Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity
APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual...
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2022-09-01
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| Online Access: | https://www.mdpi.com/1999-4915/14/9/1974 |
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| author | Fareeda M. Barzak Timothy M. Ryan Nazanin Mohammadzadeh Stefan Harjes Maksim V. Kvach Harikrishnan M. Kurup Kurt L. Krause Linda Chelico Vyacheslav V. Filichev Elena Harjes Geoffrey B. Jameson |
| author_facet | Fareeda M. Barzak Timothy M. Ryan Nazanin Mohammadzadeh Stefan Harjes Maksim V. Kvach Harikrishnan M. Kurup Kurt L. Krause Linda Chelico Vyacheslav V. Filichev Elena Harjes Geoffrey B. Jameson |
| author_sort | Fareeda M. Barzak |
| collection | DOAJ |
| description | APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes. |
| first_indexed | 2024-03-09T22:13:48Z |
| format | Article |
| id | doaj.art-e6bb7a035b1541ab907a372338d6e150 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-09T22:13:48Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-e6bb7a035b1541ab907a372338d6e1502023-11-23T19:27:22ZengMDPI AGViruses1999-49152022-09-01149197410.3390/v14091974Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and ProcessivityFareeda M. Barzak0Timothy M. Ryan1Nazanin Mohammadzadeh2Stefan Harjes3Maksim V. Kvach4Harikrishnan M. Kurup5Kurt L. Krause6Linda Chelico7Vyacheslav V. Filichev8Elena Harjes9Geoffrey B. Jameson10School of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New ZealandSAXS/WAXS, Australian Synchrotron/ANSTO, 800 Blackburn Road, Clayton, VIC 3168, AustraliaDepartment of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaSchool of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New ZealandSchool of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New ZealandSchool of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New ZealandDepartment of Biochemistry, University of Otago, P.O. Box 56, Dunedin 9054, New ZealandDepartment of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, CanadaSchool of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New ZealandSchool of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New ZealandSchool of Natural Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New ZealandAPOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes.https://www.mdpi.com/1999-4915/14/9/1974APOBEC3APOBEC3GSAXSprotein–DNA complex |
| spellingShingle | Fareeda M. Barzak Timothy M. Ryan Nazanin Mohammadzadeh Stefan Harjes Maksim V. Kvach Harikrishnan M. Kurup Kurt L. Krause Linda Chelico Vyacheslav V. Filichev Elena Harjes Geoffrey B. Jameson Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity Viruses APOBEC3 APOBEC3G SAXS protein–DNA complex |
| title | Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity |
| title_full | Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity |
| title_fullStr | Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity |
| title_full_unstemmed | Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity |
| title_short | Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity |
| title_sort | small angle x ray scattering saxs measurements of apobec3g provide structural basis for binding of single stranded dna and processivity |
| topic | APOBEC3 APOBEC3G SAXS protein–DNA complex |
| url | https://www.mdpi.com/1999-4915/14/9/1974 |
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