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Characterization of the MinD/ParA-type ATPase FlhG in Vibrio alginolyticus and implications for function of its monomeric form.
Genes to Cells ( IF 2.1 ) Pub Date : 2020-02-17 , DOI: 10.1111/gtc.12754
Seiji Kojima 1 , Yoshino Imura 1 , Hikaru Hirata 1 , Michio Homma 1
Affiliation  

FlhG is a MinD/ParA-type ATPase that works as a negative regulator for flagellar biogenesis. In Vibrio alginolyticus, FlhG functions antagonistically with the positive regulator FlhF to generate a single polar flagellum. Here, we examined the effects of ADP and ATP on the aggregation and dimerization of Vibrio FlhG. Purified FlhG aggregated after exposure to low NaCl conditions, and its aggregation was suppressed in the presence of ADP or ATP. FlhG mutants at putative ATP-binding (K31A) or catalytic (D60A) residues showed similar aggregation profiles to the wild type, but ATP caused strong aggregation of the ATPase-stimulated D171A mutant although ADP significantly suppressed the aggregation. Results of size exclusion chromatography of purified FlhG or Vibrio cell lysates suggested that FlhG exists as a monomer in solution, and ATP does not induce FlhG dimerization. The K31A and D60A mutants eluted at monomer fractions regardless of nucleotides, but ATP shifted the elution peak of the D171A mutant to slightly earlier, presumably because of a subtle conformational change. Our results suggest that monomeric FlhG can function in vivo, whose active conformation aggregates easily.

中文翻译:

溶藻弧菌中MinD / ParA型ATPase FlhG的特征及其单体形式的功能意义。

FlhG是MinD / ParA型ATP酶,可作为鞭毛生物发生的负调节剂。在溶藻弧菌中,FlhG与正调节剂FlhF具有拮抗作用,以产生单个极性鞭毛。在这里,我们检查了ADP和ATP对弧菌FlhG聚集和二聚化的影响。纯化的FlhG暴露于低NaCl条件后会聚集,在ADP或ATP的存在下其聚集受到抑制。推定的ATP结合(K31A)或催化(D60A)残基处的FlhG突变体显示出与野生型相似的聚集曲线,但是ATP引起了ATPase刺激的D171A突变体的强聚集,尽管ADP显着抑制了聚集。纯化的FlhG或弧菌细胞裂解物的尺寸排阻色谱分析结果表明,FlhG作为单体存在于溶液中,ATP不会诱导FlhG二聚化。K31A和D60A突变体的洗脱比例与核苷酸无关,但与核苷酸无关,但是ATP可能将D171A突变体的洗脱峰移到了更早的位置,这可能是由于微妙的构象变化所致。我们的结果表明,单体FlhG可以在体内发挥作用,其活性构象易于聚集。
更新日期:2020-02-17
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