Crystal Structure of the E2 Transactivation Domain of Human Papillomavirus Type 11 Bound to a Protein Interaction Inhibitor

Wang, Yong; Coulombe, René; Cameron, Dale R.; Thauvette, Louise; Massariol, Marie-Josée; Amon, Lynn M.; Fink, D; Titolo, Steve; Welchner, Ewald; Yoakim, Christiane; Archambault, Jacques; White, Peter W.

doi:10.1074/jbc.m311376200

Cited by 86 publications

(72 citation statements)

References 48 publications

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“…1B), as opposed to the outer surface, which mediates E2 · E2 interactions in the free HPV16 and HPV18 E2 structures. The absence of dimerized E2 molecules in the E1 · E2 complex, as well as in the uncomplexed HPV11 E2 structure (Wang et al 2004), supports the notion that the dimerization of E2-AD observed in some crystal forms may be fortuitous. For E2-dependent DNA replication in vivo, a single E2-binding site is sufficient in transient assays (Russell and Botchan 1995); similarly, an E2 dimer containing a single activation domain functions as wild type in vitro (Lim et al 1998).…”

Section: Structure Of the E1 · E2 Complexsupporting

confidence: 57%

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

Abbate¹,

Berger²,

Botchan³

2004

Genes Dev.

147

172

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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.[Keywords: Crystal structure E1E2 complex; papillomavirus; helicase structure; AAA+ protein; DNA replication] Papillomaviruses (PVs) are small DNA viruses that maintain latency in the stem cells of various epithelial tissues (for review, see Howley 1996). They are the etiological agents of a variety of benign lesions of the epithelium, and certain high-risk variants of the human papillomavirus (HPV), such as HPV-16, 18, and 31, are associated with cervical carcinomas (Bosch et al. 1995). The virus is maintained as an extrachromosomal nuclear plasmid that replicates in S phase with the host genome. Because PVs have few open reading frames (∼8 ORFs), the viral life cycle depends heavily on interaction with the host cell for enzymes and ancillary factors for its own gene expression and replication programs. The PVs have provided important novel insights into the regulation of cell proliferation and cancer etiology; for example, the targeted degradation of p53 directed by the HPV16 E6 protein first revealed how key cellular regulatory proteins could be specifically ubiquitinated by E3 ligases for destruction by the proteosome (Scheffner et al. 1993). The PVs also have provided models to understand how eukaryotic transcription factors coassociate to form loops between segments of DNA (Li et al. 1991) and to explore assembly of specific replication factories on defined origins of replication (Stenlund 2003b).The first two universal steps in the formation of a prereplication complex are origin recognition and subsequent distortion and separation of duplex DNA strands. The simplicity of the PV prereplication complex offers advantages for mechanistic and structural dissection of these steps. Only t...

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Section: Structure Of the E1 · E2 Complexsupporting

confidence: 57%

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

Abbate¹,

Berger²,

Botchan³

2004

Genes Dev.

147

172

View full text Add to dashboard Cite

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“…Such interdimer interactions and DNA loops would allow transcription factors that are bound near E2 at distal sites to be brought into the proximity of a transcription initiation site. However, recent reports of the structure of the HPV11 E2 transactivation domain (56) or HPV18 E2 transactivation domain (1) found no evidence of self-interaction or dimerization through the transactivation domain, so it is unclear whether the BPV1 E2 protein can also participate in interdimer interactions. Notably, the EpsteinBarr virus EBNA1 protein, which has analogous genome maintenance and segregation functions to those of E2 and associates with mitotic chromosomes, also mediates the looping of DNA (25,(34)(35)(36)59).…”

Section: Discussionmentioning

confidence: 99%

The Mitotic Chromosome Binding Activity of the Papillomavirus E2 Protein Correlates with Interaction with the Cellular Chromosomal Protein, Brd4

Baxter

McPhillips

Ozato

et al. 2005

J Virol

110

157

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The papillomavirus transcriptional activator, E2, is involved in key functions of the viral life cycle. These include transcriptional regulation, viral DNA replication, and viral genome segregation. The transactivation domain of E2 is required for each of these functions. To identify the regions of the domain that mediate binding to mitotic chromosomes, a panel of mutations has been generated and their effect on various E2 functions has been analyzed. A structural model of the bovine papillomavirus type 1 (BPV1) E2 transactivation domain was generated based on its homology with the solved structure of the human papillomavirus type 16 (HPV16) domain. This model was used to identify distinct surfaces of the domain to be targeted by point mutation to further delineate the functional region of the transactivation domain responsible for mitotic chromosome association. The mutated E2 proteins were assessed for mitotic chromosome binding and, in addition, transcriptional activation and transcriptional repression activities. Mutation of amino acids R37 and I73, which are located on a surface of the domain that in HPV16 E2 is reported to mediate self-interaction, completely eliminated mitotic chromosome binding. Mitotic chromosome binding activity was found to correlate well with the ability to interact with the cellular chromosomal associated factor Brd4, which has recently been proposed to mediate the association between BPV1 E2 and mitotic chromosomes.Papillomaviruses are small DNA viruses that infect the squamous epithelium of the skin and mucosa, causing benign tumors or papillomas (22). A key step in the viral life cycle occurs upon infection of basal epithelial cells, when the viral genome must be maintained as an extrachromosomal element to ensure persistence of the infection in dividing cells. Transient viral DNA replication requires the E2 transcriptional transactivator, the E1 helicase protein, and the origin of replication (54,55,57,61). Long-term, persistent replication requires, in addition, numerous high-affinity E2 binding sites in cis with the replication origin (47). The E2 transcriptional transactivator protein maintains the viral genomes as multicopy extrachromosomal elements by tethering them to mitotic chromosomes to ensure their nuclear retention and segregation to daughter cells (24,32,51).The E2 transactivator protein can be divided into three domains. The amino-terminal ϳ200 amino acids comprise the transactivation domain of E2, and the carboxy-terminal ϳ100 amino acids mediate DNA binding and dimerization. These domains are linked by a flexible region called the hinge. The transactivation domain and DNA binding domain are conserved among E2 proteins of different papillomaviruses, whereas the length and amino acid sequence of the hinge region vary greatly (reviewed in reference 42). The transactivation domain is critical for the segregation, DNA replication, and transcriptional regulatory functions of the E2 protein (5,41,51,58).Several groups have mapped residues important for the transactivati...

