Zoraghi Roya, Bessay Emmanuel P, Corbin Jackie D, Francis Sharron H
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
J Biol Chem. 2005 Mar 25;280(12):12051-63. doi: 10.1074/jbc.M413611200. Epub 2005 Jan 27.
The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.
环磷酸鸟苷（cGMP）结合的特异性磷酸二酯酶5（PDE5）包含一个水解cGMP的催化结构域和一个调节（R）结构域，该调节结构域含有两个GAF结构域（a和b；GAF源自哺乳动物cGMP结合磷酸二酯酶、鱼腥藻腺苷酸环化酶和大肠杆菌（FhlA）的蛋白质）。R结构域以变构方式结合cGMP，促进二聚化，并在一个受变构cGMP结合调节的位点发生磷酸化。对10种含有一个或两个GAF结构域的人PDE5A1构建体的四级结构和cGMP结合特性进行了表征。结果表明：1）GAF a模块之间（K(D)<30 nM）和GAF b模块之间（K(D)=1-20 pM）发生高亲和力同源二聚化，GAF结构域之间的序列（Thr322-Asp403）有助于二聚体稳定性；2）GAF a中的176个氨基酸（Val156-Gln331）足以结合cGMP；3）GAF a对cGMP的亲和力（K(D)<40 nM）高于分离的R结构域（K(D)=110 nM）或全酶（K(D)=200 nM），这表明包含GAF b及其侧翼氨基酸的序列在完整的R结构域中自动抑制GAF a与cGMP的结合亲和力；4）仅含有GAF a的突变体（Met1-Glu321）具有与GAF a的结构异质性一致的高亲和力、双相cGMP结合动力学，这表明在全酶或分离的R结构域中观察到的双相cGMP解离动力学不需要GAF b的存在；5）未检测到GAF b有显著的cGMP结合；6）包含GAF b及其侧翼氨基酸的序列对于环核苷酸依赖性蛋白激酶对Ser102磷酸化的cGMP刺激至关重要。这些结果为PDE5的功能提供了新的见解，进一步定义了变构cGMP结合的边界，并确定了有助于二聚化的区域。
