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Limited tolerance towards folded elements during secretion of the autotransporter Hbp
Author(s) -
Jong Wouter S. P.,
Ten HagenJongman Corinne M.,
Den Blaauwen Tanneke,
Jan Slotboom Dirk,
Tame Jeremy R. H.,
Wickström David,
De Gier JanWillem,
Otto Ben R.,
Luirink Joen
Publication year - 2007
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2007.05605.x
Subject(s) - periplasmic space , bacterial outer membrane , secretion , biology , protease , dsba , biochemistry , protein folding , secretory protein , microbiology and biotechnology , virulence , cysteine protease , biophysics , escherichia coli , enzyme , gene
Summary 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.