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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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Related in: MedlinePlus

Conformation of loops near the CDA catalytic core and their dynamics during the simulations.Ribbon representations of the A3G C-CDA starting structures (left had column) and structures after PCA analysis (right hand column) with loops near the catalytic core highlighted in colour: (A) NMR1-2K3A in green; (B) NMR2 in magenta; (C) NMR3-2K3A in blue; (D) XRAY1 in pink and (E) XRAY2-2K3A in yellow. Each Cα atom has a cone attached pointing in the direction of the motion described for the first eigenvector for that atom, with the size of the cone proportional to the amplitude of motion. Cones for residues in loops near the catalytic core are shown in blue; all others are shown in orange. Zinc ions are indicated by grey spheres. In the XRAY2-2K3A structure a second zinc ion that is remote from the catalytic core was observed. Plots for simulations with in silico-generated sequences are not shown.
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pone-0011515-g009: Conformation of loops near the CDA catalytic core and their dynamics during the simulations.Ribbon representations of the A3G C-CDA starting structures (left had column) and structures after PCA analysis (right hand column) with loops near the catalytic core highlighted in colour: (A) NMR1-2K3A in green; (B) NMR2 in magenta; (C) NMR3-2K3A in blue; (D) XRAY1 in pink and (E) XRAY2-2K3A in yellow. Each Cα atom has a cone attached pointing in the direction of the motion described for the first eigenvector for that atom, with the size of the cone proportional to the amplitude of motion. Cones for residues in loops near the catalytic core are shown in blue; all others are shown in orange. Zinc ions are indicated by grey spheres. In the XRAY2-2K3A structure a second zinc ion that is remote from the catalytic core was observed. Plots for simulations with in silico-generated sequences are not shown.

Mentions: To investigate in some detail the source of these differences, we generated ribbon models of the C-CDA on which residues R215, E259 and D316 are indicated (Figure S7). These represent the three amino acids that were commonly identified as mediating interactions with the DNA substrate by three different studies [28]–[30]. This representation of the intitial and representative MD structures highlights that there is considerable variability in the positioning of loops and side chains, which underlies the aforementioned divergence in exposed surface area and charge distribution. Indeed, the variable positioning of loops AC1, AC3 and AC7 that largely determine the accessibility of the catalytic core is evident from a comparison of the starting structures from the crystallography and NMR studies (Figure 9). These loops consistently emerged as being the most flexible parts of the A3G C-CDA throughout our set of MD simulations, as identified by a principal component analysis (PCA) (Figure 9). The considerable flexibility of these loops will also have contributed to the relatively high RMSD values of the structures during the simulations (Figure 4A). As we did not observe the formation of a common stable conformation of loops near the catalytic core during MD simulations, we conclude that there is no evidence to support the structure of any of the previously proposed DNA binding sites within the A3G C-CDA domain. This would suggest that DNA binding to the C-CDA of A3G may instead occur by an induced fit mechanism. The interaction of A3G with DNA is known to be dynamic and most likely short-lived as there is ample evidence to support that A3G can translocate along the DNA to edit multiple target sites on a single DNA substrate [24], [39], [40]. This dynamical behaviour may also underlie the current absence of high resolution structures of A3G bound to DNA.


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)

Conformation of loops near the CDA catalytic core and their dynamics during the simulations.Ribbon representations of the A3G C-CDA starting structures (left had column) and structures after PCA analysis (right hand column) with loops near the catalytic core highlighted in colour: (A) NMR1-2K3A in green; (B) NMR2 in magenta; (C) NMR3-2K3A in blue; (D) XRAY1 in pink and (E) XRAY2-2K3A in yellow. Each Cα atom has a cone attached pointing in the direction of the motion described for the first eigenvector for that atom, with the size of the cone proportional to the amplitude of motion. Cones for residues in loops near the catalytic core are shown in blue; all others are shown in orange. Zinc ions are indicated by grey spheres. In the XRAY2-2K3A structure a second zinc ion that is remote from the catalytic core was observed. Plots for simulations with in silico-generated sequences are not shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011515-g009: Conformation of loops near the CDA catalytic core and their dynamics during the simulations.Ribbon representations of the A3G C-CDA starting structures (left had column) and structures after PCA analysis (right hand column) with loops near the catalytic core highlighted in colour: (A) NMR1-2K3A in green; (B) NMR2 in magenta; (C) NMR3-2K3A in blue; (D) XRAY1 in pink and (E) XRAY2-2K3A in yellow. Each Cα atom has a cone attached pointing in the direction of the motion described for the first eigenvector for that atom, with the size of the cone proportional to the amplitude of motion. Cones for residues in loops near the catalytic core are shown in blue; all others are shown in orange. Zinc ions are indicated by grey spheres. In the XRAY2-2K3A structure a second zinc ion that is remote from the catalytic core was observed. Plots for simulations with in silico-generated sequences are not shown.
Mentions: To investigate in some detail the source of these differences, we generated ribbon models of the C-CDA on which residues R215, E259 and D316 are indicated (Figure S7). These represent the three amino acids that were commonly identified as mediating interactions with the DNA substrate by three different studies [28]–[30]. This representation of the intitial and representative MD structures highlights that there is considerable variability in the positioning of loops and side chains, which underlies the aforementioned divergence in exposed surface area and charge distribution. Indeed, the variable positioning of loops AC1, AC3 and AC7 that largely determine the accessibility of the catalytic core is evident from a comparison of the starting structures from the crystallography and NMR studies (Figure 9). These loops consistently emerged as being the most flexible parts of the A3G C-CDA throughout our set of MD simulations, as identified by a principal component analysis (PCA) (Figure 9). The considerable flexibility of these loops will also have contributed to the relatively high RMSD values of the structures during the simulations (Figure 4A). As we did not observe the formation of a common stable conformation of loops near the catalytic core during MD simulations, we conclude that there is no evidence to support the structure of any of the previously proposed DNA binding sites within the A3G C-CDA domain. This would suggest that DNA binding to the C-CDA of A3G may instead occur by an induced fit mechanism. The interaction of A3G with DNA is known to be dynamic and most likely short-lived as there is ample evidence to support that A3G can translocate along the DNA to edit multiple target sites on a single DNA substrate [24], [39], [40]. This dynamical behaviour may also underlie the current absence of high resolution structures of A3G bound to DNA.

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