Regulation of cGMP-dependent proteinkinases in Plasmodium falciparum

疟疾仍然是最具威胁性的传染病之一。然而，疟疾的治疗一直受到抗药性的阻碍，因此需要开发基于新药物靶点的创新疗法。疟疾是由疟原虫属的原生动物引起的，这些原生动物由按蚊传播。恶性疟原虫cGMP依赖蛋白激酶(PfPKG)在蚊虫和人类宿主的有性繁殖和无性繁殖中起着至关重要的作用。由于PfPKG和人(H)PKG的一级结构显著不同，PfPKG有可能通过靶向干扰来解决。然而，PfPKG的功能和调控在很大程度上是未知的。在这一应用中，我们旨在通过高分辨晶体结构在分子水平上研究PfPKG的调节和激活机制。特别是，我们将重点介绍环核苷酸结合域(CNBS)，其中PfPKG显示四个CNB，而其人类对应的CNBS显示两个。最近的结构数据(Kim等人，2015年，Huang等人，2014年，以及未发表的结构)为环核苷酸筛选选择作为选择性cGMP拮抗剂的候选者提供了合理的基础。尤其是，CNB-D对激酶的激活和活性起着至关重要的作用，对催化结构域的稳定性似乎是必不可少的。我们的初步数据表明，PfPKG和hPKG在激活机制上存在差异。基于这些初步发现，我们将研究保守的自抑制结构域的作用。此外，还将对催化区域进行详细的研究。有趣的是，我们发现PfPKG和hPKG在催化核心内高度保守的结构基序，即所谓的脊柱残基中存在变异。调节性和催化性的脊椎存在于所有的蛋白激酶中，对控制蛋白激酶的活性至关重要。我们将通过定点突变和结合研究来确定这些差异的意义。通过研究恶性疟原虫中的PKG，我们已经进入了一个新的研究领域，利用我们在环核苷酸和cNMP依赖的蛋白激酶方面的深入专业知识。我们将与结构生物学和寄生虫学方面的国际专家合作，将我们的知识应用于抗击疟疾的新战略。

Malaria is still one of the most threatening infectious diseases. However, treatment of malaria has been hampered by drug resistance and therefore requires development of innovative therapies based on novel drug targets. Malaria is caused by protozoans of the genus Plasmodium, which are transmitted by Anopheles mosquitoes. cGMP-dependent protein kinase from Plasmodium falciparum (PfPKG) is crucial in both sexual and asexual proliferation in mosquito and human hosts, respectively. Since the primary structure PfPKG and human (h)PKG differ significantly, PfPKG could potentially be addressed by targeted interference. However, the function and the regulation of PfPKG is largely unknown.In this application, we aim to investigate the regulation and activation mechanism of PfPKG on a molecular level by employing high-resolution crystal structures. In particular, we will focus on the cyclic nucleotide binding domains (CNBs), where PfPKG displays four CNBs in contrast to two in its human counterpart. Recent structural data (Kim et al., 2015, Huang et al., 2014, and unpublished structures) provide the rational basis for a cyclic nucleotide screen to select candidates that serve as selective cGMP antagonists. In particular, the CNB-D plays a critical role for kinase activation and kinase activity and appears to be essential for the stability of the catalytic domain. Our initial data demonstrate differences in the activation mechanisms between PfPKG and hPKG. Based on these preliminary findings, we will investigate the role of the conserved autoinhibitory domain. In addition, the catalytic domain will be investigated in detail. Interestingly, we identified variations between PfPKG and hPKG in highly conserved structural motifs within the catalytic core, the so-called spine residues. Regulatory and catalytic spines are found in all protein kinases and are crucial for the control of protein kinase activity. We will define the significance of these differences by site-directed mutagenesis and binding studies.By studying PKG in P. falciparum, we have entered a new research field applying our in-depths expertise with cyclic nucleotides and cNMP-dependent protein kinases. In collaboration with international specialists in structural biology and parasitology, we will apply our knowledge towards novel strategies to fight malaria.