title: Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins.
abstract: Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Denisse L
authors: DL
authors: Leyton
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Yanina R
authors: YR
authors: Sevastsyanovich
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Douglas F
authors: DF
authors: Browning
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Amanda E
authors: AE
authors: Rossiter
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Timothy J
authors: TJ
authors: Wells
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Rebecca E
authors: RE
authors: Fitzpatrick
authors: School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Michael
authors: M
authors: Overduin
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors: Adam F
authors: AF
authors: Cunningham
authors: School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom. Electronic address: i.r.henderson@bham.ac.uk.
authors: Ian R
authors: IR
authors: Henderson
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry; D001692:Biological Transport; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D001427:Bacterial Toxins; D004220:Disulfides; D029968:Escherichia coli Proteins; D003545:Cysteine