Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Human APOBEC3 (A3) proteins are cytidine deaminases involved in innate immunity against viral infections.
• APOBEC3G (A3G) is a key A3 protein that inhibits HIV-1 by deaminating viral single-stranded DNA (ssDNA).
• Characterizing the A3G-ssDNA interaction is challenging due to protein instability and loss of DNA affinity.

Purpose of the Study:

• To develop a novel method for capturing and characterizing the A3G-ssDNA interaction.
• To elucidate the structural basis of A3G's substrate recognition and catalytic mechanism.
• To understand how A3G's nucleotide-binding pockets influence DNA substrate selection.

Main Methods:

• DNA-anchoring fusion strategy using protection of telomeres protein 1 (Pot1).
• X-ray crystallography of the A3G-ssDNA complex.
• Biochemical and virological assays.

Main Results:

• A novel Pot1-A3G fusion strategy successfully captured the A3G-ssDNA interaction.
• Crystal structure revealed a unique conformation of catalytic site loops, explaining substrate scanning in the -1 pocket.
• Biochemistry and virology studies demonstrated mutual influence between A3G's nucleotide-binding pockets in substrate selection.

Conclusions:

• The study provides structural and biochemical insights into A3G's DNA substrate selection mechanism.
• Understanding A3G's substrate specificity enhances knowledge of its antiviral activity against HIV-1.
• This work defines how A3 proteins recognize specific DNA sequences, contributing to antiviral strategies.