The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2.

DNA replication of the papillomaviruses is specified by cooperative binding of two proteins to the ori site: the enhancer E2 and the viral initiator E1, a distant member of the AAA+ family of proteins. Formation of this prereplication complex is an essential step toward the construction of a functional, multimeric E1 helicase and DNA melting. To understand how E2 interacts with E1 to regulate this process, we have solved the X-ray structure of a complex containing the HPV18 E2 activation domain bound to the helicase domain of E1. Modeling the monomers of E1 to a hexameric helicase shows that E2 blocks hexamerization of E1 by shielding a region of the E1 oligomerization surface and stabilizing a conformation of E1 that is incompatible with ATP binding. Further biochemical experiments and structural analysis show that ATP is an allosteric effector of the dissociation of E2 from E1. Our data provide the first molecular insights into how a protein can regulate the assembly of an oligomeric AAA+ complex and explain at a structural level why E2, after playing a matchmaker role by guiding E1 to the DNA, must dissociate for subsequent steps of initiation to occur. Building on previously proposed ideas, we discuss how our data advance current models for the conversion of E1 in the prereplication complex to a hexameric helicase assembly.

[1]  A. Stenlund,et al.  Co‐operative interaction between the initiator E1 and the transcriptional activator E2 is required for replicator specific DNA replication of bovine papillomavirus in vivo and in vitro. , 1995, The EMBO journal.

[2]  M. Botchan,et al.  Activation of BPV-1 replication in vitro by the transcription factor E2 , 1991, Nature.

[3]  R. Tjian,et al.  Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription , 1991, Cell.

[4]  V. Moreno,et al.  Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. , 1995, Journal of the National Cancer Institute.

[5]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[6]  A. Stenlund.  Initiation of DNA replication: lessons from viral initiator proteins , 2003, Nature Reviews Molecular Cell Biology.

[7]  K. Wilson,et al.  Structure of the intact transactivation domain of the human papillomavirus E2 protein , 2000, Nature.

[8]  L. Thauvette,et al.  Characterization of Recombinant HPV6 and 11 E1 Helicases , 2001, The Journal of Biological Chemistry.

[9]  J. Kuriyan,et al.  Motors and switches: AAA+ machines within the replisome , 2002, Nature Reviews Molecular Cell Biology.

[10]  M. Botchan,et al.  Competition for DNA Binding Sites between the Short and Long Forms of E2 Dimers Underlies Repression in Bovine Papillomavirus Type 1 DNA Replication Control , 1998, Journal of Virology.

[11]  M. Stanley,et al.  A C-Terminal Helicase Domain of the Human Papillomavirus E1 Protein Binds E2 and the DNA Polymerase α-Primase p68 Subunit , 1998, Journal of Virology.

[12]  L. Joshua-Tor,et al.  Crystal structure of the DNA binding domain of the replication initiation protein E1 from papillomavirus. , 2000, Molecular cell.

[13]  K Cowtan,et al.  Modified phased translation functions and their application to molecular-fragment location. , 1998, Acta crystallographica. Section D, Biological crystallography.

[14]  H. Kowarzyk.  Structure and Function. , 1910, Nature.

[15]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[16]  J. D. Benson,et al.  Mapping and characterization of the interaction domains of human papillomavirus type 16 E1 and E2 proteins , 1997, Journal of virology.

[17]  M. Botchan,et al.  Crystal structure of the human papillomavirus type 18 E2 activation domain. , 1999, Science.

[18]  M. Botchan,et al.  E1 recognition sequences in the bovine papillomavirus type 1 origin of DNA replication: interaction between half sites of the inverted repeats , 1995, Journal of virology.

[19]  E V Koonin,et al.  A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. , 1993, Nucleic acids research.

[20]  P. Bullock.  The initiation of simian virus 40 DNA replication in vitro. , 1997, Critical reviews in biochemistry and molecular biology.

[21]  Craig M. Ogata,et al.  The structure and function of MCM from archaeal M. Thermoautotrophicum , 2003, Nature Structural Biology.

[22]  P. Bullock,et al.  Interactions Required for Binding of Simian Virus 40 T Antigen to the Viral Origin and Molecular Modeling of Initial Assembly Events , 2004, Journal of Virology.

[23]  A. Stenlund.  E1 initiator DNA binding specificity is unmasked by selective inhibition of non‐specific DNA binding , 2003, The EMBO journal.

[24]  A. Stenlund,et al.  Separate domains in E1 and E2 proteins serve architectural and productive roles for cooperative DNA binding , 2000, The EMBO journal.

[25]  M. Scheffner,et al.  The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53 , 1993, Cell.

