Multimerization-defective variants of dodecameric secretin PulD

Area of Science:

• Microbiology
• Molecular Biology
• Protein Biochemistry

Background:

• The secretin PulD from Klebsiella oxytoca is a protein component of the type II secretion system.
• The C-terminal core domain of PulD is crucial for its function and assembly.

Purpose of the Study:

• To investigate the role of amino acid residues in the C-terminal core domain of PulD in complex assembly and toxicity.
• To identify key regions involved in secretin multimerization.

Main Methods:

• In vitro transcription-translation system with liposomes.
• Random mutagenesis of the PulD gene.
• Analysis of dodecameric complex assembly in vitro and in the periplasm.
• Assessment of protein toxicity in Escherichia coli.

Main Results:

• Mutations throughout the PulD C-terminal core domain abolished toxicity and/or impaired dodecameric complex assembly.
• Most substitutions hindered in vitro and periplasmic assembly.
• Only one mutant variant could be rescued by co-production with wild-type PulD.
• A specific three-amino acid insertion created functional multimers dissociable by SDS.

Conclusions:

• Amino acid changes across the PulD C-terminal domain significantly impact secretin assembly and toxicity.
• Specific regions are critical for proper multimerization.
• The study provides insights into the structural requirements for secretin complex formation and stability.