Pertactin β-Helix Folding Mechanism Suggests Common Themes for the Secretion and Folding of Autotransporter Proteins

• Published in: Proceedings of the National Academy of Sciences - PNAS 2006-03, Vol.103 (13), p.4918-4923
• Main Authors: Junker, Mirco, Schuster, Christopher C., McDonnell, Andrew V., Sorg, Kelli A., Finn, Mary C., Berger, Bonnie, Clark, Patricia L.
• Format: Article
• Language: English
• Subjects: Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Outer Membrane Proteins - secretion; Bacterial proteins; Biological Sciences; Biology; Biophysics; Bordetella pertussis; Cell Membrane - metabolism; Crystal structure; Fluorescence; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Membrane Transport Proteins - secretion; Models, Molecular; Molecular Weight; Passengers; Periplasm; Porins; Protein Binding; Protein Denaturation - drug effects; Protein Folding; Protein refolding; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Secretion; Sequence analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin - chemistry; Trypsin - metabolism; Virulence factors; Virulence Factors, Bordetella - chemistry; Virulence Factors, Bordetella - genetics; Virulence Factors, Bordetella - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0507923103

Many virulence factors secreted from pathogenic Gram-negative bacteria are autotransporter proteins. The final step of autotransporter secretion is C → N-terminal threading of the passenger domain through the outer membrane (OM), mediated by a cotranslated C-terminal porin domain. The native structure is formed only after this final secretion step, which requires neither ATP nor a proton gradient. Sequence analysis reveals that, despite size, sequence, and functional diversity among autotransporter passenger domains, >97% are predicted to form parallel β-helices, indicating this structural topology may be important for secretion. We report the folding behavior of pertactin, an autotransporter passenger domain from Bordetella pertussis. The pertactin β-helix folds reversibly in isolation, but folding is much slower than expected based on size and native-state topology. Surprisingly, pertactin is not prone to aggregation during folding, even though folding is extremely slow. Interestingly, equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the C-terminal half of the protein. Examination of the pertactin crystal structure does not reveal any obvious reason for the enhanced stability of the C terminus. In vivo, slow folding would prevent premature folding of the passenger domain in the periplasm, before OM secretion. Moreover, the extra stability of the C-terminal rungs of the β-helix might serve as a template for the formation of native protein during OM secretion; hence, vectorial folding of the β-helix could contribute to the energy-independent translocation mechanism. Coupled with the sequence analysis, the results presented here suggest a general mechanism for autotransporter secretion.