Independent Domain Assembly in a Trapped Folding Intermediate of Multimeric Outer Membrane Secretins

• Published in: Structure (London) 2014-04, Vol.22 (4), p.582-589
• Main Authors: Guilvout, Ingrid, Chami, Mohamed, Disconzi, Elena, Bayan, Nicolas, Pugsley, Anthony P., Huysmans, Gerard H.M.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Amino acids; Assembly; Bacteria; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - ultrastructure; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - ultrastructure; Bacterial Secretion Systems - physiology; Chambers; Electron microscopy; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Klebsiella oxytoca - chemistry; Klebsiella oxytoca - metabolism; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - ultrastructure; Membranes; Protein Binding; Protein Folding; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Pseudomonas aeruginosa - chemistry; Pseudomonas aeruginosa - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - ultrastructure; Secretions; Structural Homology, Protein; Structure-Activity Relationship; Vestibules
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2014.02.009

The outer membrane portal of the Klebsiella oxytoca type II secretion system, PulD, is a prototype of a family of proteins, the secretins, which are essential components of many bacterial secretion and pilus assembly machines. PulD is a homododecamer with a periplasmic vestibule and an outer chamber on either side of a membrane-spanning region that is poorly resolved by electron microscopy. Membrane insertion involves the formation of a dodecameric membrane-embedded intermediate. Here, we describe an amino acid substitution in PulD that blocks its assembly at this intermediate “prepore” stage. Electron microscopy indicated that the prepore has an apparently normal periplasmic vestibule but a poorly organized outer chamber. A peptide loop around this amino acid appears to be important for the formation/stability of the fully folded complex. A similar assembly intermediate results from creation of the same amino acid substitution in the Pseudomonas aeruginosa secretin XcpQ. •Protein engineering traps a prepore in destabilized variants of the secretin PulD•A short peptide loop is required for stable membrane insertion/assembly•Prepores are also trapped by homologous substitutions in the secretin XcpQ•We speculate that native pore formation requires a conformational switch Guilvout et al. examine the oligomerization and pore formation by genetically destabilized variants of secretin PulD. They identify a peptide involved in membrane insertion and visualize a prepore assembly intermediate for PulD and a related secretin XcpQ, a common assembly mechanism.