Structural And Biochemical Study On GGDEF Domain Protein PA2771 From Pseudomonas Aeruginosa

C-di-GMP is an important second messenger in bacteria. It was demonstrated to play key roles in many biological processes in bacteria, such as biofilm formation, antibiotics resistance, virulence regulation, cell division and so on. C-di-GMP level in bacteria is controlled by proteins with GGDEF domain, which has DGC (diguanylate cyclase) activity, synthesizing c-di-GMP from 2 molecules of GTP, and proteins with EAL or HD-GYP domain, which has PDE (phosphodiesterase) activity, breaking c-di-GMP into linear pGpG. The signal of c-di-GMP is transmitted to down stream genes by binding to receptors with pilz domain or fleQ. GGDEF domain or EAL domain usually couples with a regulatory domain, like PAS or Che-Y or GAF domain. The activity of these hybrid proteins would be regulated by environmental signals, such as phosphorylation, ion binding, lights, NO, or heme, through the input of regulatory domains. Our work is concentrated on the research of regulatory mechanism of GGDEF proteins. In this area, two crystal structures with GGDEF domain have been tangled. Both of them contain a CheY domain and a GGDEF domain. The authors provide regulatory machnisms od these proteins in detail. But so many GGDEF domain proteins with diverse kinds of regulatory domains exist. And none of their regulatory mechanisms has been revealed. So the research on this area is still very meaningful and interesting.Our research is to illustrate the regulatory mechanism of the activity of a protein PA2771 from Pseudomonas aeruginosa Ol. PA2771 contains an N-terminal domain belonging to GAF superfamily and a C-terminal GGDEF domain. No homologous structure of fullength PA2771 exists in PDB, and the GAF domain of PA2771 shares little similarity with the known structures of GAF superfamily. GAF superfamily is well known for its ability of binding ligands and transducing signals to the active domain. Thus, we chose this PA2771 as our target proteins for studying of this kind of regulation mechanisms. In the thesis, we cloned, expressed purified native and selenomonium substituted PA2771 and the GGDEF domain of this protein using regular molecular methods and crystallized them. After obtained single crystal, we collected the data using Shanghai Synchrotron Radiation Facility and solved the structure of fullength PA2771 ang GGDEF domain by softwares like CCP4, COOT, Phenix and CNS. In addition, we use methods like HPLC, congo red plate, Ultra-visible spectroscopy, MiniQ and microscopy to test the proteins biochemical activity and its effect on biofilm formation.In this thesis, we obtained the structure of PA2771 and the complex structure of PA2771's GGDEF domain and c-di-GMP. We proposed a model on the mechanism of how the activity of GGDEF domain is regulated by GAF domain, through structural analysis and did some biochemical work to support our model. Our research shows that PA2771 exsits as a dimmer in crystal structure, with four alpha helixes from the two GAF domains as major interface. The active sites of the two mononmer stay far away from each other, forming an inactive dimer. In crystals, the inhibition site of PA2771 does not bind to c-di-GMP. After comparing with homologous structures, we supposed two possible factors blocking c-di-GMP binding. First, the loop between 119 and 126 occupied space needed in the I-site for c-di-GMP. Second, the key residue Arg251 (R of the RXXD inhibition motif) exhibiting a conformation not sutible for forming Hydrogen bond with c-di-GMP, which is critical for c-di-GMP binding. PA2771 shows high DGC activity both in vitro and in vivo and is not inhibited by product inhibition regulation. Our study shows that PA2771 exsits as a dimer in solution too, but is in the active form. Since we hypothesize that the GAF domain blocks the binding of c-di-GMP to GGDEF domain, we also solved the structure of GGDEF domain alone to see its interaction with c-di-GMP. Solo GGDEF domain has very low DGC activity and binds to c-di-GMP in the I-sties of the dimmer of GGDEF domain in crystals. Thus, without GAF domain, the protein is inactive and can be regulated by c-di-GMP. According to the results, we propose that when PA2771 does not bind to the regulatory molecules in GAF domain, the protein is of high activity because of the interference of GAF on I-Site; when the unknown molecule bind to the GAF domain, the protein would experience big structure conformation change, thus c-di-GMP would bind to the i-site and protein activity would be regulated by c-di-GMP product inhibition and has low activity. We also tried to search the ligands that bind to GAF domain through comparing our structure with other GAF structures. Although we have not found the exact molecules that binds to GAF domain, our research shows that some hydrophobic small molecules would bind to GAF domain and decrease the DGC activity of PA2771.