Minimum Activity Domain Isolation and Functional Analysis of the Antibiotic Protein CaroS2K from the LMW Bacteriocin, Carocin S2

• Main Authors: Jia-De Lin, 林佳德
• Other Authors: Duen-Yau Chuang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/92385471286829043303

碩士 === 國立中興大學 === 化學系所 === 100 === CaroS2K, which contains ribonuclease activity, is an 85kDa protein of bacteriocin protein, which produced by Pectobacterium carotovorum subsp. carotovorum (Pcc) strain 3F-3. From our previous studies, the immunity protein, caroS2I (Mw=10kDa), can associate with the CaroS2K to form a complex, and inhibit the antibiotic function. By homologous analysis from FASTA protein database, the results suggest that CaroS2K has an organization of functional domains as follows: an N-terminal part is present as a transmembrane domain; a central domain is the domain for target cell surface receptor binding; and a C-terminal domain encoding the antibiotic function. We have reported that the target for ribonuclease activity of caroS2K is bacterial ribosomal RNA. Here, to determine the exact C-terminal region of caroS2K needed for rRNA cleavage, we constructed a series of N-terminally truncated carocin S2 by Site-directed, Ligase-Independent Mutagenesis (SLIM). The deleted derivatives starting at residue Gln400, Ile450, Arg600, Gly677 and Lys691 all show the hydrolysis of RNA in vitro, while at Lys692 does not. These results suggest that the minimal ribonuclease region of caroS2K extent from C-terminal residue and locate between Lys691 and Arg783. By alanine-scanning mutagenesis experiment, we have figured out several amino acid residues that were essential for cytotoxicity of CaroS2K. In this study, we take truncated caroS2K derivative caroS2TKD677 as template, substitute its residues mentioned above with alanine. All the point mutants lose the in vitro RNase activity except Y734A and W764A. The results indicate these residues are involved in RNA hydrolysis activity, corresponding to the prediction of computational approach analysis. In order to protect carocin-producing cell itself, there is coordinate synthesis of immunity protein, CaroS2I, which associates with CaroS2K to neutralize its cytotoxicity. By co-immunoprecipitation assays, we identify that there is a interaction-site between caroS2I and C-terminus of caroS2K. In addition, the region within the uncharacterized Domain III of caroS2K resists the association between N-terminally truncated CaroS2K and CaroS2I.