Limited tolerance towards folded elements during secretion of the autotransporter Hbp.

W.S.P. Jong, C.M. ten Hagen-Jongman ten, T. den Blaauwen, D.J. Slotboom, J.R.H. Tame, D. Wickstrom, J.-W. de Gier, B.R. Otto, S. Luirink

Research output: Contribution to JournalArticleAcademic

Abstract

Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a β-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane. © 2007 The Authors.
Original languageEnglish
Pages (from-to)1524-1536
JournalMolecular Microbiology
Volume63
Issue number5
DOIs
Publication statusPublished - 2007

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Hemoglobins
Peptide Hydrolases
Disulfides
Membranes
Periplasm
Virulence Factors
Calmodulin
Gram-Negative Bacteria
Mutagenesis
Cysteine
Type V Secretion Systems
Escherichia coli
Enzymes
Proteins

Cite this

Jong, W.S.P. ; ten Hagen-Jongman ten, C.M. ; den Blaauwen, T. ; Slotboom, D.J. ; Tame, J.R.H. ; Wickstrom, D. ; de Gier, J.-W. ; Otto, B.R. ; Luirink, S. / Limited tolerance towards folded elements during secretion of the autotransporter Hbp. In: Molecular Microbiology. 2007 ; Vol. 63, No. 5. pp. 1524-1536.
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abstract = "Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a β-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane. {\circledC} 2007 The Authors.",
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Limited tolerance towards folded elements during secretion of the autotransporter Hbp. / Jong, W.S.P.; ten Hagen-Jongman ten, C.M.; den Blaauwen, T.; Slotboom, D.J.; Tame, J.R.H.; Wickstrom, D.; de Gier, J.-W.; Otto, B.R.; Luirink, S.

In: Molecular Microbiology, Vol. 63, No. 5, 2007, p. 1524-1536.

Research output: Contribution to JournalArticleAcademic

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AU - Jong, W.S.P.

AU - ten Hagen-Jongman ten, C.M.

AU - den Blaauwen, T.

AU - Slotboom, D.J.

AU - Tame, J.R.H.

AU - Wickstrom, D.

AU - de Gier, J.-W.

AU - Otto, B.R.

AU - Luirink, S.

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AB - Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a β-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane. © 2007 The Authors.

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