Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli

V. Roussel-Jazede, P. van Gelder, R. Sijbrandi, L. Rutten, B.R. Otto, J. Luirink, P. Gros, J. Tommassen, P. van Ulsen

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs. © 2011 Informa UK, Ltd.
Original languageEnglish
Pages (from-to)158-70
JournalMolecular Membrane Biology
Volume28
DOIs
Publication statusPublished - 2011

Fingerprint

IgA-specific serine endopeptidase
Neisseria meningitidis
Neisseria gonorrhoeae
Inclusion Bodies
Lipid Bilayers
Protein Sorting Signals
Gram-Negative Bacteria
Liposomes
Tryptophan
Fluorescence
Escherichia coli
Amino Acids
Peptides
Membranes
hemoglobin protease Hbp
Type V Secretion Systems
In Vitro Techniques

Bibliographical note

1464-5203 Roussel-Jazede, Virginie Van Gelder, Patrick Sijbrandi, Robert Rutten, Lucy Otto, Ben R Luirink, Joen Gros, Piet Tommassen, Jan Van Ulsen, Peter Journal Article Research Support, Non-U.S. Gov't England Mol Membr Biol. 2011 Apr;28(3):158-70. doi: 10.3109/09687688.2010.550328. Epub 2011 Feb 14.

Cite this

Roussel-Jazede, V. ; van Gelder, P. ; Sijbrandi, R. ; Rutten, L. ; Otto, B.R. ; Luirink, J. ; Gros, P. ; Tommassen, J. ; van Ulsen, P. / Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli. In: Molecular Membrane Biology. 2011 ; Vol. 28. pp. 158-70.
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abstract = "Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs. {\circledC} 2011 Informa UK, Ltd.",
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Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli. / Roussel-Jazede, V.; van Gelder, P.; Sijbrandi, R.; Rutten, L.; Otto, B.R.; Luirink, J.; Gros, P.; Tommassen, J.; van Ulsen, P.

In: Molecular Membrane Biology, Vol. 28, 2011, p. 158-70.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli

AU - Roussel-Jazede, V.

AU - van Gelder, P.

AU - Sijbrandi, R.

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AU - Otto, B.R.

AU - Luirink, J.

AU - Gros, P.

AU - Tommassen, J.

AU - van Ulsen, P.

N1 - 1464-5203 Roussel-Jazede, Virginie Van Gelder, Patrick Sijbrandi, Robert Rutten, Lucy Otto, Ben R Luirink, Joen Gros, Piet Tommassen, Jan Van Ulsen, Peter Journal Article Research Support, Non-U.S. Gov't England Mol Membr Biol. 2011 Apr;28(3):158-70. doi: 10.3109/09687688.2010.550328. Epub 2011 Feb 14.

PY - 2011

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N2 - Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs. © 2011 Informa UK, Ltd.

AB - Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs. © 2011 Informa UK, Ltd.

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DO - 10.3109/09687688.2010.550328

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JO - Molecular Membrane Biology

JF - Molecular Membrane Biology

SN - 0968-7688

ER -