Molecular Cell 21, 665–678, March 3, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.01.024

Structural and Functional Analysis of E6Oncoprotein: Insights in the Molecular Pathwaysof Human Papillomavirus-Mediated Pathogenesis

Yves Nomine,1,3 Murielle Masson,1,3

Sebastian Charbonnier,1 Katia Zanier,1

Tutik Ristriani,1 Francois Deryckere,1

Annie-Paule Sibler,1 Dominique Desplancq,1

Robert Andrew Atkinson,2 Etienne Weiss,1

Georges Orfanoudakis,1 Bruno Kieffer,2

and Gilles Trave1,*1 Equipe OncoproteineUMR CNRS 7100Ecole Superieure de Biotechnologie de StrasbourgBoulevard Sebastien BrantBP 1041367412 Illkirch CedexFrance2Laboratoire de RMNCNRS UMR 7104IGBMCRue Laurent Fries67400 IllkirchFrance

Summary

Oncoprotein E6 is essential for oncogenesis induced

by human papillomaviruses (HPVs). The solutionstructure of HPV16-E6 C-terminal domain reveals a

zinc binding fold. A model of full-length E6 is proposedand analyzed in the context of HPV evolution. E6 ap-

pears as a chameleon protein combining a conservedstructural scaffold with highly variable surfaces par-

ticipating in generic or specialized HPV functions. Weinvestigated surface residues involved in two special-

ized activities of high-risk genital HPV E6: p53 tumorsuppressor degradation and nucleic acid binding.

Screening of E6 surface mutants identified an in vivop53 degradation-defective mutant that fails to recruit

p53 to ubiquitin ligase E6AP and restores high p53levels in cervical carcinoma cells by competing with

endogeneous E6. We also mapped the nucleic acidbinding surface of E6, the positive potential of which

correlates with genital oncogenicity. E6 structure-function analysis provides new clues for understand-

ing and counteracting the complex pathways of HPV-mediated pathogenesis.

Introduction

Human papillomaviruses (HPVs) constitute a large fam-ily of small DNA viruses targeting skin or mucosal epi-thelial cells (zur Hausen, 1991). While 17 different HPVspecies, comprising more than 100 types, have beencharacterized to date, most generate limited benign hy-perproliferations (warts, papillomas). However, 99% ofcervical cancers and a significant proportion of otheranogenital cancers have been associated with infectionby a small number of ‘‘high-risk’’ genital HPV types (Dell

*Correspondence: trave@esbs.u-strasbg.fr3 These authors contributed equally to this work.

and Gaston, 2001). In addition, certain nonmelanomaskin cancers may derive from solar irradiation of papillo-mas formed by some high-risk cutaneous HPV types(Pfister, 2003). Tumorigenesis induced by high-risk gen-ital HPVs has been linked to the expression of two viraloncoproteins, E6 and E7, which cooperate in cell immor-talization and transformation (Schwarz et al., 1985). E6binds to many proteins regulating cell proliferation path-ways and often provokes their degradation (Chakrabartiand Krishna, 2003). High-risk genital HPV E6 forms withcellular ubiquitin ligase E6AP, a complex that targets thetumor suppressor p53 for degradation (Scheffner et al.,1990, 1993). E6 also represses p53-dependent tran-scription by inhibiting p300-mediated acetylation(Thomas and Chiang, 2005). By inactivating p53, the vi-rus not only prevents p53-mediated apoptosis of the in-fected cells (Chakrabarti and Krishna, 2003) and facili-tates the replication of its DNA that would otherwisebe blocked by p53 (Lepik et al., 1998), but it also favorsoncogenesis by decreasing p53-mediated control ongenomic integrity (Thomas et al., 1999). E6 also medi-ates degradation of several PDZ domain-containingproteins implicated in cellular adhesion and polaritycontrol (Massimi et al., 2004). This activity seems impor-tant for E6 contribution to cancer progression (Simon-son et al., 2005). Moreover, E6 promotes cell immortali-zation by increasing the levels of telomerase activity(Klingelhutz et al., 1996), a property apparently linkedto E6-mediated degradation of a transcriptional repres-sor of the telomerase retrotranscriptase gene (Gewinet al., 2004). In addition to its protein binding properties,high-risk genital HPV E6 is also a structure-dependentnucleic acid binding protein that recognizes four-wayDNA junctions (Ristriani et al., 2000).

