title: Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates.
abstract: The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively stable intermediate as starting point. Here, we investigated the mechanics of secreted truncated versions of the autotransporter hemoglobin protease (Hbp) of Escherichia coli using atomic force microscopy. The data obtained reveal a β-helical structure at the C terminus that is very stable. In addition, several other distinct metastable intermediates are found which are connected during unfolding by multiroute pathways. Computational analysis indicates that these intermediates correlate to the β-helical rungs in the Hbp structure which are clamped by stacked aromatic residues. Our results suggest a secretion mechanism that is initiated by a stable C-terminal structure and driven forward by several folding intermediates that build up the β-helical backbone.
authors.affiliation: Physics of Living Systems & LaserLaB Amsterdam, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Marian
authors.initials: M
authors.lastname: Baclayon
authors.affiliation: Molecular Microbiology & Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Peter van
authors.initials: Pv
authors.lastname: Ulsen
authors.affiliation: Computer Science & Bioinformatics, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.|Institute of Physical Chemistry, RWTH Aachen University , Landoltweg 2, 52056 Aachen, Germany.
authors.forename: Halima
authors.initials: H
authors.lastname: Mouhib
authors.affiliation: Physics of Living Systems & LaserLaB Amsterdam, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Maryam Hashemi
authors.initials: MH
authors.lastname: Shabestari
authors.affiliation: Computer Science & Bioinformatics, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Timo
authors.initials: T
authors.lastname: Verzijden
authors.affiliation: Computer Science & Bioinformatics, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Sanne
authors.initials: S
authors.lastname: Abeln
authors.affiliation: Physics of Living Systems & LaserLaB Amsterdam, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.|Moleculaire Biofysica, Zernike Instituut, Rijksuniversiteit Groningen , 9712 CP Groningen, The Netherlands.
authors.forename: Wouter H
authors.initials: WH
authors.lastname: Roos
authors.affiliation: Physics of Living Systems & LaserLaB Amsterdam, Vrije Universiteit Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Gijs J L
authors.initials: GJ
authors.lastname: Wuite
mesh_terms: D004926:Escherichia coli; D029968:Escherichia coli Proteins; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D000069320:Type V Secretion Systems
chemical_list: D029968:Escherichia coli Proteins; D000069320:Type V Secretion Systems
keywords: AFM; autotransporters; hemoglobin protease; mechanical unfolding; secretion