Structural Biology Study Of The Ligand Binding Domain Of Methyl-accepting Chemotaxis Protein PctA From Pseudomonas Aeruginosa

Pseudomonas aeruginosa is one of the most common opportunistic pathogen and human with low immunity are susceptible to infection.In particular,it can cause high mortality in patients with cystic fibrosis(CF).At present,Pseudomonas aeruginosa is resistant to many antibiotics,and the resistance is increasing.Therefore,it is necessary to further study it.Bacteria can adapt to a variety of living environments and be colonizied to a suitable location for their growth and reproduction.This process mainly depends on the chemotaxis of the bacteria.After bacteria detect the signals(such as amino acids and sugars),they pass the chemotaxis signaling pathway to move the bacteria near or away from the chemicals that are beneficial or harmful to them.The process of this pathway is that methyl-accepting chemotaxis proteins(MCPs)recognize chemotaxis,and then protein conformation changes.MCPs transmit the signal to the histidine kinase Che A to change the autophosphorylation of Che A.Che Y receives signals transmitted by Che A and acts on flagellum proteins to make flagella to rotate clockwise or counterclockwise.Che Z can control the phosphorylation of Che Y and control the rotation time of flagella.The ligand binding domain(LBD)of MCPs is responsible for the recognition of the chemicals.A previous study showed that the ligand binding domin of Methyl-accepting chemotaxis protein PctA can produce chemotaxis effects on 18 natural amino acids.But the specific mechanism by which PctA-LBD functions is still unclear.This work used PctA-LBD as the research object,and planned to clone,express,purify,crystallize,and use X-ray diffraction to obtain the crystal structure of PctA-LBD,and also aim to get the complex crystal structure of PctA-LBD and small molecule DPD(Dimethyl propanedioate),which would elaborate the binding mode and molecular mechanism of PctA-LBD and small molecules.In this study,firstly,the 30-278 amino acid residues fragment of PctA,i.e.,PctA-LBD,was performed through a bioinformatics analysis.The results of physicochemical properties and secondary structure showed that the protein had good properties.We performed amino acid sequence alignment of homologous proteins of PctA-LBD from different species.The results showed that the ligand binding domains of PctA from Pseudomonas aeruginosa,Cta A from Pseudomonas fluorescens and Psc A from Pseudomonas syringae had high sequence homology and some differences.It suggests that although they can recognize the same class of ligands,but PctA-LBD has its unique function.We cloned PctA-LBD into the p ET28 a vector.We used the E.coli expression system to express the proteins.We tried different purification conditions,and the protein had a slight precipitation in the buffer without glycerol.The precipitation was completely dissolved after adding 5% glycerol into the buffer,which showed that glycerol was helpful to maintain the stability of PctA-LBD.The crystals were obtained after the proteins that were acquired in two different purifications buffer were performed crystallization condition screening trials.However,crystals could not be obtained again when the experiment of the purification buffer contained 5%glycerol.We speculated that the crystals were salt crystals.The crystals size was small when proteins from the purification buffer without glycerol were performed with crystallization condition screening trials.We optimized crystallization conditions by changing the concentration and type of precipitants,p H,type and concentration of salt.But there was still no good quality crystal,and the resolution of X-ray diffraction for the crystal was low.We then tried different purification schemes,and the higher purity target protein was finally obtained by affinity chromatography with eluents of different imidazole concentrations,and subsequent ion exchange chromatography and gel filtration chromatography.We performed crystallization condition screening trials to obtain the crystals and then optimized the crystallization conditions.Finally,we get high-resolution crystals under the condition of 2.0 M Ammonium sulfate,0.1 M MES p H 5.0,and determined the three dimensional structure of PctA-LBD.In order to study the binding mode of PctA-LBD and its ligand,we tried to get the complex crystals of PctA-LBD and the small molecule compound DPD(Dimethyl Propanedioate)by soaking and co-crystallization.Although crystals were obtained after incubation of the protein and small molecules,no small molecules were found in the crystal structure.Later,we will continue our work to try to obtain the structure of the complex.In summary,high-quality protein crystals were obtained through various purification schemes and the structure of PctA-LBD was determined.Although the crystal of the compolex of PctA-LBD and DPD was not obtained,since we tried a variety schemes and obtained stable complexs.It provides a basis for determining the complex structure and developing drugs for the treatment of P.aeruginosa infections.