Introduction of a lethal redox switch that controls the opening and closing of the hydrophobic cavity in LolA.
LolA plays a critical role in the outer membrane sorting of Escherichia coli lipoproteins because it carries a hydrophobic lipoprotein from the inner membrane through the hydrophilic periplasm to the outer membrane receptor LolB. LolA has an incomplete beta-barrel structure composed of 11 beta-strands with an alpha-helical lid forming a hydrophobic cavity inside. The accompanying study revealed that the hydrophobic cavity opens and closes upon the binding and release of lipoproteins, respectively. Ile(93) in the alpha-helix and Phe(140) in the beta-strand are located close to each other in the hydrophobic cavity. These two residues were replaced by Cys to construct the I93C/F140C derivative. Expression of I93C/F140C immediately arrested growth whether wild-type LolA was present or not. However, this dominant negative phenotype was abolished by reducing agents, indicating that the intramolecular disulfide bonding between the two Cys residues is lethal. I93C/F140C was unstable, and its periplasmic level was lower than that of wild-type LolA or its single Cys derivative. Reduction of I93C/F140C was essential for the release of lipoproteins from the inner membrane. Moreover, treatment of I93C/F140C with divalent cross-linkers having different side chain lengths revealed that opening of the lid for a sufficient distance is required for the release activity. The binding of a fluorescent probe to the hydrophobic cavity of I93C/F140C also depended on reducing agents. Taken together, these results indicate that the two Cys residues introduced into LolA function as a redox switch, which regulates the opening and closing of the hydrophobic cavity.
Watanabe
Shoji
S
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
Oguchi
Yuki
Y
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
Takeda
Kazuki
K
RIKEN Spring-8 Center, Harima Institute, Hyogo 679-5148, Japan; Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Miki
Kunio
K
RIKEN Spring-8 Center, Harima Institute, Hyogo 679-5148, Japan; Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Tokuda
Hajime
H
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan. Electronic address: htokuda@iam.u-tokyo.ac.jp.
D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008074:Lipoproteins / Q000737:chemistry; D008956:Models, Chemical; D009154:Mutation; D010084:Oxidation-Reduction; D033902:Periplasmic Binding Proteins / Q000737:chemistry*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013997:Time Factors
D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C116111:LolA protein, E coli; D033902:Periplasmic Binding Proteins; D003545:Cysteine