Expression of soluble Type IV Minor Pilins and isolation of a Neisseria gonorrhoeae PilI-PilJ subcomplex

Area of Science:

• Microbiology and Molecular Biology
• Bacterial protein structure and function
• Biochemistry and Protein Purification

Background:

• Type IV pili and type II secretion systems are vital dynamic fibers in bacteria and archaea.
• Pilus fibers comprise major and minor pilin subunits with hydrophobic spines and globular heads.
• Purification of minor pilins is challenging due to their hydrophobic nature, disulfide bonds, and low abundance.

Purpose of the Study:

• To develop an efficient method for expressing and purifying soluble type IV minor pilin proteins.
• To overcome purification challenges hindering structural and functional studies of pilin protomers.
• To investigate potential protein-protein interactions among minor pilins.

Main Methods:

• Engineered signal peptidase I cleavage sites into the α-helix of pilin proteins.
• Expressed and purified soluble pilin globular domains from the periplasmic fraction of *Escherichia coli*.
• Coexpressed and purified minor pilins PilI and PilJ from *Neisseria gonorrhoeae*.

Main Results:

• Successfully obtained soluble forms of *Neisseria gonorrhoeae* minor pilins PilI and PilK.
• Purified a PilI-PilJ heterodimer, indicating a potential obligate interaction.
• Demonstrated the method's utility in identifying protein-protein interactions between minor pilins.

Conclusions:

• The engineered signal peptidase cleavage site method facilitates the purification of soluble minor pilins.
• PilI and PilJ likely form an obligate heterodimer essential for their function.
• This purification strategy advances structural and functional investigations of type IV pilins.