Structure Biology Studies On Octaprenyl Pyrophosphate Synthase And Arginase From Helicobacter Pylori

Helicobacter pylori (H. pylori) is a spirally shaped gram-negative human pathogen which is the major pathogenic factor of gastric diseases like chronic active gastritis, peptic ulceration, and was involved in the development of adeno-carcinoma and mucosa-associated lymphoid-tissue lymphoma, World Health Organization has listed it as class?of carcinogen. H. pylori is associated with a high infect rate, it is estimated that at least half of the world population is infected by this bacterium. In China, more than 600 million people were infected and in some area rate can be up to 90%. Although it has been 27 years since the discovery of H. pylori and extensive studies have been focused on pathogenic etiology, epidemiology, pathogenesis, virulence factors, diagnosis, treatment and prevention strategies of the bacterium, some problems remain unclear, such as the mechanism involved in persistent and chronic infection, the relationship between H. pylori and gastric cancer, the reason between the high infection and low incidence rate, et al. Thus, it is in urged need to fully understand its pathogenesis, which will guide the research and development of new drugs, vaccines and treatments strategies for H. pylori infection.A large number of pathogenic factors have played a key role in the successful colonization of H. pylori to the special physiological environment of the host stomach, adhesion of gastric epithelial cells and development of a variety of gastric diseases. For example: virulence factors such as CagA?VacA?Urease?Hsp60?NAP?OipA?DupA are directly related to the virulence of the pathogen; Some adhesions such as BabA?SabA?SabB?AlpA?AlpB?IceA?HpaA and the recently identified proteins HP1188 and HP1430 are involved in interaction with host gastric epithelial cells to facility the adhesion of the bacterial; Two different Carbonic anhydrate and the two component system proteins CrdS/CrdR?ArsR/ArsS are involved in acid resistance. These effectors are closely associated with the infection and pathogenic mechanism of H. pylori.Proteins function through its unique three dimensional structure and fold pattern. In order to shed lights on the pathogenic mechanism of some pathogenic factors, we there for have embarked on structure determination of some important virulence factors and some functional proteins use method of macromolecular crystallography. We have selected more than 20 targets from virulence factors, adhesions, acid resistance associated proteins and some enzymes involved in fetal physiological function, 2 structures were determined and 3 proteins were crystallized for the moment, some work were still in progress.This thesis is a brief summary of the main results we obtained in the past 3 years; it is constituted with three parts.The first part is the crystallographic and functional studies on octaprenyl pyrophosphate synthase (OPPs) from H. pylori. OPPs catalyzes consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C40 octaprenyl pyrophosphate (OPP), which constitutes the side chain of bacterial ubiquinone or menaquinone, the latter sever as a component of the electron transport chain. Studies also reveal that OPP is essential for the growth of bacterial, indicting the importance of the protein. Here we have cloned the gene of H. pylori OPPs, expressed, purified, and crystallized the protein, and collected a diffraction data set to 2.00? resolution. The structure of OPPs was determined by MAD method. Based on the structure, we show the fold mode of the monomer and the assembly of the dimer, and the differences between homology structures. We have proposed the mode for substrate recruit and product release, and identified the key residue for product chain length determination. Besides, we have gain the structure of OPPs in complex with substrate IPP, FPP and also the intermediate GGPP by either molecular docking or modeling, based on these structures, we propose here the possible catalytic mechanism of enzymes from this family. These results significantly extend our understanding on the biosynthesis of polyprenyl molecules.The second part is structure and enzymatic studies on arginase RocF from H. pylori. Arginase is a metalloenzyme of urea cycle that hydrolyses arginine to ornithine and urea, it require divalent cations for catalytic activity. RocF is an important component involved in acid resistance and plays key roles of adaptive adhesion of H. pylori. It compete the common substrate L-arginine with host inducible nitric oxide synthase to reduce the synthesis of NO, and it is able to inhibit human T cell proliferation, thus efficiently reduces host cellular immune response. As a result, RocF was thought to be involved in escape from host immune system and contribute to persistence infection of H. pylori. Both in vivo and in vitro studies reveal that knockout of rocf gene resulting in significantly inhibit of the colonization of H. pylori to host gastric mucosa, indicating the importance of RocF in the pathogenicity of the bacterial. In contrast to other arginases, RocF shows several unique features such as optimal activity with Co²⁺ instead of Mn²⁺ as a metal cofactor, highest activity at low pH (6.1 instead of 9.5), presence of a unique motif with 13 residues and considerable differences at the N- and C-terminal sites. This suggests that there could be a difference in the active site architecture compared to the other arginases. In this study, RocF was purified and crystallized in complex with Mn²⁺ and a diffraction data set was collected to 2.2 (A|?) resolution. The structure of RocF was determined by MR method, we have identified the key residues involved in ion binding and located the active site, and analyzed the difference on sequence and structure of RocF and other arginases. We also established the enzymatic assay system for RocF, we show that reducing agent has a significant influence on the enzyme activity of RocF and RocF has an ion optimum of Co²⁺>Ni²⁺>Mn²⁺. Now we are focused on crystallizing RocF in complex with substrate analogues nor-NOHA and BEC, which will extend our understanding of the enzymatic mechanism of arginase.The last part are some of other work that listed as follows.1) Primary crystallographic studies of H. pylori adhesion A (HpaA). HpaA is the most important adhesions of H. pylori and play key roles in interaction with host's gastric epithelial cells. Besides, it shows strong immunogenicity and was able to elicit protective immune response in mouse mode, so it was widely used in vaccine development. In this paper, HpaA was expressed and purified, we have obtained its crystal, but they are of poor quality and was not able to used for data collection, now we are optimize the condition in order to get better crystals of HpaA. 2) Primary crystallographic studies on HspA, CagA, and ArsS from H. pylori. HspA is a chaperone and involved in correctly folding of other proteins, it adopts a unique C-domain as compared with other homologous proteins, and it forms different oligomeric state when binding different ions. CagA is the most important virulence factor of H. pylori; it is secreted into host gastric epithelial cells by type?secretion system, then it will interact with host proteins, resulting in the change of the polarity and cell motility of gastric epithelial cells, tight junction dysfunction, actin cross-linking and elongation, these change are directly related to the development of some stomach disease. ArsS is a protein of two component system, it was able to sense the change of pH of the inner environment of the bacterium, and then regulate the transcription and expression of some proteins of some proteins involved in acid resistance. We have clone, express and purify the intact form all domains of these proteins and screened the crystallization conditions, only HspA was able to form tiny crystals with poor quality, CagA and ArsS was not able to crystallize due to some reason. These studies are still in progress. 3) The properties of the fusion protein FsS1' constructed during my master degree was further studied, domain prediction reveal that there are two independent globular domain corresponding to the two subunits in the fusion protein separated by a disordered linker, this result indicate that the spatial structure of the two domains was not interfered by each other, which ensure that their immunogenicity will not be affected. The protein was successfully refolded by dilution and purified by gel filtration, production of polyclonal antiserum against FsS1' show that the antiserum had a high titer of specific IgG, and FsmS1 is of great immunogenicity, these results indicate that FsS1 ' is a promising candidate antigen for the development of genetic pertussis vaccines.