Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity
Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity
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DOI：
10.1042/bcj20160969
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发表时间：
2017-07-15
影响因子：
4.1
通讯作者：
Herberg, Friedrich W.
中科院分区：
文献类型：
--
作者：
Lorenz, Robin;Moon, Eui-Whan;Herberg, Friedrich W.
环磷酸腺苷和环磷酸鸟苷是普遍存在的第二信使，在所有形式的生命中调节效应蛋白的活性。主要的效应蛋白，3 '，5'-环腺苷一磷酸（cAMP）依赖性蛋白激酶（PKA）和3 '，5'-环鸟苷一磷酸（cGMP）依赖性蛋白激酶（PKG），分别优先被cAMP和cGMP激活。然而，这种环核苷酸选择性的分子基础仍然没有完全理解。对PKA调节亚基I型α（RI α）的分离的环核苷酸结合（CNB）结构域的分析表明，C-末端CNB-B比N-末端CNB-A具有更高的cAMP亲和力和选择性。在这里，我们表明，使用定点诱变引入cGMP特异性残基降低了CNB-B的选择性，而两个突变（G316 R/A336 T）的组合导致cGMP选择性结合结构域。此外，将相应的突变（T192 R/A212 T）引入PKA RI alpha CNB-A中将该结构域转变为高度cGMP选择性结构域，强调了这些接触对于实现cGMP特异性的重要性。与通用嘌呤核苷酸3 '，5'-环肌苷一磷酸（cIMP）的结合数据显示，引入的精氨酸残基与核碱基的6位氧相互作用。分离的CNB-B G316 R/A336 T双突变体与cAMP或cGMP的共晶体结构揭示，引入的苏氨酸和精氨酸残基保持其保守接触，如在PKG I CNB-B中所见。这些结果提高了我们对环核苷酸结合和环核苷酸特异性的分子基础的理解。
Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type I alpha (RI alpha) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RI alpha CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity.