Structure of the double-stranded DNA-binding type IV secretion protein TraN from Enterococcus

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Conjugative transfer via type IV secretion systems (T4SS) is a primary mechanism for antimicrobial resistance dissemination.
• Plasmid pIP501 is a Gram-positive conjugative plasmid capable of transfer to Gram-negative bacteria, a significant factor in resistance spread.
• The pIP501-encoded TraN protein is involved in plasmid transfer, localizing to the cytoplasm and exhibiting DNA-binding properties.

Purpose of the Study:

• To elucidate the function and structure of the pIP501 TraN protein in the context of conjugative transfer.
• To identify the specific DNA-binding site of TraN within the pIP501 plasmid.
• To understand TraN's role in the initiation of plasmid DNA transfer.

Main Methods:

• Novel exonuclease digestion and sequencing-based footprinting technique to identify DNA-binding sites.
• X-ray crystallography to determine the three-dimensional structure of TraN at 1.35 Å resolution.
• Bioinformatic analysis to compare TraN structure with known proteins.

Main Results:

• A specific DNA-binding site for TraN upstream of the pIP501 origin of transfer (oriT) was identified.
• The crystal structure of TraN revealed an internal dimer fold with helix-turn-helix (HTH) motifs at both ends.
• TraN shares structural homology with only one half of known excisionases and MerR family transcriptional regulators.

Conclusions:

• TraN is likely an accessory protein to the pIP501 relaxase TraA, playing a role in the early stages of conjugative transfer.
• TraN may facilitate the recruitment of the relaxosome to the mating pore, potentially activating TraA relaxase activity.
• The unique dimeric structure of TraN suggests a novel mechanism for initiating plasmid transfer in Gram-positive bacteria.