Structure-function Of Phage Persistence Related Protein RnlA-Dmd, Of RRNA Methylation Protein RsmH, Of Metabolite Regulator Protein Crc

Due to the abuse of antibiotics, the drug resistance of pathogenic bacteria is growing, producing a large number of multiple drug-resistant strains, even the super bacteria with vast majority of antibiotic resistance. More and more scientists are interested in the research of the mechanism of antibiotics, developing new drug target and new methods to replace the traditional treatment.This thesis contains there parts:the structural and functional studies of RnlA from Escherichia coli and Dmd from T4phage providing theoretical basis for phagotherapy, of methyltransferase RsmH from Escherichia coli(as the most important target, methylation of ribosome affect antibiotics designment), of global metabolic regulator Crc from Pseudomonas aeruginosa(as the new target, the structural information providing theoretical basis for drug designment).Escherichia coli RnlA-RnlB is a newly identified toxin-antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA-Dmd represents a novel TA system. Here, we have determined the high resolution crystal structure of RnlA and Dmd. Dmd is composed of one domain. Two molecules in one unit indicating that Dmd may be a dimer in solution. RnlA is composed of three independent domains:NTD (N-terminal domain), NRD (N repeated domain) and DBD (Dmd binding domain), which is an organization not previously observed among known toxin structures. Small-angle X-ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C-terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure-function relationship.RsmH is a specific AdoMet-dependent methyltransferase (MTase) responsible for N4-methylation of C1402in16S rRNA and conserved in almost all species of bacteria. The methylcytidine interacts with the P-site codon of the mRNA and increases ribosomal decoding fidelity. In this study, high resolution crystal structure (2.25A) of Escherichia coli RsmH in complex with AdoMet and cytidine (the putative rRNA binding site) was determined. The structural analysis demonstrated that the complex consists of two distinct but structurally related domains:the typical MTase domain and the putative substrate recognition and binding domain. A deep pocket was found in the conserved AdoMet binding domain. It was also found that the cytidine of the putative rRNA binding site bound far from AdoMet with the distance of25.9A. It indicates that the complex is not in a catalytically active state. Further analysis of small-angle X-ray scattering (SAXS) data revealed that in contrast to the crystal state the RsmH forms a dimer in solution. It implies that an active status of the RsmH in vivo is achieved by a formation of the dimeric architecture. In general, crystal and solution structural analysis provides new information on the mechanism of the methylation of the fine-tuning ribosomal decoding center by the RsmH.The global metabolic regulator catabolite repression control(Crc) has recently been found to modulate the susceptibility to antibiotics and virulence in the opportunistic pathogen Pseudomonas aeruginosa and been suggested as a nonlethal target for novel antimicrobials. In P. aeruginosa, Crc couples with the CA motifs from the small RNA CrcZ to form a posttranscriptional regulator system and is removed from the5-end of the target mRNAs. In this study, we first reported the crystal structure of Crc from P. aeruginosa refined to2.20A. The structure showed that it consists of two halves with similar overall topology and there are11β strands surrounded by13helices, forming a four-layered α/β-sandwich. The circulardichroism spectroscopy revealed that it is thermostable in solution and shares similar characteristics to that in crystal. Comprehensive structural analysis and comparison with the homologies of Crc showed high similarity with several known nucleases and consequently may be classified into a member exodeoxyribonuclease Ⅲ. However, it shows distinct substrate specificity (RNA as the preferred substrate) compared to these DNA endonucleases. Structural comparisons also revealed potential RNA recognition and binding region mainly consisting of five flexible loops. Our structure study provided the basis for the future application of Crc as a target to develop new antibiotics.