担当区分：筆頭著者   出版者・発行元：Cold Spring Harbor Laboratory  

ABSTRACT

The bacterial condensin MukB facilitates proper chromosome segregation inEscherichia coli. A portion of the MukB proteins localize at a specific chromosome region, binding to DNA in a non-sequence-specific manner. However, it is unclear how MukB localizes at a particular site without sequence specificity. Like other structural maintenance of chromosome (SMC) proteins, MukB topologically loads onto DNA, and It has an intrinsic property of preferential topological loading onto the single-stranded DNA (ssDNA). We consider it crucial for the localization of a specific region. To investigate the property of MukB, we attempted to identify positively charged amino acid residues responsible for ssDNA binding. We created a series of mutated MukB proteins in which a single positively charged amino acid was replaced with a negatively charged one. The results showed that some substitutions located on the inner surface of the MukB head domain impacted ssDNA-binding activity, leading to deficiencies in cell growth and nucleoid segregation. The efficiency of topological loading onto ssDNA was also decreased when the positive charges were replaced with negative ones. These amino acid residues align with and bind to ssDNA when the MukB dimer secures ssDNA within its ring, thereby likely strengthening the ssDNA-binding ability of MukB.

DOI： 10.1101/2023.09.21.558748

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掲載種別：論文集(書籍)内論文  

DOI： 10.1007/978-1-4939-9520-2_14

PubMed

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

RseP, an Escherichia coli S2P family intramembrane cleaving protease, is involved in regulation of the extracytoplasmic stress response and membrane quality control through specific cleavage of substrates. Recent research suggested that the PDZ domains and the MRE beta-loop (membrane-reentrant beta-loop) are involved in substrate discrimination; the former would serve to prevent cleavage of substrates with a large periplasmic domain, whereas the latter would directly interact with the substrate's transmembrane segment and induce its conformational change. However, the mechanisms underlying specific substrate recognition and cleavage by RseP are not fully understood. Here, the roles of the N-terminal part of the first cytoplasmic loop region (C1N) of RseP that contains a highly conserved GFG motif were investigated. A Cys modifiability assay suggested that C1N is partly membrane-inserted like the MRE beta-loop. Pro, but not Cys, substitutions in the GFG motif region compromised the proteolytic function of RseP, suggesting the importance of a higher order structure of this motif region. Several lines of evidence indicated that the GFG motif region directly interacts with the substrate and also aids the function of the MRE beta-loop that participates in substrate recognition by RseP. These findings provide insights into the substrate recognition mechanisms of S2P proteases.

DOI： 10.1111/mmi.13659

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記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：ELSEVIER ACADEMIC PRESS INC  

Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-sE protein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins. Here, we describe several methods for characterization of the proteolytic functions and structure of RseP mainly in vivo, including a proteolytic activity assay using model substrates, an in vitro analysis of cleavage of signal peptides in a detergent solution and in the membrane vesicles, structural analysis of membrane-embedded RseP based on the thiol modifiability of introduced cysteine residues, and the protein interaction analysis by in vivo cross-linking protocols.

DOI： 10.1016/bs.mie.2016.09.044

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.7554/eLife.08928

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