Solution structure of the cGMP binding GAF domain from phosphodiesterase 5 - Insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change
Solution structure of the cGMP binding GAF domain from phosphodiesterase 5 - Insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change
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DOI：
10.1074/jbc.m801577200
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发表时间：
2008-08-15
影响因子：
4.8
通讯作者：
Klevit, Rachel E.
中科院分区：
文献类型：
--
作者：
Heikaus, Clemens C.;Stout, Joseph R.;Klevit, Rachel E.
磷酸二酯酶5（PDE 5）通过调节cGMP水解来控制细胞内cGMP水平。cGMP与N-末端GAF A结构域的结合增加了C-末端催化结构域的水解活性。我们给出了cGMP结合的PDE 5A GAF A结构域的NMR溶液结构。通过37个分子间的核Overhauser效应来定义掩埋结合口袋中的cGMP取向。与来自PDE 2A和腺苷酸环化酶cyaB 2的GAFdomains的比较揭示了一个保守的六链β折叠和四个α螺旋的整体结构域折叠，形成了一个明确的cGMP结合口袋。然而，核苷酸配位与一系列改变的结合接触是不同的。该结构表明核苷酸结合特异性由Asp-196提供，其定位为与cGMP的鸟嘌呤环形成两个氢键。Asp-196的丙氨酸突变破坏cGMP结合并增加仅含有GAF A的构建体中的cAMP亲和力，从而导致改变的cAMP结合结构构象。对串联GAF结构域的NMR研究揭示了在不存在cGMP的情况下的柔性GAF A结构域，并且表明在配体结合后的大的构象变化。此外，我们确定了一个区域的类似的20个残基直接N-末端的GAF A作为关键的串联GAF结构域的紧密二聚化。本文揭示的PDE 5调控结构域的特征为今后研究PDE 5的调控机制和设计PDE 5功能的GAF特异性调控因子提供了初步的结构基础。
Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased bycGMP binding to the N-terminal GAF A domain. We present theNMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison withGAFdomains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMRstudies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of similar to 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.