TY - JOUR
T1 - Asymmetric assembly of merkel cell polyomavirus large T-antigen origin binding domains at the viral origin
AU - Harrison, Celia J.
AU - Meinke, Gretchen
AU - Kwun, Hyun Jin
AU - Rogalin, Henry
AU - Phelan, Paul J.
AU - Bullock, Peter A.
AU - Chang, Yuan
AU - Moore, Patrick S.
AU - Bohm, Andrew
N1 - Funding Information:
We wish to thank Howard Robinson of the NSLS for data collection. We also thank the following for access and assistance during the ITC experiments: Knut Langsetmo, David Jeruzalmi, Michael Durney, Rachel Nager, Gillian Henry, and Jim Baleja. We also thank Brian Schaffhausen for critical reading of the manuscript. This work was supported by National Institutes of Health grant R21A1082496 to A.B. P.A.B. was supported by National Institutes of Health grant R01GM055397 . Y.C. and P.S.M. were supported by National Institutes of Health grants CA136363 and CA120726 and were also supported as American Cancer Society professors.
PY - 2011/6/17
Y1 - 2011/6/17
N2 - The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ∼ 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.
AB - The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ∼ 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.
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U2 - 10.1016/j.jmb.2011.03.051
DO - 10.1016/j.jmb.2011.03.051
M3 - Article
C2 - 21501625
AN - SCOPUS:79957673618
SN - 0022-2836
VL - 409
SP - 529
EP - 542
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -