Vectorial Transport and Folding of an Autotransporter Virulence Protein During Outer Membrane Secretion

Abstract

Many virulence factors secreted from pathogenic Gram-negative bacteria are autotransporter proteins. The final step of autotransporter secretion is passage across the outer membrane (OM), mediated by a cotranslated C-terminal porin domain. Sequence analysis reveals that, despite size, sequence, and functional diversity, >97% of autotransporter passenger domains are predicted to form parallel β-helices, suggesting this structure is important for secretion. We report the folding behavior of pertactin, an autotransporter passenger domain from Bordetella pertussis. Despite slow but reversible folding in vitro, the β-helix is not prone to aggregation. Interestingly, equilibrium denaturation results in formation of a partially folded structure, with a stable core comprising the C-terminal half of pertactin. Examination of the crystal structure does not reveal any obvious reason for the enhanced stability of the C-terminus. Crystallographic data of the partially folded state shows native like structure for the C-terminus. Interestingly, the C-terminus forms a dimer with a non-native interface with a relatively low KD ∼ 0.3 μM. In vivo, slow folding would prevent premature folding in the periplasm, before OM secretion. Moreover, the extra stability of the C-terminal rungs might serve as a template for the β-helix formation during secretion; hence, vectorial folding of the β-helix could contribute to the energy-independent translocation. We show here that the C-terminus is the first part of the pertactin passenger domain reaching the OM, and that the C-terminus can adopt a stable structure outside the cell, prior to the completion of OM secretion. Coupled with the sequence analysis, these results suggest a general mechanism for autotransporter secretion. The combination of this data, including the lack of pertactin aggregation, could lead to new insights into the formation and prevention of protein aggregation in vivo.