Optimized method for TAG protein homology modeling: In silico and experimental structural characterization

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• DNA glycosylases are crucial enzymes that initiate DNA repair by cleaving the N-glycosyl bond, releasing a free base and creating abasic sites.
• The function and structure of DNA glycosylases in the pathogenic bacterium Acinetobacter baumannii remain poorly understood.
• Targeting these enzymes presents a promising drug design strategy against A. baumannii infections.

Purpose of the Study:

• To elucidate the structural scaffold of the TAG enzyme from A. baumannii using molecular modeling.
• To characterize the oligomeric state, secondary structure, and substrate binding of the recombinant TAG protein.
• To identify potential inhibitors through molecular docking and virtual screening.

Main Methods:

• Optimized molecular modeling and in silico approaches for structural analysis.
• Expression and purification of recombinant TAG protein.
• Size exclusion chromatography to determine oligomeric state.
• Circular Dichroism (CD) spectroscopy for secondary structure and substrate binding analysis.
• Molecular docking and virtual screening for inhibitor identification.

Main Results:

• The TAG enzyme exists as a monomer with a molecular weight of approximately 21 kDa.
• CD spectroscopy results align with in silico predictions, confirming secondary structure and substrate binding.
• Near UV-CD spectra indicate the involvement of Tyrosine residues in substrate recognition.
• Molecular docking studies revealed crucial interactions for DNA binding and catalysis, reconfirmed by in silico mutational studies.

Conclusions:

• The study provides a detailed structural understanding of the TAG enzyme from A. baumannii.
• Key residues involved in DNA binding and catalytic activity have been identified.
• This knowledge facilitates the rational design of potent inhibitors as potential therapeutic agents against A. baumannii.