Study On The Structure And Chemical Regulation Of Serine/threonine Phosphatase Stp1

Staphylococcus aureus is a kind of important pathogenic bacteria which cause serious damage to the human life and health,it can cause local tissue fester disease such as infections,pneumonia and sepsis.Recentlly,the emergence of multidrug resistance poses major difficulty to clinical treatment.People died from methicillin-resistant staphylococcus aureus(MRSA)over the same period people died from the infection of HIV/AIDS in the U.S.The infection with MRSA,HBV and AIDS are listed as the toughest treatment infectious diseases in the world.Current clinical drugs are antibiotics used to treat bacterial infections,antibiotics are by directly inhibit the growth of pathogenic bacteria,or directly kill pathogenic bacteria to reach the effect of clinical treatment.The inherent mechanism of bacterium bestow on some bacterial robust selection pressure(selective pressure),catalysted and further enriched the drug-resistant strains.In the face of increasingly severe antibiotic resistance of bacteria,someone began to think about a new strategy to fight infection,such as reducing bacterial virulence,rather than directly kill bacteria and thus to achieve the effect of the treatment.By reducing bacterial virulence to reduce pathogenic bacteria,effectively prevent and treat a variety of infections.Serine/threonine protein phosphatase Stp1 and Stk1 are only conservative phosphate kinase and phosphatase pair in staphylococcus aureus.Stk1 / Stp1 rely on phosphorylation/dephosphorylation substrates as global regulatory factor in bacteria cell.Recent research implied Stp1 has the effect on toxicity and virulence of S.aureus strain.Deletions of phosphatase gene Stp1 results in a decreased virulence up to 1000-10000 in newman stain,suggested that Stp1 play an key role in regulation of virulence.Therefore,we aimed at Stp1 carrying on further study,expected to develop a new type of small molecules to resist to staphylococcus aureus virulence.Because of the crystal structure of Stp1 has not been parsed,the specific mechanism is unclear.Herein,we get protein crystal structure by expression Stp1 in prokaryotes,purification,crystallization and X-Ray diffraction,etc.Besides,we designed a series of new antibacterial small molecule compounds to evaluate the activity of inhibition activity,based on the structure of Stp1 crystal.Moreover,we designed some mutation to verify their activity whether validated on the protein active sites through the analysis of Stp1 crystal structure.We proved that His12 has a combination with Stp1 in ITC method,detected Stp1 has the effect of dephosphorylation peptides which has been phosphorylation in serine/threonine residues by LC—MS,there is no signification difference on peptide activity when we changed the phosphorylated peptide amino acid sequence.At last,we tried to cultivate crystal Stp1-His12 complexes to reveal the action mechanism of dephosphorylation.In this study,we expressed protein in prokaryotic,purified,cultivated crystal to parse the crystal of Stp1,we have screened 517 small molecule compounds from Specs compounds library based on the crystal structure,at the same time,we screened more than 10,000 small molecular compounds by the high throughput screening,and evaluated their activity.We revealed the Stp1 how to identify the phosphorylated peptide substrates from the structure simulation,verified Stp1 is the selective serine and threonine phosphorylase by the biochemical experiment,at the same time,we test the peptides which involved in Mgr A is the substrate of Stp1,to replace the nonpolypeptide substrate to provide the basis.