Structure And Function Research On Pseudomonas Aeruginosa Derived RNASE E Activity Inhibitor A(rraa) And Flagella HOOK Cap Protein FLGD

PartⅠSRUCTURE AND FUNCTIONAL RESEARCH ON RNASE E ACTIVITY INHIBITOR A OF PSEUDOMONAS AERUGINOSA.RNase E functions as the rate-limiting enzyme in the global mRNA metabolism as well as in the maturation of functional RNAs. The endoribonuclease, binding to the PNPase trimer, the RhlB monomer, and the enolase dimer, assemble into an RNA degradosome necessary for effective RNA processing. The processing of target RNAs by RNase E is found to be negative regulated by the protein modulator RraA which appears to work by interacting with the non-catalytic region of the endoribonuclease and significantly reduce the interaction of PNPase, RhlB and enolase within the RNA degradosome.Here we report the crystal structure of PaRraA from Pseudomonas aeruginosa to a resolution of 2.0 ?. The overall architecture of PaRraA is very similar to other known RraAs, which are highly structurally conserved. Gel filtration and dynamic light scattering experiments suggest that the protein regulator is arranged as a hexamer, consistent with the crystal packing of"a dimer of a trimer"arrangement. Structure and sequence conservation analysis suggests that the hexamer PaRraA contains six putative charged protein-protein interaction sites which may serve as binding sites for RNase E. The richly charged residues of PaRraA may allow it to compete and hamper the PNPase trimer, the RhlB monomer, and the enolase dimer for binding to RNaseE. Our structural data in combination with sequence analysis supports the idea that PaRraA contains six putative charged protein-protein interaction sites which may serve as binding sites with strong force to bind to RNase E. The dimensional hamper and rivalrous interaction from hexamer RraA may be the reasons resulting in the remodel of RNase E degradosome.PartⅡCRYSTAL STRUCTURE DETERMINATION OF PSEUDOMONAS AERUGINOSA DERIVED FLLAGELA HOOK CAP PROTEIN FLGD.Bacterial flagella, which are related to the virulence of opportunistic pathogens, have been examined in several animal models of infection. Different bacterial species have distinctive arrangements of flagella and unique flagellar antigens. These characteristics can be exploited in the identification of the infectious agents that may be involved in a given disease process. The bacterial flagellum, a complicated nanostructure protein machine which requires about 50 proteins for regulation and assembly, consists of three major substructures: the filament, the hook and the basal body. FlgD functions as the hook scaffolding protein and is also considered to be the hook-capping protein and the basal body rod-modification protein. In addition, together with FliK, FlgD regulates the assembly of the hook cap structure to prevent the leakage of hook monomers into the medium and hook monomer polymerization as well playing a role in determination of the correct hook length. Studies of flagellar biosynthesis in Escherichia coli and Salmonella enterica suggest that the hook cap is a transient intermediate structure in hook biosynthesis and FlgD is not found in the mature flagellum. Because FlgD isrequired for hook regulation and assembly and therefore plays a critical role in bacterial pathogenesis. To date, only the crystal structure of the C-terminal domain (residues 87–220) of FlgD from Xanthomonas campestris (XcFlgD; PDB code 3c12) has been determined. This structure comprises a novel hybrid fold consisting of a tudor-like domain interdigitated with a fibronectin type III domain. However, the FlgD C-terminal domain has not provided any clues about the function of the intact protein in the assembly and the regulation of this hook construction. In order to better understand the biological role of FlgD in flagellar hook biosynthesis, we have got the Se-Met PaFlgD and collected a set of diffraction data with resolution 2.4? in Shanghai Synchrontron Radiation Facility(SSRF) and use the method of Multi-wavelength Anomalous Dispersion(MAD) to determined the crystal structure of PaFlgD. The molecular model construction and analysis are in processing.