Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24044
標題: 細菌第二型分泌系統組成蛋白EpsL與汞抗性操作組轉錄調控因子MerR1之結構與功能分析
Structural studies of bacterial type II secretion system component EpsL and transcription regulator MerR1 of mercury resistance operon TnMERI1
作者: 林麗瑩
Lin, Li-Ying
關鍵字: bacterial type II secretion system
細菌第二型分泌系統
transcription regulator MerR1
汞抗性操作組轉錄調控因子
出版社: 生物化學研究所
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摘要: (Part I: Structural studies of bacterial type II secretion system component EpsL) The type II secretion system (T2SS) is used for the translocation of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria. At least 12 distinct protein components are required for the functioning of T2SS by forming a secretion nano-machine that spans both the outer and inner membranes, providing a direct connection between the cytoplasm and outer membrane. Such a trans-envelop assembly couples ATP hydrolysis, taking place exclusively in the cytoplasm, to protein translocation. In T2SS, the cytosolic secretion ATPase GspE is recruited to the membrane-associated secretion complex by interacting with the cytoplasmic membrane proteins GspL. Therefore, GspL appears to serve as a critical link between ATP utilization and exoprotein secretion. Full-length GspL is known to form dimers in vivo, yeast two-hybrid analyses further suggested that both the cytoplasmic and periplasmic domain of GspL can form homomeric interactions. From crystallographic data, cyto-EpsL and N1-EpsE/cyto-EpsL structures of Vibrio cholerae also exist as dimers in the crystals. The existence of homodimer may be important for GspL function. To obtain additional insights on GspL function, we have determined the crystal structure of cytoplasmic domain of GspL from Vibrio parahaemolyticus (cyto-EpsL) at 2.67 Å resolution by the multiwavelength anomalous diffraction method. The cyto-EpsL adopts a dimeric architecture that was first observed in the V. cholerae protein as well as its complex with the N1 domain of EpsE protein. Results from cross-linking assay showed that dimer interface affected cyto-EpsL dimerization efficiency, indicating the crystallographic dimer may indeed exist in solution. It remains to be determined whether this interface is important for protein secretion. Together with the results from gel filtration analysis, a possible mechanism by which the cyto-EpsL dimer interacts with the secretion ATPase was proposed. (Part II: Structural studies of bacterial transcription regulator MerR1 of mercury resistance operon TnMERI1) The mercurial compounds are best known for their extreme toxicity to living organisms due to their high affinity towards all thio-containing proteins and a tendency to substitute and block the functions of essential metals. For some bacteria, carrying a suite of cotranscribed genes, termed the mercury resistance mer operon, allows them to survive in environments contain mercurial compounds. The mer operon is consisted of MerP, MerT, MerC, MerE, MerF, MerA, and MerR genes, which encode proteins capable of converting inorganic (Hg(II)) and organiomercurial compounds (such as methylmercury, MeHg) to less toxic form (Hg(0)). The mer operon transcription is regulated by mercury resistance operon repressor (MerR) protein. Although its name suggests a repressive role during transcription regulation, MerR may also function as a transcription activator. In the absence of Hg(II), MerR binds and represses the transcription of mer operon. However, MerR is converted into a transcription activator upon Hg(II) binding. To understand how MerR regulates the transcription of mer operon, we have determined the structure of MerR protein from Gram-positive bacteria Bacillus megaterium MB1 by the multiwavelength anomalous diffraction method. The MerR1 monomer contains a DNA-binding domain, a dimerization helix and a metal-binding motif. Like most other transcription factors, dimerization of MerR1 is required for function. A total of four MerR1 dimers are present in the asymmetric unit, all exhibiting similar quaternary structure. Compared with the structures of other MerR family members, the metal binding domain of one MerR1 monomer winds around the dimerization helix of the other monomer, suggesting that Hg(II) binding may alter the quaternary structure of MerR1. Such a structural transition may reposition the two DNA-binding domains, thus allows the promoter DNA to interact productively with the RNA polymerase to turn on transcription.
URI: http://hdl.handle.net/11455/24044
其他識別: U0005-1508201023362000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1508201023362000
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