In vitro multimerization and membrane insertion of bacterial outer membrane secretin PulD

Area of Science:

• Molecular Biology
• Structural Biology
• Protein Biochemistry

Background:

• Outer membrane secretins are large protein complexes essential for secreting various molecules across the bacterial outer membrane.
• Understanding the assembly and membrane insertion mechanisms of secretins is key to deciphering their biological roles.

Purpose of the Study:

• To investigate the intrinsic properties of Klebsiella oxytoca secretin PulD regarding its multimerization and membrane insertion.
• To determine if these properties are independent of the cellular environment.

Main Methods:

• In vitro transcription-translation system supplemented with liposomes.
• Negative-stain and cryo-electron microscopy.
• Differential urea extraction.
• Bacterial growth assays.

Main Results:

• Secretin PulD and its core domain spontaneously formed dodecameric rings and inserted into liposomes in vitro.
• Monomeric PulD was observed only in the presence of detergent (Brij-35).
• Synthesis of PulD in E. coli without a signal peptide led to rapid cell death, with observed PulD forming multimers.

Conclusions:

• Multimerization and membrane insertion are intrinsic properties of secretin PulD, not dependent on specific membrane environments or accessory factors.
• These properties appear conserved in closely related secretins (e.g., Erwinia chrysanthemi OutD) but not in more distantly related ones (e.g., Neisseria meningitidis PilQ).
• This study reports the first instance of spontaneous in vitro assembly and membrane insertion of a large outer membrane protein complex.