutations at D143 and D147 drastically affect the stability of the trimeric structure of PfuPCNA. The PfuPCNA(D143A) still retained the activity to stimulate the DNA polymerase reaction, but PfuPCNA(D143A/D147A) lost the activity. Crystal structures of the mutant PfuPCNAs were determined. Although the wild-type PCNA forms a toroidal trimer with intermolecular hydrogen bonds between the N- and C-terminal domains, the mutant PfuPCNAs exist as V-shaped dimers through intermolecular hydrogen bonds between the two C-terminal domains in the crystal. Because the mutated residues are involved in the intermolecular ion pairs through their side chains in the wild-type PfuPCNA, these ion pairs seem to play a key role in maintaining the toroidal structure of the PfuPCNA trimer. The comparison of the crystal structures revealed intriguing conformational. flexibility of each domain in the PfuPCNA subunit. This structural versatility of PCNA may be involved in the mechanisms for ring opening and closing.