The essential transfer protein TraM binds to DNA as a tetramer

Area of Science:

• Molecular Biology
• Microbiology
• Structural Biology

Background:

• TraM proteins are essential for conjugative DNA transfer mediated by F-like plasmids.
• These proteins are sequence-specific DNA binding proteins.

Purpose of the Study:

• To investigate the quaternary structure and DNA binding properties of the TraM wild-type protein from resistance plasmid R1 and its mutants.
• To elucidate the structural domains responsible for TraM dimerization and tetramerization.

Main Methods:

• Size-exclusion chromatography and differential scanning calorimetry to determine protein quaternary structure.
• Chemical cross-linking and light scattering to corroborate structural findings.
• Band-shift and fluorescence spectroscopy to analyze TraM-DNA interactions.
• Circular dichroism (CD) spectroscopy to assess secondary structure changes upon DNA binding.

Main Results:

• Purified TraM (amino acids 2-127) forms stable tetramers in solution; a truncated version (TraMM26, amino acids 2-56) forms dimers.
• Dimerization is associated with the N-terminal domain, and tetramerization with the C-terminal domain.
• TraM remains tetrameric when bound to its minimal DNA binding site.
• TraM exhibits very strong DNA binding (Kd ~ 10^8 M^-1).
• DNA binding induces an increase in TraM's alpha-helicity (48% to 58%), suggesting an induced fit mechanism.

Conclusions:

• TraM protein structure is predominantly tetrameric, essential for its function in DNA binding.
• The N-terminal and C-terminal domains are responsible for dimerization and tetramerization, respectively.
• TraM utilizes an induced fit mechanism upon DNA binding, enhancing its affinity and function in conjugative DNA transfer.