Detailed structural and assembly model of the type II secretion pilus from sparse data

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Gram-negative bacteria utilize type II secretion systems (T2SS) for secreting proteins.
• T2SS share similarities with archaeal flagella and type IV pilus (T4P) systems in filament assembly.
• The Klebsiella oxytoca T2SS involves pseudopili essential for pullulanase (PulA) secretion.

Purpose of the Study:

• To determine the detailed structure of the PulG pilus from Klebsiella oxytoca.
• To understand the molecular interactions and assembly of T2SS pili.
• To provide a refined model for T2SS pseudopilus structure.

Main Methods:

• Molecular modeling integrating crystallographic and electron microscopy data.
• Biochemical analysis using single and complementary charge inversions.
• Site-directed mutagenesis with double-cysteine substitutions for cross-linking studies.

Main Results:

• A pseudoatomic resolution model of the PulG pilus was generated.
• Two critical intermolecular salt bridges essential for pilus function were identified.
• Position-specific cross-linking confirmed residue distances within the assembled pili.
• The PulG pilus exhibits a right-handed helical organization of subunits.

Conclusions:

• The study provides a refined structural model for T2SS pili, specifically the PulG pilus.
• Key hydrophobic and electrostatic interactions are conserved within major pseudopilins, suggesting broad relevance.
• The findings enhance understanding of protein secretion mechanisms in gram-negative bacteria via T2SS.