Despite its small size (151 residues for HPV16 E6), E6has proven difficult to isolate in a stable folded form (No-mine et al., 2001a). A monodispersity-based strategywas applied to optimize production of folded E6, anda mutant of HPV16 E6 was designed with six noncon-served cystein residues changed to serine to minimizeoxidation problems (Nomine et al., 2001b). This mutant,E6 6C/6S, was purified as a soluble and biologically ac-tive form yet remained unsuitable for structural work.However, E6 was shown to contain two stable foldeddomains, E6N and E6C, of about 75 residues each (Lipariet al., 2001; Nomine et al., 2003). A 15N-labeled sample ofthe E6C domain of E6 6C/6S was produced, and its 1Hand 15N frequencies were fully assigned (Nomine et al.,2005). Here, we present the three-dimensional structureof E6C domain and a derived model of full-length E6.Structure-based sequence analysis provides new cluesfor analyzing the diversity of E6 functions.

Results

The Structure of E6C Reveals a Novel ZincBinding Fold

Severe line broadening linked to conformational ex-change had initially hindered the assignment of (1H-1H)2D-NOESY experiments (Nomine et al., 2003), but the

Molecular Cell666

Figure 1. Solution Structure of E6C

(A) Superimposition of the family of ten final structures of E6C.

(B) Ribbon representation of E6C.

(C) Exposed hydrophobic surfaces. Two van der Waals surface plots of the structured part of E6C (residues 80–140), rotated 180º with respect to

each other, are shown with ribbon plots in the same orientations. In surface plots, residues are colored as follows: F, W, L, I, V, cyan; Y, green; H,

blue; other residues, white.

(D) Surface charge potential. Two van der Waals surface plots, rotated 90º with respect to those in (C), are colored according to positive (blue) or

negative (red) potential.

obtention of 15N-labeled E6C samples allowed us to as-sign all backbone 1H and 15N frequencies of the domain(Nomine et al., 2005). Subsequently, a complete andconsistent set of interproton distances could be col-lected and interpreted as a single set of conformations(Figure 1A and see Figure S1 in the Supplemental Dataavailable with this article online). The structure is, intrin-sically, relatively imprecise due to unusual backbone dy-namics within particular regions (Nomine et al., 2005). Infull-length E6, these movements may be suppressed byinteraction with E6N. The experimental NOE data set in-cluded 941 interresidue contacts and resulted in a finalprecision of 0.93 A for the structured part of the domain(Table 1). The C-terminal residues R141-L151, encom-passing the PDZ binding motif (Thomas et al., 2002),adopt a random conformation.

The E6C construct contains four Cys-Ser mutations,but we expect its structure to be identical to the wild-type for the following reasons: (1) the mutated cysteinswere not conserved in E6 alignments, (2) the Cys/Ser

change is relatively isosteric, (3) the mutations did notprevent the domain from adopting a stable fold, (4) allmutated positions turn out to be exposed on the surfaceof the calculated structure, and (5) these mutations arealso present in the full-length E6 6C/6S mutant display-ing in vitro and in vivo p53 degradation properties indis-tinguishable from those of wild-type E6 (Nomine et al.,2001b) (see also Figure 6).

E6C represents a novel a/b zinc binding fold consist-ing of a three-stranded b sheet (S1, S2, and S3) withtwo short helices (H1 and H2) packed on one of its sides(Figure 1B). The zinc binding site, involving the long loop(L2) connecting H1 to H2 and a short C-terminal helix(H3), is peripheric and protrudes away from the b sheet.However, the zinc binding site is connected to the maincore via several buried residues: I101, K115, L110, andR135 (see Figure 2B). The main hydrophobic core, lo-cated between the b sheet and helices H1 and H2, iscentered on the aromatic side chain of W132, sur-rounded by several fully buried residues: L83, L96,

Structure-Function Analysis of HPV E6 Oncoprotein667

L119, F125, and N127. Additional core residues Y81,Y92, L99, and L100 are partly exposed and, with two fullyexposed hydrophobic residues (Y84 and L88), they forma large hydrophobic patch on one face of the domain op-posite to the zinc binding site (Figure 1C).

