Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.
Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.
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DOI：
10.1126/scisignal.aad4805
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发表时间：
2016-03-01
影响因子：
7.3
通讯作者：
Zhang YJ
中科院分区：
文献类型：
--
作者：
Irani S;Yogesha SD;Mayfield J;Zhang M;Zhang Y;Matthews WL;Nie G;Prescott NA;Zhang YJ
RNA聚合酶II（RNAPII）羧基末端结构域（CTD）磷酸化状态的变化与真核生物转录过程相关。酵母蛋白质转录调节因子1（Rtr 1）和人类同源RNAPII相关蛋白2（RPAP 2）可能作为CTD磷酸酶发挥作用;然而，乳酸克鲁维酵母Rtr 1的晶体结构缺乏共有活性位点。我们确定了一个磷酰基转移结构域在酿酒酵母Rtr 1通过获得和表征的2.6 μ m分辨率的晶体结构。我们确定了一个假定的底物结合口袋之间的锌指结构域和一对螺旋，其中包含一个被困的硫酸根离子的深沟。由于硫酸盐模拟磷酸基团的化学性质，因此结构数据表明该沟代表磷酰基转移活性位点。该沟内衬的残基的突变破坏了用荧光化学底物在体外测定的酶的催化活性，并且突变的Rtr 1的表达未能挽救缺乏Rtr 1的酵母的生长。RPAP 2和保守的推定的催化位点的突变体在相同的化学测定中的磷酸酶活性的表征表明保守的反应机制。我们的数据表明，磷酰基转移结构域的结构和反应机制的磷酰基转移活性的Rtr 1是不同于其他磷酸酶家族。
Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.