A tandem motif-based and structural approach can identify hidden functional phosphodiesterases.
A tandem motif-based and structural approach can identify hidden functional phosphodiesterases.
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DOI：
10.1016/j.csbj.2021.01.036
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发表时间：
2021
影响因子：
6
通讯作者：
Jaworski K
中科院分区：
文献类型：
--
作者：
Kwiatkowski M;Wong A;Kozakiewicz A;Gehring C;Jaworski K
环核苷酸单磷酸 (cNMP) 越来越被认为是控制原核生物和真核生物许多生理和发育过程的重要信号分子。 cNMP 的降解与其生成同样重要，因为它提供了瞬时和动态细胞水平调节的能力，但与它们的生成酶不同，降解酶环核苷酸磷酸二酯酶 (PDE) 在高等植物中有些难以捉摸。基于序列分析和典型偏微分方程催化中心的结构特性，我们开发了一个共有序列搜索基序，并用它来识别候选偏微分方程。其中之一是拟南芥 K+-摄取渗透酶 (AtKUP5)。结构和分子对接分析表明，所鉴定的 PDE 结构域占据了该蛋白的 C 末端，形成了一个暴露于溶剂的独特口袋，可以在空间上容纳环单磷酸腺苷 (cAMP) 底物，重要的是，cAMP 呈现出有利于与共有基序中的关键氨基酸相互作用的结合姿势。 PDE 活性通过灵敏的液相色谱串联质谱 (LC-MS/MS) 方法进行确认。值得注意的是，这种活性是由 Ca2+/CaM 复合物刺激的，表面等离子共振 (SPR) 证实了 Ca2+/CaM 复合物与 PDE 中心的结合。由于 AtKUP5 还具有 K+ 转运所必需的腺苷酸环化酶 (AC) 活性，因此我们提出，这种双重兼职 AC-PDE 架构可提供调节作用，对 cAMP 水平进行复杂的分子内调节，从而实现 K+ 稳态中 cAMP 信号的微调。
Cyclic nucleotide monophosphates (cNMPs) are increasingly recognized as essential signaling molecules governing many physiological and developmental processes in prokaryotes and eukaryotes. Degradation of cNMPs is as important as their generation because it offers the capability for transient and dynamic cellular level regulation but unlike their generating enzymes, the degrading enzymes, cyclic nucleotide phosphodiesterases (PDEs) are somewhat elusive in higher plants. Based on sequence analysis and structural properties of canonical PDE catalytic centers, we have developed a consensus sequence search motif and used it to identify candidate PDEs. One of these is an Arabidopsis thaliana K+-Uptake Permease (AtKUP5). Structural and molecular docking analysis revealed that the identified PDE domain occupies the C-terminal of this protein forming a solvent-exposed distinctive pocket that can spatially accommodate the cyclic adenosine monophosphate (cAMP) substrate and importantly, cAMP assumes a binding pose that is favorable for interactions with the key amino acids in the consensus motif. PDE activity was confirmed by the sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Notably, this activity was stimulated by the Ca2+/CaM complex, the binding of which to the PDE center was confirmed by surface plasmon resonance (SPR). Since AtKUP5 also has adenylate cyclase (AC) activity that is essential for K+ transport, we propose that this dual moonlighting AC-PDE architecture, offers modulatory roles that afford intricate intramolecular regulation of cAMP levels thereby enabling fine-tuning of cAMP signaling in K+ homeostasis.
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影响因子：
4.6
作者：
Kasahara M;Suetsugu N;Urano Y;Yamamoto C;Ohmori M;Takada Y;Okuda S;Nishiyama T;Sakayama H;Kohchi T;Takahashi F
通讯作者：
Takahashi F
影响因子：
4.8
作者：
Ludidi, N;Gehring, C
通讯作者：
Gehring, C
影响因子：
11.6
作者：
Shen, Qingwen;Zhan, Xinqiao;Hong, Yueyun
通讯作者：
Hong, Yueyun
DOI：
10.1186/s12964-016-0133-2
发表时间：
2016-05-11
期刊：
Cell communication and signaling : CCS
影响因子：
--
作者：
Donaldson L;Meier S;Gehring C
通讯作者：
Gehring C
影响因子：
5.7
作者：
Wong A;Gehring C;Irving HR
通讯作者：
Irving HR