title: Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli.
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.
authors.affiliation: Department of Molecular Microbiology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.
authors.forename: Virginie
authors.initials: V
authors.lastname: Roussel-Jazédé
authors.forename: Patrick
authors.initials: P
authors.lastname: Van Gelder
authors.forename: Robert
authors.initials: R
authors.lastname: Sijbrandi
authors.forename: Lucy
authors.initials: L
authors.lastname: Rutten
authors.forename: Ben R
authors.initials: BR
authors.lastname: Otto
authors.forename: Joen
authors.initials: J
authors.lastname: Luirink
authors.forename: Piet
authors.initials: P
authors.lastname: Gros
authors.forename: Jan
authors.initials: J
authors.lastname: Tommassen
authors.forename: Peter
authors.initials: P
authors.lastname: Van Ulsen
mesh_terms: D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008081:Liposomes / Q000737:chemistry; D009345:Neisseria meningitidis / Q000201:enzymology; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D008081:Liposomes; D010450:Endopeptidases; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase; C114676:hemoglobin protease Hbp