S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Master's Degree_농생명공학부)
Structural analysis of HlyU from Vibrio vulnificus, a toxin-inducing transcriptional activator
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 농업생명과학대학 농생명공학부, 2017. 8. 하남출.
- Vibrio vulnificus is a foodborne pathogenic bacterium that is present in marine environments. The infection results from uptake of contaminated food or direct contacts on the injured skin, causing severe gastroenteritis or fatal septicemia in a mortality rate of more than 50% within a couple of days. The most important virulence factor of V. vulnificus is a RtxA toxin that can kill the host cells by forming pores on the membrane. Induction of RtxA toxin is mediated by a transcriptional regulator HlyU, which belongs to ArsR/SmtB protein family that operates as a homodimer. To date, the crystal structures of HlyU from Vibrio cholera and V. vulnificus have been determined. However, it remains elucidated how HlyU is activated and is participated in transcription of rtxA at a molecular level. To reveal the mechanism of action of HlyU in V. vulnificus, I first determined a minimal DNA sequence for binding of HlyU, indicating that at least two units of the HlyU dimer is bound to the promoter region of rtxA1. Subsequently, the structural determination of the complex of DNA-VvHlyU was tried by x-ray crystallography. The crystals of complex of HlyU and the DNA fragment were obtained, and the optimization of the crystallization conditions is now in progress to determine the crystal structure.
Recently, a research group in this department discovered a synthetic chemical compound called CM2660 inhibits the function of HlyU when the compound was treated to the bacteria. In order to identify mechanism of how CM2660 inhibits the function of HlyU, I determined crystal structure of HlyU from V. vulnificus with a protein sample treated with CM2660. A significant change of the inter-subunit orientation by the compound was found with the reduced interval of the two DNA binding α-helices in the HlyU dimer. This conformational change in the dimer is expected to be associated with a lower binding ability to DNA, leading to inactivation of the HlyU function. Although the compound itself was not observed in the crystal structure, the distance between two cysteine, Cys30 and Cys96 in each subunit, was shortened when compared to the compound-free structure previously reported. In addition, I further identified that CM2660 did not disrupt the dimeric form of HlyU and the presence of extra electron density map between Cys30 and Cys96. Taken together, it is expected that CM2660 may promote the interaction or modification at Cys30 and consequently result in conformational change in the dimer. In order to analyze modification of the cysteine residues, mass spectrometric analysis is under way. This study contributes to the understanding of the HlyU molecular mechanism, which is directly related to the pathogenicity of the fatal foodborne bacterium V. vulnificus.