Comparison of Iron-Binding Ability Between Thr70-NapA and Ser70-NapA of Helicobacter pylori.
BACKGROUND
The neutrophil-activating protein (NapA) of Helicobacter pylori (H. pylori), with DNA-binding and iron seizing properties, is a fundamental virulence factor involved in H. pylori-related diseases. Compared with Ser70-NapA strain, Thr70-NapA strain is more intimately correlated with iron-deficiency anemia.


METHODS
To investigate whether two types of proteins differ in iron-binding ability, mutated Thr70-NapA and Ser70-NapA strains were established. Isothermal titration calorimetry (ITC) method was conducted to measure the binding between the NapA protein and Fe(2+) . The structural changes of NapA protein were also tested during iron interaction by fast protein liquid chromatography (FPLC) and circular dichroism (CD) methods. DNA-binding assay was performed for evaluate the affinity of both mutated and wild types of NapA with DNA.


RESULTS
Mutated Thr70-NapA had higher iron-binding ability than wild Ser70-NapA. The structural stability of Thr70-NapA was disrupted and became more active along with the rising concentration of Fe(2+) , whereas no similar association was observed between Ser70-NapA and Fe(2+) level. When the iron/protein molar ratio ranged from 10 to 20, both Ser70-NapA and Thr70-NapA displayed weaker DNA-binding ability.


CONCLUSIONS
Thr70-NapA has much stronger ability to sequester ferrous ion compared with Ser70-NapA in H. pylori. In addition, the DNA-binding property of NapA is dependent upon the Fe(2+) concentration.
Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou, China.
Weiran
W
Shan
State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, China.
Hsiang-Fu
HF
Kung
Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou, China.
Ruiguang
R
Ge
D018798:Anemia, Iron-Deficiency / Q000382:microbiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000235:genetics*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D033862:Iron-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D013045:Species Specificity
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D033862:Iron-Binding Proteins; C092412:neutrophil-activating protein A, Helicobacter pylori; D007501:Iron
DNA binding; Helicobacter pylori; ferrous ions; neutrophil-activating protein