Phosphoryl transfer by protein kinase A is captured in a crystal lattice.
Phosphoryl transfer by protein kinase A is captured in a crystal lattice.
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DOI：
10.1021/ja312237q
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发表时间：
2013-03-27
影响因子：
15
通讯作者：
Taylor, Susan S.
中科院分区：
文献类型：
--
作者：
Bastidas, Adam C.;Deal, Michael S.;Steichen, Jon M.;Guo, Yurong;Wu, Jian;Taylor, Susan S.
cAMP 依赖性蛋白激酶 (PKA) 的催化 (C) 亚基是一种丝氨酸/苏氨酸激酶，负责 cAMP 信号传导的大部分作用，PKA 是整个激酶家族的原型。尽管对 PKA 进行了多项研究，但磷酰基转移所涉及的步骤、催化必需的镁离子的作用以及控制 ADP 释放限速步骤的过程尚未解决。在这里，我们确定了γ-磷酸盐从通常不可水解的ATP类似物腺苷-5'-(β,γ-亚氨基)三磷酸盐(AMP-PNP)缓慢磷酰基转移到蛋白质晶体内的底物肽上的条件。通过将两种产物捕获在晶格中，我们现在拥有所有催化步骤的完整分辨率曲线。一种精细至 1.55 Å 分辨率的晶体结构显示了蛋白质的两种状态，其中 55% 显示完整的 AMP-PNP 和未磷酸化底物，45% 显示 AMP-PNP 的 γ-磷酸盐转移到底物肽上，产生 AMP-PN 和磷酸化底物。另一种结构精炼至 2.15 Å 分辨率，显示磷酰基完全转移至基底。这些结构除了将两种产物捕获在晶格中之外，还表明一种镁离子（以前称为 Mg2）是更稳定的结合离子。磷酰基转移后，Mg2 招募水分子以保留八面体配位几何形状，表明该镁离子的强结合特性，并且在完成磷酰基转移后 Mg2 保留在活性位点，而 Mg1 被排出。因此，Mg1 的损失可能是 ADP 释放限速步骤的重要组成部分。
The catalytic (C) subunit of cAMP-dependent protein kinase (PKA) is a serine/threonine kinase responsible for most of the effects of cAMP signaling, and PKA serves as a prototype for the entire kinase family. Despite multiple studies of PKA, the steps involved in phosphoryl transfer, the roles of the catalytically essential magnesium ions, and the processes that govern the rate-limiting step of ADP release are unresolved. Here we identified conditions that yielded slow phosphoryl transfer of the γ-phosphate from the generally non-hydrolyzable analog of ATP, adenosine-5'-(β,γ-imido)triphosphate (AMP-PNP), onto a substrate peptide within protein crystals. By trapping both products in the crystal lattice, we now have a complete resolution profile of all the catalytic steps. One crystal structure refined to 1.55 Å resolution shows two states of the protein with 55% displaying intact AMP-PNP and unphosphorylated substrate and 45% displaying transfer of the γ-phosphate of AMP-PNP onto the substrate peptide yielding AMP-PN and phosphorylated substrate. Another structure refined to 2.15 Å resolution displays complete phosphoryl transfer to the substrate. These structures, in addition to trapping both products in the crystal lattice, implicate one magnesium ion, previously termed Mg2, as the more stably bound ion. Following phosphoryl transfer, Mg2 recruits a water molecule to retain an octahedral coordination geometry suggesting strong binding character of this magnesium ion, and Mg2 remains in the active site following complete phosphoryl transfer while Mg1 is expelled. Loss of Mg1 may thus be an important part of the rate-limiting step of ADP release.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4.8
作者：
Iyer, GH;Moore, MJ;Taylor, SS
通讯作者：
Taylor, SS
DOI：
10.1038/nsb780
发表时间：
2002-04-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Madhusudan;Akamine, P;Taylor, SS
通讯作者：
Taylor, SS
影响因子：
5.7
作者：
Bao, Zhao Qin;Jacobsen, Douglas M.;Young, Matthew A.
通讯作者：
Young, Matthew A.
影响因子：
2.9
作者：
Herberg, FW;Doyle, ML;Taylor, SS
通讯作者：
Taylor, SS