Crystal Structure And Biological Function Of JHP933 From Helicobacter Pylori Strain J99 Plasiticity Region

There are servaral regions in Helicobacter pylori genome who have been termed“plasiticity region”.The prominent characteristics of these regions genes are their lower G+C content(35% vs 39% of the rest of H. pylori genome) and inserted sequences. The fact that nearly half of the strain-specific genes of H. pylori are located in the plasticity region indicates that they may play important roles in H.pylori’s pathogenesis.jhp0933 is one of the plasiticity region genes and encodes a putative protein with 267 amino acids.A blast search of this protein sequences indicated that it might be classified into the nucleotidyltransferase(NTase)superfamily,but the certain function remains unknown.In the first part of this study,we expressed,purified and crystalized JHP933. Then we determined the crystal structure of JHP933 at a resolution of 2.1 ? by Single wavelength anomalous dispersion.The structure reveals that JHP933 is a new member of nuclotidyltransferase superfamily proteins with a characteristic αβαβαβα topology.A Dali search for structural homology identified lincosamide-resistance protein Lin A as closest related structure though it shares 16% homologous sequence with JHP933.Therefore, we hypothesise that JHP933 may play the role of lincosamide-resistance.In addition,we pointed out the active sites of substrate-binding located in the N-terminal conservative catalytic residues by the structure superposition with Lin B/Mg2+/clindamycin complexes.In the second part of this study,firstly,we examined the presence of jhp0933 of 133 H.pylori strains including 63 with gastritis,51 with ulter and 19 with gastric cancer.The prevalence of jhp0933 were 58.7%,54.9% and 84.2%, respectively. The prevalence of jhp0933 of H.pylori strains from gastric caner patients were significantly greater than those from patients with gastritis and ulter(p﹤ 0.05).We also confirmed the expression of jhp0933 gene by RT-PCR.Secondly,we constructed a jhp0933-deleted mutant of H.pylori strain J99 by DNA Homologus Recombination Method. Lincosamide susceptibility tests were performed in H.pylori wild strain J99,jhp0933-deleted mutant and H.pylori isolates strains.The results of susceptibility tests don’t support the hypothesis that JHP933 may play the role of lincosamide-resistance.Finally, we have studied the binding relationship between JHP933 and NTPs including ATP,GTP,CTP,TTP and UTP by using Isothermal Titration Calorimetry( ITC).The ITC’s data reveal that JHP933 can bind to UTP specifically which provides a valuable clue for the screen of JHP933’s catalytic substrates.In summary, The crystal structure reveals that JHP933 is a new member of nuclotidyltransferase superfamily proteins from H.pylori. JHP933 has the catalytic residues for substrate binding within conservative active site and can bind to UTP specifically.Although a Dali search for structural homology identified lincosamide-resistance protein Lin A as closest related JHP933’s structure, the susceptibility tests don’t support that JHP933 is a lincosamide-resistance protein. In addition,the overall rate of jhp0933 in H.pylori isolates is 60.9% and it is significantly higher in H.pylori isolates from gastric cancer patients than others.