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A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding.

Whelan F, Stead JA, Shkumatov AV, Svergun DI, Sanders CM, Antson AA - Nucleic Acids Res. (2011)

Bottom Line: Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis.We argue that these interactions impart processivity to DNA unwinding.Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

View Article: PubMed Central - PubMed

Affiliation: York Structural Biology Laboratory, The University of York, York YO10 5DD, UK.

ABSTRACT
The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

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SAXS ab initio and rigid body modelling of the E1HD/ATP hexamer (A) SAXS profile at infinite dilution for the E1HD hexamer (Experimental data, black circles), overlaid with the crystal structure, ab initio and combined ab initio/rigid body model fits. The linear Guinier plot shown inset confirmed the sample was monodisperse. Predicted scattering for the crystal structure of the E1HD hexamer 2V9P chains A–F (blue), GASBOR ab initio model (green), ab initio/rigid body BUNCH model (red) are shown as solid lines. (B) Distance distribution P(r) function calculated using GNOM for E1HD/ATP hexamer, Dmax = 13 nm. (C) The GASBOR model (χ = 1.34) (grey) was aligned with 2V9P chains A–F (cartoon representation, (left) and translated along the x-axis, and both viewed from the N-terminal oligomerization domain (top), and rotated about the x-axis by 90° (bottom). The hexamer is coloured lime, green, cyan, blue, pink and purple from chain A–F, respectively. (D) BUNCH symmetrical hexameric model constructed using 2V9P chain A, refined using Normal Mode Analysis (χ = 1.57) (top view) shows ‘dummy’ residue (DRs) C-terminal tails (spheres) are located between monomers. N-terminal and loop DRs are also indicated (side view). Figures were prepared using PyMOL (42).
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gkr906-F3: SAXS ab initio and rigid body modelling of the E1HD/ATP hexamer (A) SAXS profile at infinite dilution for the E1HD hexamer (Experimental data, black circles), overlaid with the crystal structure, ab initio and combined ab initio/rigid body model fits. The linear Guinier plot shown inset confirmed the sample was monodisperse. Predicted scattering for the crystal structure of the E1HD hexamer 2V9P chains A–F (blue), GASBOR ab initio model (green), ab initio/rigid body BUNCH model (red) are shown as solid lines. (B) Distance distribution P(r) function calculated using GNOM for E1HD/ATP hexamer, Dmax = 13 nm. (C) The GASBOR model (χ = 1.34) (grey) was aligned with 2V9P chains A–F (cartoon representation, (left) and translated along the x-axis, and both viewed from the N-terminal oligomerization domain (top), and rotated about the x-axis by 90° (bottom). The hexamer is coloured lime, green, cyan, blue, pink and purple from chain A–F, respectively. (D) BUNCH symmetrical hexameric model constructed using 2V9P chain A, refined using Normal Mode Analysis (χ = 1.57) (top view) shows ‘dummy’ residue (DRs) C-terminal tails (spheres) are located between monomers. N-terminal and loop DRs are also indicated (side view). Figures were prepared using PyMOL (42).

Mentions: Next, we induced hexamerization of E1HD with ATP and collected the SAXS data (Figure 3A, black circles). The Guinier plot of the data extrapolated to infinite dilution indicated monodisperse particles (Figure 3A, inset) with a radius of gyration (Rg) of 3.9 ± 0.1 nm. Molecular weight (MW) estimation using Porod analysis indicated that the MW equals 230 ± 20 kDa, in agreement with the calculated 209 kDa mass of the hexamer. Further, as described below, ab initio modelling using DAMMIN identified an excluded volume, Vp = 380 ± 40 nm3, which is consistent with a hexameric assembly. Finally, given Dmax = 13 nm (Figure 3B), the SAXS MoW applet (23) was used to approximate the MW of the E1HD complex, yielding 210 ± 20 kDa, the equivalent of six monomers per assembly. X-ray scattering intensity predicted on the basis of the crystal structure (residues 301–579; PDB code 2V9P), gave Rg = 3.5 nm, Dmax = 11.4 nm and Vp = 300 nm3, all values being smaller than those observed by SAXS. Computed scattering for the E1HD hexamer (residues 308–577, PDB code 2GXA) yielded similar results (Rg = 3.5 nm, Dmax = 11.3 nm and Vp = 300 nm3). This size difference was likely due to the absence of the C-terminal extensions present in the protein used for SAXS analysis. Further, scattering calculated for the crystal structures of E1HD showed a discrepancy in fit to the experimental data (s range from 0.2 to 2.5 nm−1), where χ = 2.79 and χ = 2.77 for 2V9P (Figure 3A, blue line) and 2GXA, respectively. Scattering of a hybrid model of 2V9P and 2GXA (see ‘Materials and Methods’ section) was also computed, resulting in a similar size particle with a slightly improved fit to the experimental data (data not shown). All scattering parameters and χ fits are summarized in Table 1.Figure 3.


A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding.

