Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins |
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Authors: | Leyton Denisse L Sevastsyanovich Yanina R Browning Douglas F Rossiter Amanda E Wells Timothy J Fitzpatrick Rebecca E Overduin Michael Cunningham Adam F Henderson Ian R |
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Affiliation: | School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom. |
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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. |
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Keywords: | Bacterial Toxins Escherichia coli Microbiology Protein Folding Protein Secretion Autotransporter BAM Complex Cysteine Pairs Disulfide-bonded Loops |
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