Department of Biochemistry and Molecular Biology, Thomas Jefferson University , 233 South 10th Street, Philadelphia, Pennsylvania 19107, United States.
Biochemistry. 2014 Feb 11;53(5):862-71. doi: 10.1021/bi401240x. Epub 2014 Jan 31.
PIR1 is an atypical dual-specificity phosphatase (DSP) that dephosphorylates RNA with a higher specificity than phosphoproteins. Here we report the atomic structure of a catalytically inactive mutant (C152S) of the human PIR1 phosphatase core (PIR1-core, residues 29-205), refined at 1.20 Å resolution. PIR1-core shares structural similarities with DSPs related to Vaccinia virus VH1 and with RNA 5'-phosphatases such as the baculovirus RNA triphosphatase and the human mRNA capping enzyme. The PIR1 active site cleft is wider and deeper than that of VH1 and contains two bound ions: a phosphate trapped above the catalytic cysteine C152 exemplifies the binding mode expected for the γ-phosphate of RNA, and ∼6 Å away, a chloride ion coordinates the general base R158. Two residues in the PIR1 phosphate-binding loop (P-loop), a histidine (H154) downstream of C152 and an asparagine (N157) preceding R158, make close contacts with the active site phosphate, and their nonaliphatic side chains are essential for phosphatase activity in vitro. These residues are conserved in all RNA 5'-phosphatases that, analogous to PIR1, lack a "general acid" residue. Thus, a deep active site crevice, two active site ions, and conserved P-loop residues stabilizing the γ-phosphate of RNA are defining features of atypical DSPs that specialize in dephosphorylating 5'-RNA.
PIR1 是一种非典型的双特异性磷酸酶 (DSP)，它对 RNA 的去磷酸化特异性高于对磷酸化蛋白。在这里，我们报告了人类 PIR1 磷酸酶核心 (PIR1-core，残基 29-205) 的催化失活突变体 (C152S) 的原子结构，分辨率为 1.20 Å。PIR1-core 与痘苗病毒 VH1 相关的 DSP 以及杆状病毒 RNA 三磷酸酶和人类 mRNA 加帽酶等 RNA 5'-磷酸酶具有结构相似性。PIR1 活性位点裂缝比 VH1 更宽更深，并且包含两个结合离子：位于催化半胱氨酸 C152 上方的磷酸基团捕获了 RNA γ-磷酸的结合模式的例子，并且在大约 6 Å 处，一个氯离子与通用碱基 R158 配位。PIR1 磷酸结合环 (P 环) 中的两个残基，C152 下游的组氨酸 (H154) 和 R158 前的天冬酰胺 (N157)，与活性位点磷酸紧密接触，它们的非脂族侧链对于体外磷酸酶活性是必需的。这些残基在所有 RNA 5'-磷酸酶中都是保守的，与 PIR1 类似，它们缺乏“通用酸”残基。因此，深的活性位点裂缝、两个活性位点离子和保守的 P 环残基稳定 RNA 的 γ-磷酸是专门用于去磷酸化 5'-RNA 的非典型 DSP 的特征。
