Related Experiment Videos
Structural basis of pilus subunit recognition by the PapD chaperone
M J Kuehn1, D J Ogg, J Kihlberg
1Department of Molecular Microbiology, Washington University, St. Louis, MO 63110.
Insights
Gram-negative bacteria use periplasmic chaperones like PapD for assembling pili. Researchers determined the crystal structure of PapD with a pilus subunit peptide, revealing key interactions essential for bacterial surface fiber assembly.
Area of Science:
- Microbiology
- Structural Biology
- Bacterial Pathogenesis
Background:
- Gram-negative bacteria assemble virulence-associated pili using periplasmic chaperones.
- PapD is a key chaperone in this process, and its subunit interactions are crucial for pilus assembly.
Purpose of the Study:
- To investigate the structural basis of PapD interactions with pilus subunits.
- To understand the molecular mechanisms underlying pilus assembly in Gram-negative bacteria.
Main Methods:
- X-ray crystallography was used to determine the structure of the PapD chaperone complexed with a pilus subunit peptide.
- Site-directed mutagenesis was employed to validate the role of specific residues in PapD-subunit interactions.
Main Results:
- The crystal structure revealed that a pilus subunit peptide binds to PapD in an extended conformation, with its C-terminus anchored in the chaperone's interdomain cleft.
- Hydrogen bonds to invariant residues Arg8 and Lys112, along with hydrophobic interactions, stabilize the complex.
- Mutations in Arg8 and Lys112 disrupted PapD's ability to bind subunits and mediate pilus assembly in vivo.
Conclusions:
- The determined PapD-peptide structure accurately reflects critical interactions involved in pilus subunit binding.
- Invariant residues Arg8 and Lys112 are essential for PapD function in pilus assembly.
- This structural insight provides a foundation for understanding chaperone-mediated protein assembly in bacteria.
Abstract:
The assembly of different types of virulence-associated surface fibers called pili in Gram-negative bacteria requires periplasmic chaperones. PapD is the prototype member of the periplasmic chaperone family, and the structural basis of its interactions with pilus subunits was investigated. Peptides corresponding to the carboxyl terminus of pilus subunits bound PapD and blocked the ability of PapD to bind to the pilus adhesin PapG in vitro. The crystal structure of PapD complexed to the PapG carboxyl-terminal peptide was determined to 3.0 A resolution. The peptide bound in an extended conformation with its carboxyl terminus anchored in the interdomain cleft of the chaperone via hydrogen bonds to invariant chaperone residues Arg8 and Lys112. Main chain hydrogen bonds and contacts between hydrophobic residues in the peptide and the chaperone stabilized the complex and may play a role in determining binding specificity. Site-directed mutations in Arg8 and Lys112 abolished the ability of PapD to bind pilus subunits and mediate pilus assembly in vivo, an indication that the PapD-peptide crystal structure is a reflection of at least part of the PapD-subunit interaction.