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“…Although the role of this dimerization has not been determined, it seemed to be of major importance for protein function, as most mutations in HPV-16 E2 that compromise transactivation without affecting replication were found to map in the dimerization interface (28). Although the HPV-11 E2 protein crystal structure has since been determined to be monomeric, probably due to different crystallization conditions (45), dimer formation in the HPV-16 E2 TAD was verified in solution, using biophysical techniques (5).…”

Section: Discussionmentioning

confidence: 99%

“…The structure of the intact E2 protein closely resembles that of other mammalian transcription factors, consisting of a DNA-binding/dimerization domain (DBD) connected by a flexible linker to a multiple-protein-binding transactivation domain (TAD). The three-dimensional structures of the C-terminal DBD (11,16,17) and N-terminal TAD (1,5,15,30,45) of several E2 proteins have been reported, both alone and in complex, revealing a tight dimer of the DBD bound to DNA and a characteristic L-shaped TAD structure. In the case of HPV-16, two TAD domains form a dimer; this additional dimerization interface has been proposed to link two E2 dimers bound via the DBD to two distant E2BS within the URR (5).…”

mentioning

confidence: 99%

Dimerization of the Human Papillomavirus Type 16 E2 N Terminus Results in DNA Looping within the Upstream Regulatory Region

Hernandez-Ramon

Burns

Zhang

et al. 2008

J Virol

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Papillomavirus E2 proteins play a central role in regulating viral gene expression and replication. DNAbinding activity is associated with the C-terminal domain of E2, which forms a stable dimer, while the N-terminal domain is responsible for E2's replication and transactivation functions. The crystal structure of the latter domain revealed a second dimerization interface on E2 which may be responsible for DNA loop formation in the regulatory region of the human papillomavirus (HPV) genome. We investigated the biological significance of the N-terminal dimerization by introducing single amino acid substitutions into the dimerization interface. As expected, these substitutions did not influence the C-terminal dimerization and DNA-binding functions of E2. However, the mutations led to reduced transactivation of a synthetic E2-responsive reporter gene, while HPV DNA replication was unaffected. The effect of the mutations on DNA looping was visualized by atomic force microscopy. While wild-type E2 was able to generate DNA loops, all three mutant E2 proteins were defective in this ability. Our results suggest that N-terminal dimerization plays a role in E2-mediated transactivation, probably via DNA looping, a common mechanism for remote regulation of gene transcription.Several members of the human papillomavirus (HPV) family have been strongly implicated in the development of human cancers, particularly cervical cancer; of these, HPV type 16 (HPV-16) is the most common type found in tumors (25). Indeed, the presence of HPV DNA has been identified as the major risk factor for cervical carcinoma and is increasingly implicated in a number of other human tumors (4, 14). The E2 proteins of HPVs are central regulatory proteins in the viral life cycle. While considered to be a typical transcription factor, the E2 protein is equally important in the initiation of viral DNA replication. The loss or deletion of the E2 open reading frame (ORF) occurs frequently in cervical carcinoma cells when the genome of highrisk HPV types, such as HPV-16, becomes integrated into the cell genome (10). This has resulted in a hypothesis which states that the removal of E2 control results in deregulated expression of the HPV oncogenes E6 and E7. The presumed mode of action of HPV E2 is mediated by its binding to the consensus sequence AGGCN 4 GCCT, which is normally present in four copies in the upstream regulatory region (URR) of the genomes of genital HPVs at highly conserved locations relative to the transcriptional start site and origin of viral DNA replication (26). It has been proposed that binding to these sites has a hierarchical priority which at low concentrations of E2 results in transcriptional transactivation, whereas increasing amounts result in repression of the HPV early promoter (18,29,38). However, it has recently been shown that, in the case of HPV-16, E2 binding sites (E2BS) 1 and 2 (and 3 to a certain degree) are important for transcriptional repression, independent of binding affinities (37). Hence, the precise mechanism of...

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Crystal Structure of the E2 Transactivation Domain of Human Papillomavirus Type 11 Bound to a Protein Interaction Inhibitor

Cited by 86 publications

References 48 publications

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

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

The Mitotic Chromosome Binding Activity of the Papillomavirus E2 Protein Correlates with Interaction with the Cellular Chromosomal Protein, Brd4

Dimerization of the Human Papillomavirus Type 16 E2 N Terminus Results in DNA Looping within the Upstream Regulatory Region

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