PDF(Pubmed)
Abstract：
Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.
摘要：
环核苷酸结合域（CNB）通过cAMP或cGMP赋予许多信号蛋白的变构调节，包括PKA和PKG。系统发育远处锥虫的PKA是第一个例外，因为它不依赖环核苷酸并对核苷类似物有反应（Bachmaier等人。,2019)。这里,我们发现天然核苷肌苷，鸟苷和腺苷是重要的热带病原体布鲁氏锥虫的PKA直系同源物的纳摩尔亲和力CNB配体和活化剂，克氏锥虫,还有利什曼尼亚.结合亲和力的序列和结构决定因素，通过结构-活性关系（SAR）分析建立PKAR的特异性和激酶激活，共晶结构和诱变。在结合位点中2至3个氨基酸的取代足以将CNB结构域从核苷转化为环核苷酸特异性。此外,与CNB-B高亲和力结合需要锥虫特异性C末端螺旋(αD)。αD螺旋用作将配体与溶剂屏蔽的结合位点的盖子。鸟苷对CNB-B的选择性和腺苷对CNB-A的选择性导致在低纳摩尔浓度下的协同激酶活化。PKA从快速裂解拉下来建立鸟苷作为主要的配体在体内的T.brucei血流形式，而鸟苷和腺苷似乎在昆虫媒介的前周期发育阶段协同作用。我们讨论了CNB结构域在PKA的进化和募集中用于新型核苷介导的信号传导的用途。
