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Rationalisation of the differences between APOBEC3G structures from crystallography and NMR studies by molecular dynamics simulations.

Autore F, Bergeron JR, Malim MH, Fraternali F, Huthoff H - PLoS ONE (2010)

Bottom Line: In the course of these simulations, we observed a general trend towards increased definition of the beta2 strand for those structures that have a distorted starting conformation of beta2.We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core.We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

ABSTRACT
The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal beta-strand, beta2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the beta2 strand for those structures that have a distorted starting conformation of beta2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the beta2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

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Alignment of the β1-β2 region from A2 and A3 proteins.Amino acid sequence alignment of the β1-β2 region from the human A2 and A3 proteins as generated with T-coffee, with the most conserved residues indicated by grey shading. The positions of β1 and β2 as present in the A2 crystal structure are indicated by arrows above the alignment. H-bonds between back-bone atoms in the β1-β2 sheet of A2 are indicated with red lines. Purple lines indicate H-bonds shared by the two crystal structures of the A3G C-CDA domain. The following H-bonds indicated by purple lines are also present in the NMR structures: L220-L242 in NMR1-2K3A and NMR3-2K3A; Y222-G240 in all NMR structures. Green lines indicate H-bonds unique to XRAY1 and blue lines indicate H-bonds observed in XRAY2-2K3A. Except for the absence of H-bonds between Y225-L235 in NMR2 and H228-T231 in NMR3-2K3A, H-bonds indicated by blue lines are also present in the NMR structures.
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pone-0011515-g003: Alignment of the β1-β2 region from A2 and A3 proteins.Amino acid sequence alignment of the β1-β2 region from the human A2 and A3 proteins as generated with T-coffee, with the most conserved residues indicated by grey shading. The positions of β1 and β2 as present in the A2 crystal structure are indicated by arrows above the alignment. H-bonds between back-bone atoms in the β1-β2 sheet of A2 are indicated with red lines. Purple lines indicate H-bonds shared by the two crystal structures of the A3G C-CDA domain. The following H-bonds indicated by purple lines are also present in the NMR structures: L220-L242 in NMR1-2K3A and NMR3-2K3A; Y222-G240 in all NMR structures. Green lines indicate H-bonds unique to XRAY1 and blue lines indicate H-bonds observed in XRAY2-2K3A. Except for the absence of H-bonds between Y225-L235 in NMR2 and H228-T231 in NMR3-2K3A, H-bonds indicated by blue lines are also present in the NMR structures.

Mentions: The distorted conformation of the β2 that is observed in the majority of A3G C-CDA structures represents a unique feature among CDA enzymes, as all other structures of these proteins show a defined and continuous β2 strand [34]. Indeed, the structure of the closely related A2 protein also shows an intact β2 region that is continuous with β1 and furthermore supports oligomerisation via β2-β2 interactions [33]. To determine whether this difference may be due to divergent primary sequences, we generated an alignment of the β1-β2 region from A2 and the human A3 proteins, indicating the β1-β2 interactions observed in the crystal structures of A2 and the various structures of the A3G C-CDA (Figure 3). From the alignment, it is apparent that the sequence of the β1 region contains a strongly conserved L-C-F/Y motif, which in the A2 structure interacts with a G-Y-L motif in the β2 region of A2. The latter motif is partly conserved in the human A3 proteins, and in A3G corresponds to G-F-L at positions 240 to 242. Importantly, the interaction between these motifs is evidenced by both crystal structures and is to variable extents also evident from the NMR data (Figure 3).


Rationalisation of the differences between APOBEC3G structures from crystallography and NMR studies by molecular dynamics simulations.

Autore F, Bergeron JR, Malim MH, Fraternali F, Huthoff H - PLoS ONE (2010)

Alignment of the β1-β2 region from A2 and A3 proteins.Amino acid sequence alignment of the β1-β2 region from the human A2 and A3 proteins as generated with T-coffee, with the most conserved residues indicated by grey shading. The positions of β1 and β2 as present in the A2 crystal structure are indicated by arrows above the alignment. H-bonds between back-bone atoms in the β1-β2 sheet of A2 are indicated with red lines. Purple lines indicate H-bonds shared by the two crystal structures of the A3G C-CDA domain. The following H-bonds indicated by purple lines are also present in the NMR structures: L220-L242 in NMR1-2K3A and NMR3-2K3A; Y222-G240 in all NMR structures. Green lines indicate H-bonds unique to XRAY1 and blue lines indicate H-bonds observed in XRAY2-2K3A. Except for the absence of H-bonds between Y225-L235 in NMR2 and H228-T231 in NMR3-2K3A, H-bonds indicated by blue lines are also present in the NMR structures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902501&req=5

pone-0011515-g003: Alignment of the β1-β2 region from A2 and A3 proteins.Amino acid sequence alignment of the β1-β2 region from the human A2 and A3 proteins as generated with T-coffee, with the most conserved residues indicated by grey shading. The positions of β1 and β2 as present in the A2 crystal structure are indicated by arrows above the alignment. H-bonds between back-bone atoms in the β1-β2 sheet of A2 are indicated with red lines. Purple lines indicate H-bonds shared by the two crystal structures of the A3G C-CDA domain. The following H-bonds indicated by purple lines are also present in the NMR structures: L220-L242 in NMR1-2K3A and NMR3-2K3A; Y222-G240 in all NMR structures. Green lines indicate H-bonds unique to XRAY1 and blue lines indicate H-bonds observed in XRAY2-2K3A. Except for the absence of H-bonds between Y225-L235 in NMR2 and H228-T231 in NMR3-2K3A, H-bonds indicated by blue lines are also present in the NMR structures.
Mentions: The distorted conformation of the β2 that is observed in the majority of A3G C-CDA structures represents a unique feature among CDA enzymes, as all other structures of these proteins show a defined and continuous β2 strand [34]. Indeed, the structure of the closely related A2 protein also shows an intact β2 region that is continuous with β1 and furthermore supports oligomerisation via β2-β2 interactions [33]. To determine whether this difference may be due to divergent primary sequences, we generated an alignment of the β1-β2 region from A2 and the human A3 proteins, indicating the β1-β2 interactions observed in the crystal structures of A2 and the various structures of the A3G C-CDA (Figure 3). From the alignment, it is apparent that the sequence of the β1 region contains a strongly conserved L-C-F/Y motif, which in the A2 structure interacts with a G-Y-L motif in the β2 region of A2. The latter motif is partly conserved in the human A3 proteins, and in A3G corresponds to G-F-L at positions 240 to 242. Importantly, the interaction between these motifs is evidenced by both crystal structures and is to variable extents also evident from the NMR data (Figure 3).

Bottom Line: In the course of these simulations, we observed a general trend towards increased definition of the beta2 strand for those structures that have a distorted starting conformation of beta2.We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core.We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

ABSTRACT
The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal beta-strand, beta2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the beta2 strand for those structures that have a distorted starting conformation of beta2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the beta2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

Show MeSH
Related in: MedlinePlus