[26]  C. M. Sanders,et al.  Transcription Factor-dependent Loading of the E1 Initiator Reveals Modular Assembly of the Papillomavirus Origin Melting Complex* , 2000, The Journal of Biological Chemistry.

[27]  N. Cozzarelli,et al.  Characterization of Simian Virus 40 T-antigen Double Hexamers Bound to a Replication Fork , 2002, The Journal of Biological Chemistry.

[28]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[29]  M. Botchan,et al.  Genetic analysis of the activation domain of bovine papillomavirus protein E2: its role in transcription and replication , 1996, Journal of virology.

[30]  T. R. Broker,et al.  Chaperone Proteins Abrogate Inhibition of the Human Papillomavirus (HPV) E1 Replicative Helicase by the HPV E2 Protein , 2002, Molecular and Cellular Biology.

[31]  J. Tainer,et al.  Structure and mechanism of the RuvB Holliday junction branch migration motor. , 2001, Journal of molecular biology.

[32]  M. Botchan,et al.  The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[33]  R. Knippers,et al.  Simian virus 40 large tumor antigen on replicating viral chromatin: tight binding and localization on the viral genome , 1983, Journal of virology.

[34]  J. Hurwitz,et al.  Bovine papilloma virus (BPV)-encoded E2 protein enhances binding of E1 protein to the BPV replication origin. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[35]  D. Simmons,et al.  SV40 large T antigen functions in DNA replication and transformation. , 2000, Advances in virus research.

[36]  M. Botchan,et al.  Biochemical and Electron Microscopic Image Analysis of the Hexameric E1 Helicase* , 1999, The Journal of Biological Chemistry.

[37]  E V Koonin,et al.  AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. , 1999, Genome research.

[38]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[39]  W. Rocque,et al.  Replication-associated activities of purified human papillomavirus type 11 E1 helicase. , 2000, Protein expression and purification.

[40]  A. Stenlund,et al.  Characterization of the DNA-Binding Domain of the Bovine Papillomavirus Replication Initiator E1 , 1998, Journal of Virology.

[41]  Yong Wang,et al.  Crystal Structure of the E2 Transactivation Domain of Human Papillomavirus Type 11 Bound to a Protein Interaction Inhibitor* , 2004, Journal of Biological Chemistry.

[42]  M. Botchan,et al.  cis-Acting components of human papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin , 1995, Journal of virology.

[43]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[44]  C. M. Sanders,et al.  Recruitment and loading of the E1 initiator protein: an ATP‐dependent process catalysed by a transcription factor , 1998, The EMBO journal.

[45]  Julian Peto,et al.  Prevalence of Human Papillomavirus in Cervical Cancer: a Worldwide Perspective , 1995 .

[46]  J. Decaprio,et al.  Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen , 2003, Nature.

[47]  V S Lamzin,et al.  Automated refinement for protein crystallography. , 1997, Methods in enzymology.

[48]  L. Joshua-Tor,et al.  Crystal structures of two intermediates in the assembly of the papillomavirus replication initiation complex , 2002, The EMBO journal.

[49]  R. Huber,et al.  Mutational studies on HslU and its docking mode with HslV. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[50]  T. R. Broker,et al.  Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[51]  M. Botchan,et al.  Site-specific DNA-binding proteins important for replication and transcription have multiple activities. , 1993, Cold Spring Harbor symposia on quantitative biology.

[52]  M. Botchan,et al.  Transcription factor E2 regulates BPV-1 DNA replication in vitro by direct protein-protein interaction. , 1991, Cold Spring Harbor symposia on quantitative biology.

[53]  K. Wilson,et al.  Efficient anisotropic refinement of macromolecular structures using FFT. , 1999, Acta crystallographica. Section D, Biological crystallography.

[54]  A. Aggarwal,et al.  Crystal structure of the SF3 helicase from adeno-associated virus type 2. , 2003, Structure.

[55]  P. Winokur,et al.  The transactivation and DNA binding domains of the BPV-1 E2 protein have different roles in cooperative origin binding with the E1 protein. , 1996, Virology.

[56]  M. Botchan,et al.  Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator. , 1990, Science.

[57]  P. Lambert,et al.  Bovine papillomavirus type 1 E1 and simian virus 40 large T antigen share regions of sequence similarity required for multiple functions , 1997, Journal of virology.

[58]  D. Sanford,et al.  Solution structure of the origin DNA-binding domain of SV40 T-antigen , 1996, Nature Structural Biology.

[59]  J. Hurwitz,et al.  The bovine papillomavirus E2 protein modulates the assembly of but is not stably maintained in a replication-competent multimeric E1-replication origin complex. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[60]  J. D. Benson,et al.  Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions , 1996, Journal of virology.