Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing

Area of Science:

• Molecular Biology
• Microbiology
• Structural Biology

Background:

• Type 4 secretion systems (T4SS) are crucial for spreading antibiotic resistance and virulence factors.
• Conjugative T4SS utilize relaxases to process DNA for horizontal gene transfer.
• TraI from plasmid pKM101 is a key relaxase involved in this process.

Purpose of the Study:

• To elucidate the DNA-binding mechanism of conjugative relaxases.
• To characterize the conformational changes in TraI upon DNA binding.
• To investigate the enzymatic activities of TraI domains and full-length protein.

Main Methods:

• X-ray crystallography to determine the structure of TraI's trans-esterase domain with and without DNA.
• Biochemical assays to characterize DNA binding, nicking, and religation activities.
• Comparative analysis of TraI with homologous relaxases.

Main Results:

• The crystal structure of TraI's trans-esterase domain bound to oriT DNA reveals a conserved DNA-binding mechanism.
• An apo structure of the trans-esterase domain shows a flexible thumb subdomain that undergoes significant conformational change upon DNA binding.
• Biochemical characterization confirms conserved enzymatic activity of the TraI trans-esterase domain.

Conclusions:

• The TraI trans-esterase domain from pKM101 exhibits conserved DNA-binding and enzymatic functions similar to homologs from R388 and F plasmids.
• Structural and biochemical data provide new insights into the mechanism of DNA processing by conjugative relaxases.
• This study clarifies the functional behavior of TraI, contributing to our understanding of horizontal gene transfer.