Department of Pharmacology, Baylor College of Medicine, Houston, Texas, United States of America.
PLoS One. 2011 Apr 19;6(4):e18413. doi: 10.1371/journal.pone.0018413.
Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively.
METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A) of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP.
CONCLUSIONS/SIGNIFICANCE: Our findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.
环鸟苷酸依赖性蛋白激酶（PKG）是一氧化氮-cGMP 信号通路的核心介质，可磷酸化下游底物，这些底物对调节平滑肌张力、血小板激活、疼痛感知和记忆形成至关重要。作为 cGMP 的主要受体之一，PKG 介导 cGMP 升高药物的大部分作用，例如一氧化氮供体和磷酸二酯酶抑制剂，分别用于治疗心绞痛和勃起功能障碍。
方法/主要发现：我们通过确定与人 PKG I 的氨基末端环核苷酸结合域（CNBD-A）结合的 cGMP 或 cAMP 的晶体结构，研究了环核苷酸与 PKG 结合的机制。我们还确定了无结合核苷酸的 CNBD-A 结构。结合 cAMP 或 cGMP 的 CNBD-A 的晶体结构表明，cAMP 以顺式或反式构型结合，而 cGMP 仅以顺式构型结合，保守的苏氨酸残基锚定环磷酸和鸟嘌呤部分。无结合环核苷酸的 CNBD-A 结构与结合环核苷酸的结构相似。令人惊讶的是，等温滴定量热法实验表明，CNBD-A 以相对较高的亲和力结合 cGMP 和 cAMP，对 cGMP 的亲和力约为 cAMP 的两倍。
结论/意义：我们的发现表明，CNBD-A 通过与 Thr193 的相互作用以及不寻常的顺式肽形成残基 Leu172 和 Cys173 将 cGMP 结合在顺式构象中。尽管这些研究首次提供了环核苷酸与 PKG 结合的结构见解，但我们的 ITC 结果仅显示对 cGMP 的两倍偏好，表明其他结构域需要先前报道的环核苷酸选择性。
