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Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded
Fareeda M Barzak1, Timothy M Ryan2, Nazanin Mohammadzadeh3
1School of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand.
Viruses
|September 23, 2022
Summary
The study reveals the structure of APOBEC3G bound to single-stranded DNA (ssDNA) using SAXS. This provides insights into how APOBEC3G interacts with DNA, explaining its antiviral activity and enzyme processivity.
Area of Science:
- Biochemistry
- Structural Biology
- Innate Immunity
Background:
- APOBEC3 enzymes are crucial for innate immunity, acting as DNA/RNA deaminases against viruses and retrotransposons.
- APOBEC3G, a key enzyme, restricts HIV replication but its full-length structure bound to ssDNA remains poorly understood.
- Existing structural data is limited to individual domains or apo-forms, lacking insight into the complete enzyme-ssDNA complex.
Purpose of the Study:
- To determine the solution-state structure of full-length APOBEC3G in complex with modified ssDNA.
- To elucidate the structural basis for APOBEC3G's interaction with ssDNA and its implications for enzyme function.
- To provide a structural model for understanding APOBEC3-ssDNA interactions and enzyme processivity.
Main Methods:
- Small-angle X-ray scattering (SAXS) was employed to study the structure of full-length APOBEC3G with and without a 40-mer modified ssDNA.
- Size-exclusion chromatography (SEC) was used prior to SAXS for partial separation of protein-nucleic acid complexes.
- A modified ssDNA containing 2'-deoxyzebularine was used to inhibit cytosine deamination during structural analysis.
Main Results:
- Full-length APOBEC3G exists as multiple multimeric species, with tetramers being the most prominent in the absence of ssDNA.
- Upon ssDNA addition, tetrameric and dimeric forms of APOBEC3G dissociate, leading to a dominant monomeric species bound to ssDNA.
- The SAXS data revealed a structural model of the full-length APOBEC3G-ssDNA complex, highlighting how both domains interact with the nucleic acid.
Conclusions:
- The study presents the first structural model of full-length APOBEC3G bound to ssDNA in solution.
- The observed structural changes explain the shift from multimeric to monomeric forms upon DNA binding and provide mechanistic insights into enzyme processivity.
- This work lays the foundation for further investigations into APOBEC3-ssDNA interactions and their role in antiviral defense.
