Probing the roles of active site residues in the 3′-5′ exonuclease of the Werner syndrome protein
Probing the roles of active site residues in the 3′-5′ exonuclease of the Werner syndrome protein
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DOI：
10.1074/jbc.m609657200
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发表时间：
2007-03-30
影响因子：
4.8
通讯作者：
Cho, Yunje
中科院分区：
文献类型：
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作者：
Choi, Jung Min;Kang, Sung Yun;Cho, Yunje
Werner综合征是一种由WS基因突变和Werner蛋白（WRN）功能缺陷引起的早衰性疾病。WRN的缺乏导致基因组不稳定的细胞表型。WRN属于RecQ DNA解旋酶家族，但与其他RecQ家族成员不同，它具有功能性核酸外切酶结构域。我们确定了与Zn 2+和硫酸根离子结合的mWRNexo（残基31-238）的晶体结构。与人WRNexo（hWRNexo）的结构相比，在mWRNexo的H5-H6环和螺旋H6和H7中的几个活性位点残基中观察到显著的构象变化，这可能是因为硫酸根的存在，其模拟底物DNA的磷酸根。特别地，Lys（185）和Tyr（206）的侧链朝向Zn 2+离子重新取向，而Arg（190）的侧链指向远离活性位点中心。这些保守残基的突变分析废除了WRN核酸外切酶活性，表明这些残基在WRNexo活性中起关键作用。底物建模和突变分析的基础上，我们提出了一种机制，WRNexo成为激活后，底物DNA结合。我们还描述了小鼠WRNexo（mWRNexoL，残基31-330）的低分辨率三聚体结构，如小角X射线散射（SAXS）分析所阐明的。
Werner syndrome is a premature aging disease caused by mutations in the WS gene and a deficiency in the function of Werner protein (WRN). The lack of WRN results in a cellular phenotype of genomic instability. WRN belongs to the RecQ DNA helicase family, but unlike other RecQ family members it possesses a functional exonuclease domain. We determined the crystal structure of mWRNexo (residues 31-238) bound to Zn2+ and the sulfate ion. Compared with the structure of human WRNexo (hWRNexo), notable conformational changes were observed in several active site residues in an H5-H6 loop and in helices H6 and H7 of mWRNexo, presumably because of the presence of sulfate, which mimics the phosphate of substrate DNA. In particular, the side chains of Lys(185) and Tyr(206) were reoriented toward the Zn2+ ion, whereas the side chain of Arg(190) pointed away from the active site center. Mutational analysis of these conserved residues abolished WRN exonuclease activity, suggesting that these residues play a critical role in the WRNexo activity. Based on substrate modeling and mutational analyses, we propose a mechanism by which WRNexo becomes activated upon substrate DNA binding. We also describe the low resolution trimeric structure of mouse WRNexo (mWRNexoL, residues 31-330), as elucidated by small angle x-ray scattering (SAXS) analyses.