The surface charge potential of E6C is mostly positive(Figure 1D), except for a neutral area corresponding tothe hydrophobic patch and a few negative spots at thetop of helices H1 and H2.

Helix H1 exhibits a sharp 60º kink in its middle (Fig-ure 1B), at the level of residues K94-P95. This may resultfrom the combined effects of P95 acting as a helixbreaker and of bulky Y92 pulling H1 away from theb sheet. In most HPV types, P95 is replaced by helix-compatible residues and Y92 by smaller residues (seeFigure 2A), suggesting that, in such E6C domains, H1adopts a continuous helical conformation.

Structure-Based Alignment of E6C and E6NSequences and Homology Modeling of E6N Domain

HPV sequences were classified in 17 main species: A(1–11), B1a(1–2), B1b(1–2), B2, and E1 (Chan et al., 1995)(http://www.stdgen.lanl.gov/stdgen/virus/). Alignmentof a subset of E6C sequences comprising one memberof each HPV species (Figure 2A) shows that most buriedresidues of E6C have a conserved hydrophobic charac-ter, except highly conserved K115 and nonconservedN127 and R135 (Figure 2B). E6N domains from thesame HPV species are readily aligned with E6C domainsby introducing a two-residue gap within loop L1 (Fig-ure 2A). This gap places L12 of E6N in register withL83, a buried residue of E6C that anchors strand S1 tothe main hydrophobic core. In this alignment, all buriedhydrophobic positions of E6C remain hydrophobic inE6N, and even the buried hydrophilic positions of E6C

Table 1. Structural Statistics of E6C

Numbers of Restraints for Final Calculation (Numbers of NOEs

Affected by Exchange in Brackets)

Total NOE restraints 941

Sequential (|i–j| = 1) 357 (139)

Medium range (1 < |i–j| % 4) 229 (95)

Long range (|i–j| > 4) 355 (137)

Hydrogen bonds 11

Dihedral angle restraints (F,J) 63

Structure Statistics

Rms deviations from idealized covalent

geometry

Bonds (A) 0.0052 6 0.0002

Bond angles (º) 0.78 6 0.04

Improper torsions (º) 0.77 6 0.03

NOE restraints (A) 0.044 6 0.0017

Dihedral angle restraints (º) 0.25 6 0.17

Ramachandran Plot

Residues in most favorable regions (%) 89.7

Residues in additional favorable regions (%) 7.8

Residues in generously favorable regions (%) 0.7

Residues in disallowed regions (%) 1.9

Coordinate Precision (Rmsd in A)

Backbone atoms in secondary structures 0.51

Backbone atoms (80–139) 0.93

All atoms (80–139) 1.66

(K115, N127, and R135) are substituted by conservedhydrophobic positions in E6N. The two domains weresuggested to adopt a similar fold based on early se-quence analysis (Cole and Danos, 1987) as well as oncircular dichroism (CD) experiments detecting compa-rable a/b secondary structure in E6N, E6C, and full-length E6 (Lipari et al., 2001; Nomine et al., 2001b,2003). Using the backbone fold of E6C as a template,we built a plausible model of E6N in which all buried res-idues correspond to conserved hydrophobic positions(Figure 2C). However, slight differences between thefolds of E6C and E6N are to be expected. In E6C, thecentral core aromatic (W132) is provided by strand S3,while, in E6N, the conserved buried aromatic appearsto be provided by strand S2 (Y54) (Figure 2A). In addi-tion, the conformation of helix H1 may be modulatedby substitutions at positions 19 and 22 (correspondingto Y92 and P95 of E6C).

A number of residues conserved in all HPV species areexposed on the surface of both E6C and the model ofE6N (Figures 2D and 2E). Some may participate in ge-neric functions shared by all HPVs. Others may havea structural role: E41, R48, E89, and E114, for instance,may be involved in helix capping. Finally, residues in-volved in the exposed hydrophobic surface of HPV16E6C (Figure 1C) are conserved in all E6C and E6N se-quences, suggesting that, within full-length E6, thesehydrophobic patches are not exposed to water butrather interact with each other.

