Schultz Joachim E
Pharmazeutisches Institut der Universität Tübingen, Morgenstelle 8, Tübingen, 72076, Germany.
Handb Exp Pharmacol. 2009(191):93-109. doi: 10.1007/978-3-540-68964-5_6.
The GAF domain is a small-molecule-binding-domain (SMBD) identified in >7400 proteins. However, mostly the ligands are unknown. Here we mainly deal with regulatory N-terminal tandem GAF domains, GAF-A and GAF-B, of four mammalian phosphodiesterases (PDEs) and of two cyanobacterial adenylyl cyclases (ACs) which bind cyclic nucleotides. These tandem GAFs are preceded by N-terminal sequences of variable lengths and a function of their own. In mammals, GAF domains are found only in cyclic nucleotide PDEs 2, 5, 6, 10, and 11. cAMP is the ligand for phosphodiesterase 10, cGMP for the others. Two cyanobacterial ACs, CyaB1 and 2, carry regulatory cAMP-binding tandem GAF domains which are similar in sequence to the mammalian ones. These tandem GAF domains have a prominent NKFDE motif which contributes to ligand binding in an as yet unknown manner. Contradicting structures (parallel vs. antiparallel) are available for the tandem GAF domains of PDE 2 and AC CyaB2. In addition, the structures of phosphodiesterase 5 and 10 GAF monomers with bound ligands have been solved. In all instances, cyclic nucleotide binding involves specific protein-ligand interactions within a tightly closed binding pocket and minimal solvent exposure of the ligand. The PDE tandem GAF domains can functionally substitute for the tandem of the cyanobacterial AC CyaB1; e.g. cGMP-regulation is grafted onto the AC using tandem GAFs from PDEs 2, 5 and 11. Studies of GAF domain-regulated PDEs are hampered by the identities of regulator and substrate molecules. Using AC CyaB1 as a reporter which uses ATP as a substrate solves this issue and makes the tandem GAF domains of mammalian PDEs available for detailed kinetic and mechanistic studies. In addition, drugs which potentially act on PDE regulatory domains may be assayed with such a novel test system.
GAF结构域是在7400多种蛋白质中鉴定出的一种小分子结合结构域（SMBD）。然而，大多数情况下其配体尚不清楚。在此，我们主要研究四种哺乳动物磷酸二酯酶（PDE）和两种蓝藻腺苷酸环化酶（AC）的调节性N端串联GAF结构域GAF-A和GAF-B，它们可结合环核苷酸。这些串联GAF结构域之前是长度可变的N端序列且具有自身功能。在哺乳动物中，GAF结构域仅存在于环核苷酸磷酸二酯酶2、5、6、10和11中。cAMP是磷酸二酯酶10的配体，cGMP是其他磷酸二酯酶的配体。两种蓝藻AC，即CyaB1和2，携带调节性cAMP结合串联GAF结构域，其序列与哺乳动物的相似。这些串联GAF结构域有一个突出的NKFDE基序，它以一种尚不清楚的方式有助于配体结合。PDE 2和AC CyaB2的串联GAF结构域存在相互矛盾的结构（平行与反平行）。此外，已解析出结合配体的磷酸二酯酶5和10 GAF单体的结构。在所有情况下，环核苷酸结合涉及紧密封闭的结合口袋内特定的蛋白质-配体相互作用以及配体最小程度的溶剂暴露。PDE串联GAF结构域在功能上可替代蓝藻AC CyaB1的串联结构域；例如，使用来自PDE 2、5和11的串联GAF结构域可将cGMP调节嫁接到AC上。对GAF结构域调节的PDE的研究因调节分子和底物分子的特性而受阻。使用以ATP为底物的AC CyaB1作为报告分子解决了这个问题，并使哺乳动物PDE的串联GAF结构域可用于详细的动力学和机制研究。此外，可能作用于PDE调节结构域的药物可用这样一种新型测试系统进行检测。
