Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada.
INRS-Institut Armand-Frappier, Laval, QC H7V 1B7, Canada.
Proc Natl Acad Sci U S A. 2019 Jul 9;116(28):14164-14173. doi: 10.1073/pnas.1905558116. Epub 2019 Jun 25.
The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from and , revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.
环鸟苷酸 3',5'-单磷酸 (cGMP)-依赖性蛋白激酶 (PKG) 是在 25 年前被发现的；然而，从任何物种中获得整个 PKG 酶或催化结构域的结构的努力都失败了。在疟原虫中，PKG 的 cGMP 依赖性激活触发了整个复杂生命周期中的关键发育转变。我们已经确定了 和 中的无 cGMP 的晶体结构，揭示了当酶处于非活性状态时，关键结构成分（包括 N 端自动抑制片段（AIS）、四个预测的环核苷酸结合结构域（CNB）和激酶结构域（KD））的排列方式。四个 CNB 和 KD 呈五边形排列，AIS 与 KD 的底物结合位点对接，呈交换结构域二聚体排列。我们表明，尽管该蛋白主要以单体形式存在（二聚体不太可能代表生理形式），但 AIS 的结合对于保持 PKG 处于非活性状态是必要的。一个主要特征是一条充当 KD 的 N 端螺旋以及相邻 CNB 的帽状螺旋的双重作用的螺旋。相邻 CNB 之间的连接螺旋网络有助于将激酶保持在非活性构象。我们提出了一个方案，其中 cGMP 的协同结合从最接近 KD 的 CNB 开始，通过结构中继机制在五边形分子周围传递构象变化，使 PKG 能够协调快速、高度调节的发育转变，以响应寄生虫中环鸟苷酸水平的动态调节。