A Pseudodimeric Model of E6N-E6C Assembly

within Full-Length E6We explored the possible modes of assembly of E6Nand E6C that would mask their hydrophobic patchesand allow an optimal fit of secondary structures withoutsteric clashes. We found that these criteria are fulfilledonly if the two domains face each other symmetricallyin a pseudodimeric fashion (Figure 3A). Antiparallel as-sembly of S1 b strands from E6N and E6C leads toa six-stranded b sheet with the two H1 helices packingin a classical manner and the zinc binding sites locatedat opposite edges of the molecule (Figure 3B). In sucha model, exposed hydrophobic side chains of the indi-vidual domains establish a network of interactions (Fig-ure 3C). The highly conserved solvent-exposed residueL88 of E6C comes into close proximity of I26, I27, andY54 of E6N, and a similar fit is observed symmetricallybetween L15 of E6N and L99, L100, and N127 of E6C.

Three short regions flanking E6N and E6C domainsare not predicted by the model (Figure 3D). The N-termi-nal and C-terminal tails are highly variable in both lengthand sequence, but the interdomain linker has a fixedlength in all HPV species and contains several highlyconserved hydrophobic residues presenting the period-icity of a helix (Figure 2A). Such a helix could establishhydrophobic interactions with the b sheet (Figure 3E),possibly rigidifying the relative positions of the two do-mains.

HSQC-NMR mapping experiments were performedon mixtures of nonlabeled E6N with 15N-labeled E6C(Figure S2). E6N was prone to aggregation, limiting sam-ple concentration and subsequent data quality. Never-theless, the data indicate that the two domains forma complex involving residues S80–S82 of b strand S1

Molecular Cell668

Figure 2. Conservation of Buried and Exposed Residues in E6C and E6N

(A) Alignment of E6C (upper half) and E6N (lower half) domains from 17 E6 sequences representative of the main HPV species. Plotted above the

alignment is the percentage exposure to solvent of each structured residue of E6C, calculated as described by Chothia (1984) and using the

Structure-Function Analysis of HPV E6 Oncoprotein669

Figure 3. The Pseudodimeric Model of E6

(A) E6N model and E6C structure positioned symmetrically with exposed hydrophobic patches facing each other.

(B) Proposed pseudodimeric arrangement of E6N and E6C. The gray closed line indicates the DNA binding region of E6C, mapped in the present

work.

(C) Contact map of hydrophobic residues involved in the interface of the pseudodimeric model.

(D) Position of the three flanking regions not predicted by the model.

(E) A view of the core model highlighting surface residues conserved in all E6 proteins. Previously mutated residues are indicated with underlined

red labels.

and residue L100, in accord with the pseudodimericmodel that predicts close contacts between backboneamides of these residues and E6N.

E6 from Different HPV Species Have DistinctSequence Signatures Correlated with Defined

Clusters of Exposed ResiduesThe strict conservation of structural residues suggeststhat all HPV E6 proteins adopt the proposed pseudodi-meric scaffold. This assumption allows us to predict sur-

face properties of different HPV E6 proteins. Such ananalysis, performed on E6 from HPV 16, 11, and 5, re-veals a chameleon protein with highly variable surfacepatterns (Figure 4A). This observation led us to examinethe ‘‘specialized’’ positions conserved only in E6 fromparticular HPV species (in contrast to the ‘‘generic’’ po-sitions conserved in all HPV species). Specialized posi-tions identified in sequences of E6 from high-risk genitalspecies A7 and A9 (Figure 4B) form a corolla of exposedresidues around the E6 model (Figure 4C). The three

WHATIF program (Vriend, 1990). Arrows and cylinders denote b strands and a helices, respectively. The first and last residues of each element of

secondary structure are indicated. Residues are numbered as in the shorter transcript of HPV 16 E6 (151 residues). Positions exhibiting con-

served physicochemical characteristics are colored as follows: magenta, hydrophobic (W, F, Y, L, I, V, M); green, basic (K, R, H); red, acidic

(E, D); orange, polar (Q, N, T, S); brown, cystein (C); cyan, small (G, A, P).

(B) Ribbon plot of E6C structure showing all residues found to be buried according to low solvent exposure values (below 20%) confirmed by

visual inspection. Underlined red labels indicate residues mutated in publications cited in main text. Circled labels indicate nonconserved buried

hydrophilic residues R135 and N127.

