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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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Continuum electrostatics of the BPV1 E1 hexamer. Calculations were made for a hybrid E1 hexamer generated using PDB structures 2V9P and 2GXA. Electrostatic potentials were contoured at +0.25 kT/e (blue) and −0.25 kT/e (red). E1 is shown along the oligomer axis (left) and rotated by 90°about the X-axis (right). The C-terminal ‘dummy’ residues are shown as black circles, overlaid on the electrostatic continuum images.
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gkr906-F5: Continuum electrostatics of the BPV1 E1 hexamer. Calculations were made for a hybrid E1 hexamer generated using PDB structures 2V9P and 2GXA. Electrostatic potentials were contoured at +0.25 kT/e (blue) and −0.25 kT/e (red). E1 is shown along the oligomer axis (left) and rotated by 90°about the X-axis (right). The C-terminal ‘dummy’ residues are shown as black circles, overlaid on the electrostatic continuum images.

Mentions: As the C-terminus contains a conserved segment of negatively charged residues, we reasoned that its role could be to stabilize the hexamer through electrostatic interactions. We investigated this possibility using electrostatic potential calculations, focusing on continuum electrostatic effects (46) (Figure 5). This analysis has highlighted the positive potential of the central tunnel, previously characterized as the region of the hexamer that binds ssDNA. Surrounding the central tunnel, at the top and the base of the hexamer, are areas of negative potential framing large extended areas of positive potential emerging at the surface of each subunit (Figure 5, right). This positive potential is a dominant feature around the outside of the hexamer. Notably, the electrostatic potential has asymmetric features at each subunit interface, possibly conferring a variable effect on the position of the C-terminal peptide. We note that the BUNCH modelling positioned the conserved acidic C-termini between extended basic potentials of adjacent subunits (Figure 5, black circles).Figure 5.


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)

Continuum electrostatics of the BPV1 E1 hexamer. Calculations were made for a hybrid E1 hexamer generated using PDB structures 2V9P and 2GXA. Electrostatic potentials were contoured at +0.25 kT/e (blue) and −0.25 kT/e (red). E1 is shown along the oligomer axis (left) and rotated by 90°about the X-axis (right). The C-terminal ‘dummy’ residues are shown as black circles, overlaid on the electrostatic continuum images.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3300016&req=5

gkr906-F5: Continuum electrostatics of the BPV1 E1 hexamer. Calculations were made for a hybrid E1 hexamer generated using PDB structures 2V9P and 2GXA. Electrostatic potentials were contoured at +0.25 kT/e (blue) and −0.25 kT/e (red). E1 is shown along the oligomer axis (left) and rotated by 90°about the X-axis (right). The C-terminal ‘dummy’ residues are shown as black circles, overlaid on the electrostatic continuum images.
Mentions: As the C-terminus contains a conserved segment of negatively charged residues, we reasoned that its role could be to stabilize the hexamer through electrostatic interactions. We investigated this possibility using electrostatic potential calculations, focusing on continuum electrostatic effects (46) (Figure 5). This analysis has highlighted the positive potential of the central tunnel, previously characterized as the region of the hexamer that binds ssDNA. Surrounding the central tunnel, at the top and the base of the hexamer, are areas of negative potential framing large extended areas of positive potential emerging at the surface of each subunit (Figure 5, right). This positive potential is a dominant feature around the outside of the hexamer. Notably, the electrostatic potential has asymmetric features at each subunit interface, possibly conferring a variable effect on the position of the C-terminal peptide. We note that the BUNCH modelling positioned the conserved acidic C-termini between extended basic potentials of adjacent subunits (Figure 5, black circles).Figure 5.

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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