Whelan F, Stead JA, Shkumatov AV, Svergun DI, Sanders CM, Antson AA - Nucleic Acids Res. (2011)

SAXS ab initio and rigid body modelling of the E1HD/ATP hexamer (A) SAXS profile at infinite dilution for the E1HD hexamer (Experimental data, black circles), overlaid with the crystal structure, ab initio and combined ab initio/rigid body model fits. The linear Guinier plot shown inset confirmed the sample was monodisperse. Predicted scattering for the crystal structure of the E1HD hexamer 2V9P chains A–F (blue), GASBOR ab initio model (green), ab initio/rigid body BUNCH model (red) are shown as solid lines. (B) Distance distribution P(r) function calculated using GNOM for E1HD/ATP hexamer, Dmax = 13 nm. (C) The GASBOR model (χ = 1.34) (grey) was aligned with 2V9P chains A–F (cartoon representation, (left) and translated along the x-axis, and both viewed from the N-terminal oligomerization domain (top), and rotated about the x-axis by 90° (bottom). The hexamer is coloured lime, green, cyan, blue, pink and purple from chain A–F, respectively. (D) BUNCH symmetrical hexameric model constructed using 2V9P chain A, refined using Normal Mode Analysis (χ = 1.57) (top view) shows ‘dummy’ residue (DRs) C-terminal tails (spheres) are located between monomers. N-terminal and loop DRs are also indicated (side view). Figures were prepared using PyMOL (42).
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gkr906-F3: SAXS ab initio and rigid body modelling of the E1HD/ATP hexamer (A) SAXS profile at infinite dilution for the E1HD hexamer (Experimental data, black circles), overlaid with the crystal structure, ab initio and combined ab initio/rigid body model fits. The linear Guinier plot shown inset confirmed the sample was monodisperse. Predicted scattering for the crystal structure of the E1HD hexamer 2V9P chains A–F (blue), GASBOR ab initio model (green), ab initio/rigid body BUNCH model (red) are shown as solid lines. (B) Distance distribution P(r) function calculated using GNOM for E1HD/ATP hexamer, Dmax = 13 nm. (C) The GASBOR model (χ = 1.34) (grey) was aligned with 2V9P chains A–F (cartoon representation, (left) and translated along the x-axis, and both viewed from the N-terminal oligomerization domain (top), and rotated about the x-axis by 90° (bottom). The hexamer is coloured lime, green, cyan, blue, pink and purple from chain A–F, respectively. (D) BUNCH symmetrical hexameric model constructed using 2V9P chain A, refined using Normal Mode Analysis (χ = 1.57) (top view) shows ‘dummy’ residue (DRs) C-terminal tails (spheres) are located between monomers. N-terminal and loop DRs are also indicated (side view). Figures were prepared using PyMOL (42).
Mentions: Next, we induced hexamerization of E1HD with ATP and collected the SAXS data (Figure 3A, black circles). The Guinier plot of the data extrapolated to infinite dilution indicated monodisperse particles (Figure 3A, inset) with a radius of gyration (Rg) of 3.9 ± 0.1 nm. Molecular weight (MW) estimation using Porod analysis indicated that the MW equals 230 ± 20 kDa, in agreement with the calculated 209 kDa mass of the hexamer. Further, as described below, ab initio modelling using DAMMIN identified an excluded volume, Vp = 380 ± 40 nm3, which is consistent with a hexameric assembly. Finally, given Dmax = 13 nm (Figure 3B), the SAXS MoW applet (23) was used to approximate the MW of the E1HD complex, yielding 210 ± 20 kDa, the equivalent of six monomers per assembly. X-ray scattering intensity predicted on the basis of the crystal structure (residues 301–579; PDB code 2V9P), gave Rg = 3.5 nm, Dmax = 11.4 nm and Vp = 300 nm3, all values being smaller than those observed by SAXS. Computed scattering for the E1HD hexamer (residues 308–577, PDB code 2GXA) yielded similar results (Rg = 3.5 nm, Dmax = 11.3 nm and Vp = 300 nm3). This size difference was likely due to the absence of the C-terminal extensions present in the protein used for SAXS analysis. Further, scattering calculated for the crystal structures of E1HD showed a discrepancy in fit to the experimental data (s range from 0.2 to 2.5 nm−1), where χ = 2.79 and χ = 2.77 for 2V9P (Figure 3A, blue line) and 2GXA, respectively. Scattering of a hybrid model of 2V9P and 2GXA (see ‘Materials and Methods’ section) was also computed, resulting in a similar size particle with a slightly improved fit to the experimental data (data not shown). All scattering parameters and χ fits are summarized in Table 1.Figure 3.

Bottom Line: Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis.We argue that these interactions impart processivity to DNA unwinding.Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

View Article: PubMed Central - PubMed

Affiliation: York Structural Biology Laboratory, The University of York, York YO10 5DD, UK.

ABSTRACT
The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

Show MeSH
Related in: MedlinePlus