Escherichia coli primase zinc is sensitive to substrate and cofactor binding

Area of Science:

• Biochemistry
• Structural Biology
• Metalloprotein Chemistry

Background:

• Primase from Escherichia coli is essential for DNA replication initiation.
• The enzyme contains a single zinc site crucial for its activity.
• Understanding the zinc site's coordination environment is key to elucidating primase function.

Purpose of the Study:

• To investigate the ligation state of the zinc site in E. coli primase.
• To determine how substrate and cofactor binding affects zinc coordination.
• To map the proximity of the catalytic metal-binding site to the zinc site.

Main Methods:

• X-ray absorption spectroscopy (XAS) was employed to analyze the zinc site.
• Primase was studied in its native state and in complexes with various substrates and cofactors (ATP, (dT)17, magnesium acetate, manganese acetate).
• Analysis focused on the distances and types of ligands coordinated to the zinc atom.

Main Results:

• The native and low-magnesium primase zinc sites are tetrahedrally ligated by three sulfurs and one histidine nitrogen.
• Binding of ATP and/or (dT)17 introduces additional nitrogen/oxygen ligands to the zinc site.
• High magnesium acetate leads to a distinct ligation state, while the catalytic metal site appears to be >5 A from the zinc.

Conclusions:

• The ligation state of the primase zinc site is dynamic and responsive to substrate binding.
• These structural changes are likely critical for primase catalytic activity.
• XAS is a powerful tool for characterizing metalloprotein active sites and their interactions.