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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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Sequence and disorder conservation. (A) E1 and LTag helicase structures are shown side by side. The acidic residues of the C-terminal segment of E1 (BUNCH model) are shown as red spheres (left). The corresponding C-terminal extension of LTag is represented as a dotted line (right), with the acidic region represented as a red dotted line. The structurally characterized C-terminal residues are shown as spheres. (B) Alignment of E1 helicase sequences (BPV1 and HPVs 1, 2, 8, 9, 11, 16, 17, 18, 31, 57) and SV40 LTag shows C-terminal sequence conservation following the last α-helix. The secondary structure in this region of BPV1 E1 is illustrated above the alignment. A 16 residue insertion in this region of LTag, not present in the E1 sequences, is represented by a dotted line. The underlined section indicates the position of the sequence logo. (C) A sequence logo covering the region 572–589 (BPV 1 E1) of the MSA produced using WebLogo (45). This representation of the alignment in (B) shows maintenance of residues typically associated with disorder. (D) Disorder prediction (PONDR) based on aligned C-terminal sequences of HPV 11, 16, 18; BPV1 E1 and LTag illustrates homologous disorder potential. The sequence numbering corresponds to BPV1 E1.
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gkr906-F4: Sequence and disorder conservation. (A) E1 and LTag helicase structures are shown side by side. The acidic residues of the C-terminal segment of E1 (BUNCH model) are shown as red spheres (left). The corresponding C-terminal extension of LTag is represented as a dotted line (right), with the acidic region represented as a red dotted line. The structurally characterized C-terminal residues are shown as spheres. (B) Alignment of E1 helicase sequences (BPV1 and HPVs 1, 2, 8, 9, 11, 16, 17, 18, 31, 57) and SV40 LTag shows C-terminal sequence conservation following the last α-helix. The secondary structure in this region of BPV1 E1 is illustrated above the alignment. A 16 residue insertion in this region of LTag, not present in the E1 sequences, is represented by a dotted line. The underlined section indicates the position of the sequence logo. (C) A sequence logo covering the region 572–589 (BPV 1 E1) of the MSA produced using WebLogo (45). This representation of the alignment in (B) shows maintenance of residues typically associated with disorder. (D) Disorder prediction (PONDR) based on aligned C-terminal sequences of HPV 11, 16, 18; BPV1 E1 and LTag illustrates homologous disorder potential. The sequence numbering corresponds to BPV1 E1.

Mentions: The E1 helicase is structurally similar to the SV40 LTag helicase (Figure 4A). Notably, the C-terminus of neither helicase has been characterized structurally by X-ray crystallography, but both feature a conserved acidic C-terminal region immediately following the last α-helix defined in both structures (Figure 4B and C). Sequence-based analysis identified potential disorder in BPV1 E1 encompassing residues 577–603 (RONN), 573–605 (DisEMBL), 579–593 and 598–605 (Disopred), 577–593 and 602–605 (PONDR). In particular, a sequence motif comprising amino acids 572–589 associated with the intrinsic disorder (44) appears to be conserved, as illustrated in Figure 4C. The position of the predicted disorder is conserved in other HPV E1 proteins and also in SV40 LTag helicase (Figure 4D) and other polyomavirus LTag helicases, aligning with the previously identified sequence motif (BPV1 E1 572–589, Figure 4C). In agreement with this analysis, in the crystal structure of E1 helicase (2V9P), there was no electron density observed for residues 580–605 and there was no difference between E1HD and E1HDΔC26 CD spectra. Taken together, the data indicate that the negatively charged, conserved C-terminus has a flexible character.Figure 4.


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)

Sequence and disorder conservation. (A) E1 and LTag helicase structures are shown side by side. The acidic residues of the C-terminal segment of E1 (BUNCH model) are shown as red spheres (left). The corresponding C-terminal extension of LTag is represented as a dotted line (right), with the acidic region represented as a red dotted line. The structurally characterized C-terminal residues are shown as spheres. (B) Alignment of E1 helicase sequences (BPV1 and HPVs 1, 2, 8, 9, 11, 16, 17, 18, 31, 57) and SV40 LTag shows C-terminal sequence conservation following the last α-helix. The secondary structure in this region of BPV1 E1 is illustrated above the alignment. A 16 residue insertion in this region of LTag, not present in the E1 sequences, is represented by a dotted line. The underlined section indicates the position of the sequence logo. (C) A sequence logo covering the region 572–589 (BPV 1 E1) of the MSA produced using WebLogo (45). This representation of the alignment in (B) shows maintenance of residues typically associated with disorder. (D) Disorder prediction (PONDR) based on aligned C-terminal sequences of HPV 11, 16, 18; BPV1 E1 and LTag illustrates homologous disorder potential. The sequence numbering corresponds to BPV1 E1.
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gkr906-F4: Sequence and disorder conservation. (A) E1 and LTag helicase structures are shown side by side. The acidic residues of the C-terminal segment of E1 (BUNCH model) are shown as red spheres (left). The corresponding C-terminal extension of LTag is represented as a dotted line (right), with the acidic region represented as a red dotted line. The structurally characterized C-terminal residues are shown as spheres. (B) Alignment of E1 helicase sequences (BPV1 and HPVs 1, 2, 8, 9, 11, 16, 17, 18, 31, 57) and SV40 LTag shows C-terminal sequence conservation following the last α-helix. The secondary structure in this region of BPV1 E1 is illustrated above the alignment. A 16 residue insertion in this region of LTag, not present in the E1 sequences, is represented by a dotted line. The underlined section indicates the position of the sequence logo. (C) A sequence logo covering the region 572–589 (BPV 1 E1) of the MSA produced using WebLogo (45). This representation of the alignment in (B) shows maintenance of residues typically associated with disorder. (D) Disorder prediction (PONDR) based on aligned C-terminal sequences of HPV 11, 16, 18; BPV1 E1 and LTag illustrates homologous disorder potential. The sequence numbering corresponds to BPV1 E1.
Mentions: The E1 helicase is structurally similar to the SV40 LTag helicase (Figure 4A). Notably, the C-terminus of neither helicase has been characterized structurally by X-ray crystallography, but both feature a conserved acidic C-terminal region immediately following the last α-helix defined in both structures (Figure 4B and C). Sequence-based analysis identified potential disorder in BPV1 E1 encompassing residues 577–603 (RONN), 573–605 (DisEMBL), 579–593 and 598–605 (Disopred), 577–593 and 602–605 (PONDR). In particular, a sequence motif comprising amino acids 572–589 associated with the intrinsic disorder (44) appears to be conserved, as illustrated in Figure 4C. The position of the predicted disorder is conserved in other HPV E1 proteins and also in SV40 LTag helicase (Figure 4D) and other polyomavirus LTag helicases, aligning with the previously identified sequence motif (BPV1 E1 572–589, Figure 4C). In agreement with this analysis, in the crystal structure of E1 helicase (2V9P), there was no electron density observed for residues 580–605 and there was no difference between E1HD and E1HDΔC26 CD spectra. Taken together, the data indicate that the negatively charged, conserved C-terminus has a flexible character.Figure 4.

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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