Functional dissection of the conjugative coupling protein TrwB

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• TrwB is a conjugative coupling protein crucial for plasmid R388 DNA transfer.
• It connects the relaxosome to the type IV secretion system, transporting the relaxase TrwC.
• TrwB possesses ATPase activity, likely involved in DNA pumping during conjugation.

Purpose of the Study:

• To investigate the functional domains of TrwB, particularly its cytoplasmic domain and N-terminal transmembrane domain.
• To identify key residues and structural features essential for TrwB's role in DNA and protein transport.
• To explore TrwB's interaction with the type IV secretion apparatus and related proteins like TrwE.

Main Methods:

• Development of a conjugation assay to quantify TrwB function.
• Construction and analysis of point mutants in the TrwB cytoplasmic domain targeting the NTP-binding region, surface, and internal channel.
• Random mutagenesis of the TrwB N-terminal transmembrane domain followed by a two-hybrid screen for enhanced TrwE interaction.

Main Results:

• The integrity of the nucleotide-binding domain is essential for TrwB function and monomer-monomer interactions.
• Polar residues in the internal channel and at its entrance are important for TrwB function and interactions with relaxosomal components.
• Mutations in transmembrane helices, particularly a proline residue, were identified that enhance TrwB-TrwE protein-protein interactions.

Conclusions:

• TrwB's nucleotide-binding domain and specific polar residues are critical for its conjugative coupling activity.
• Interactions within the TrwB hexamer and with relaxosomal components are vital for DNA and protein transport.
• Specific mutations in TrwB's transmembrane domain can modulate its interaction with the type IV secretion system, potentially via key proline residues.