Estimating the size of the active translocation pore of an autotransporter.
Autotransporters (ATs) are large virulence factors secreted by Gram-negative bacteria. The passenger domain, carrying the virulence functions, is transported across the bacterial outer membrane in a step that is facilitated by a C-terminal β-domain. This domain folds into a β-barrel with a central aqueous pore of ∼1 nm inner diameter according to crystal structures. However, these static dimensions are not compatible with the observed secretion of passengers that may contain natural short-spaced disulfide bonds or artificially fused folded elements. Here, we have systematically analyzed the dimensions of the active AT passenger translocator by inserting peptides of different length and structural complexity in the passenger of the AT hemoglobin protease. The peptides were introduced in a short loop protruding from the main structure and flanked by two single cysteines. Our results show that the attained secondary structure may be more critical for secretion than the length of peptide inserted. Furthermore, the data suggest that, during passenger translocation, at least four extended polypeptides or an extended polypeptide and an α-helix are accommodated in the translocator, indicating that the diameter of the active translocation pore is up to 1.7 nm. If the β-domain functions as the translocator, it must be forced into an expanded conformation during passenger translocation.
Section of Molecular Microbiology, Department of Molecular Cell Biology, VU University, 1081 HVAmsterdam, The Netherlands. a.m.sauriperis@vu.nl
Ana
A
Saurí
Corinne M
CM
Ten Hagen-Jongman
Peter
P
van Ulsen
Joen
J
Luirink
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001692:Biological Transport; D010450:Endopeptidases / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D021381:Protein Transport
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D010450:Endopeptidases; C114676:hemoglobin protease Hbp