Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria
Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria
复制标题
蓝藻中 SasA-KaiC 复合物形成的原子力显微镜分析涉及从 KaiABC 时钟机制到输出途径的信息传递
DOI：
10.1111/gtc.12574
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发表时间：
2018
期刊：
影响因子：
2.1
通讯作者：
Ishiura Masahiro
中科院分区：
文献类型：
--
作者：
Murakami Reiko;Hokonohara Hitomi;Che Dock-Chil;Kawai Tomoji;Matsumoto Takuya;Ishiura Masahiro
蓝藻生物钟振荡器由三种时钟蛋白组成：KaIA、Kaib和KaiC。SAA是一种与KaiC结合的EnvZ样正统组氨酸激酶，参与主要的时钟输出途径，在体外主要以三聚体(SasA3聚体)存在，偶尔以六聚体(SasA6聚体)存在。此前，凝胶过滤层析估计Sasa-KaiCDD复合体的分子质量大于670kDa。KaiCDD是一个突变的KaiC，在两个磷酸化位点上有两个Asp取代。这个值与分子比为1：1的SasA3聚体-KaiC六聚体(KaiC6聚体)络合物的理论估计值480 kDa不一致。为了弄清SASA-KAIC复合体的结构，我们用原子力显微镜分析了含有0.1kMol/L ATP和5 mmo1/L氯化镁(mg-ATP)的KaiCDD、SASA以及含有Sasa和KaiCDD6的混合物与MG-ATP.KaiCDD图像分为两种类型，高度分布分别对应于KaiCDD单体(KaiCDD1mer)和KaiCDD6mer。SAA图像分为两类，高度分别对应于SasA3mer和SasA6mer。SASA-KaiCDD混合物的AFM图像不仅显示了KaiCDD1mer、KaiCDD6mer、SasA3merand SasA6mer，而且还显示了更大区域的“岛”，表明存在Sasa-KaiCDD复合体的聚合形式。
The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC‐binding EnvZ‐like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer) and occasionally as a hexamer (SasA6mer) in vitro. Previously, the molecular mass of the SasA‐KaiCDDcomplex, where KaiCDDis a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel‐filtration chromatography to be larger than 670 kDa. This value disagrees with the theoretical estimation of 480 kDa for a SasA3mer‐KaiC hexamer (KaiC6mer) complex with a 1:1 molecular ratio. To clarify the structure of the SasA‐KaiC complex, we analyzed KaiCDDwith 0.1 mmol/L ATP and 5 mmol/L MgCl2(Mg‐ATP), SasA and a mixture containing SasA and KaiCDD6merwith Mg‐ATP by atomic force microscopy (AFM). KaiCDDimages were classified into two types with height distribution corresponding to KaiCDDmonomer (KaiCDD1mer) and KaiCDD6mer, respectively. SasA images were classified into two types with height corresponding to SasA3merand SasA6mer, respectively. The AFM images of the SasA‐KaiCDDmixture indicated not only KaiCDD1mer, KaiCDD6mer, SasA3merand SasA6mer, but also wider area “islands,” suggesting the presence of a polymerized form of the SasA‐KaiCDDcomplex.