TY - JOUR
T1 - Limited tolerance towards folded elements during secretion of the autotransporter Hbp.
AU - Jong, W.S.P.
AU - ten Hagen-Jongman ten, C.M.
AU - den Blaauwen, T.
AU - Slotboom, D.J.
AU - Tame, J.R.H.
AU - Wickstrom, D.
AU - de Gier, J.-W.
AU - Otto, B.R.
AU - Luirink, S.
PY - 2007
Y1 - 2007
N2 - 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. © 2007 The Authors.
AB - 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. © 2007 The Authors.
U2 - 10.1111/j.1365-2958.2007.05605.x
DO - 10.1111/j.1365-2958.2007.05605.x
M3 - Article
SN - 0950-382X
VL - 63
SP - 1524
EP - 1536
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 5
ER -