Limited tolerance towards folded elements during secretion of the autotransporter Hbp

Molecular Microbiology - Tập 63 Số 5 - Trang 1524-1536 - 2007
Wouter S. P. Jong1, Corinne M. ten Hagen‐Jongman1, Tanneke den Blaauwen2, Dirk Jan Slotboom3, Jeremy R. H. Tame4, David Wickström5, Jan‐Willem de Gier5, Ben R. Otto1, Joen Luirink1
1Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, 1081 HV Amsterdam, the Netherlands.
2Department of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, the Netherlands.
3Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, the Netherlands
4Protein Design Laboratory, Yokohama City University, Yokohama 230-0045, Japan
5Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, SE-106 91 Stockholm, Sweden

Tóm tắt

SummaryMany 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.

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Molecular Microbiology

Tập 63 Số 5

1524-1536

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