Molecular basis for the control of K(+) uptake via KtrAB and KimA by cyclic di-AMP

钾稳态是细菌生存所必需的，并由各种K（+）输入者和输出者的协调功能控制。虽然不同的配体和刺激物被认为调节这些，但最近，环二AMP已被确定为不同革兰氏阳性菌中的支配性调节信号分子。环二磷酸腺苷的产生似乎取决于K（+）（可用性。在过量的外部钾增加细胞内的环二-AMP水平被检测到。反之亦然，钾的吸收和释放被证明是由环二AMP的基因表达和蛋白质活性的控制调制。在枯草芽孢杆菌中，环状二AMP直接与K（+）导入剂KtrAB和KimA结合，导致其抑制。然而，蛋白质失活的潜在机制仍然难以捉摸。在这里，我们将阐明环二AMP结合和转运抑制的分子原理。由于K（+）通道KtrAB和钾质子同向转运体KimA被认为不具有任何结构相似性，因此预期不同的结合基序和调节机制。环二AMP结合的结合位点和结构后果将通过单粒子冷冻电子显微镜和X射线晶体学来解决。脉冲EPR光谱将阐明KtrAB和KimA的核苷酸依赖性构象动力学。互补突变研究结合功能测定将鉴定核苷酸结合和随后活性调节所需的残基。

The potassium homeostasis is essential for bacterial survival and controlled by the orchestrated function of various K(+) importers and exporters. While different ligands and stimuli have been thought to regulate those, recently, cyclic di-AMP has been identified as overarching regulatory signaling molecule in different Gram-positive bacteria. The production of cyclic di-AMP appears to depend on the K(+) (availability. At an excess of external potassium an increased intracellular cyclic di-AMP level was detected. Vice versa, the uptake and release of potassium was shown to be modulated both by the control of gene expression and protein activity by cyclic di-AMP. In Bacillus subtilis cyclic di-AMP directly binds to K(+) importers KtrAB and KimA leading to their inhibition. However, the underlying mechanism of protein deactivation remains elusive. Here, we will shed light on the molecular principles of cyclic di-AMP binding and transport inhibition. Since K(+) channel KtrAB and potassium-proton symporter KimA supposedly do not share any structural similarities, different binding motifs and regulatory mechanisms are expected. The binding sites and structural consequences of cyclic di-AMP binding will be solved by single particle cryo-electron microscopy and X-ray crystallography. Pulsed EPR spectroscopy will elucidate the nucleotide-dependent conformational dynamics of KtrAB and KimA. Complementary mutational studies in combination with functional assays will identify residues required for nucleotide binding and the subsequent regulation of activity.