title: Limited tolerance towards folded elements during secretion of the autotransporter Hbp.
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 beta-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.
authors.affiliation: Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, 1081 HV Amsterdam, the Netherlands.
authors.forename: Wouter S P
authors.initials: WS
authors.lastname: Jong
authors.forename: Corinne M
authors.initials: CM
authors.lastname: ten Hagen-Jongman
authors.forename: Tanneke
authors.initials: T
authors.lastname: den Blaauwen
authors.forename: Dirk Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.forename: Jeremy R H
authors.initials: JR
authors.lastname: Tame
authors.forename: David
authors.initials: D
authors.lastname: Wickström
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: de Gier
authors.forename: Ben R
authors.initials: BR
authors.lastname: Otto
authors.forename: Joen
authors.initials: J
authors.lastname: Luirink
mesh_terms: D003545:Cysteine / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D033903:Periplasmic Proteins / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; D010450:Endopeptidases; C061682:DegP protease; D012697:Serine Endopeptidases; C114676:hemoglobin protease Hbp; D019704:Protein Disulfide-Isomerases; D003545:Cysteine