(C) Ribbon plot of E6N model, oriented slightly differently than E6C in (B) to show all buried residues. Note that all buried residues correspond to

conserved hydrophobic positions in the E6N alignment.

(D and E) Ribbon plots of E6C structure and E6N model, respectively, showing conserved surface positions. Previously mutated residues are

indicated with underlined red labels.

Molecular Cell670

Figure 4. Examples of Residue Specialization in HPV Species

(A) Surface properties of E6 from HPV 16, 11, and 5 were analyzed by coloring residues of the pseudodimeric model according to the physico-

chemical properties of the corresponding residues in the strain considered. Color codes: A, G, P, white; R, K, H, green; D, E, red; N, Q, S, T, yellow;

M, V, L, I, F, W, Y, purple. The model is oriented as in Figure 3B with an additional rotation of 90º.

(B) Alignment of E6 sequences from four representative strains from high-risk genital HPV species A9 and A7, four strains from low-risk genital

species A10, and four strains from high-risk cutaneous species B1A1. The positions that exhibit specialized conservation in species A9 and A7

are highlighted (same color code as in Figure 2).

(C) The specialized positions of high-risk genital HPVs are shown on the model of E6. Each domain presents two surface clusters of specialized

residues, labeled R1 and R2 for E6C and R10 and R20 for E6N.

flanking regions absent from the E6 model also containnumerous high-risk genital specialized positions (Fig-ure 4B), the general hydrophilic nature of which sug-gests that they are also exposed to solvent.

We examined the fate of the high-risk genital special-ized positions within other HPV species. A10 is a low-risk genital species considered a close neighbor of thehigh-risk genital species, while B1a1 is a species ofcutaneous HPVs associated to Epidermodysplasia ver-ruciformis and malignant skin carcinomas, classifiedat the other extreme of the phylogenetic tree (see Fig-ure 5C). Interestingly, most specialized positions of E6from high-risk genital species are also conserved withinspecies A10 and B1a1 but often with different physico-chemical characteristics (Figure 4B). For instance, posi-tion 107 is either a conserved Q, a conserved H, or a con-served L in species A7/A9, A10, or B1a1, respectively.Such clusters of specialized E6 surface residues, con-served within species but differing from one species toanother, may confer on E6 proteins of particular HPVspecies the ability to recognize distinct sets of cellulartargets.

E6 DNA Binding Site Is Mapped to the Vicinity

of the Zinc Atom of E6C, where ElectrostaticPotential Correlates with HPV Agressivity

E6C was previously shown to retain selective binding toDNA junctions (Nomine et al., 2003; Ristriani et al.,2001).The DNA binding surface of E6 was analyzed byserial HSQC measurements performed on 15N-labeledE6C mixed with increasing concentrations of a syntheticcruciform DNA junction (Figure S3). Changes in chemi-cal shift observed at a low DNA:protein ratio (1:10)allowed us to map E6C residues influenced by high-affinity binding to the junction core (Figure 5A). On themodel of E6 (Figure 3B), these residues are localized op-posite to the E6N/E6C interface, in an area presentinga pointed shape that must facilitate insertion of E6 intothe bulky junction core. At higher DNA:protein ratios,most peaks in the spectrum progressively disappeared,suggesting that E6C started to form heterogeneouscomplexes with the DNA by binding to secondary low-affinity sites on the double-stranded arms of the junc-tion, as has been observed for other junction bindingproteins (Bonnefoy et al., 1994).

Structure-Function Analysis of HPV E6 Oncoprotein671

Figure 5. DNA Binding Surface of E6C Domain

(A) (Upper plot) Changes in 1H-15N chemical shifts observed against E6C sequence upon addition of cruciform DNA at a 10:1 molar ratio. The gray

color for R124 indicates uncertainty due to low signal intensity and spectral overlap. Colored residues in the sequence indicate point mutations

that affect DNA binding (Ristriani et al., 2002). The ‘‘minimal DNA binding construct’’ is the ZD2 construct shown to retain cruciform binding (Ris-

triani et al., 2001).

(B) Charge potential of E6C domains in the context of HPV evolution. The phylogenetic tree is derived from the classification by Chan et al. (1995)

of HPV types into 17 main species: A(1–11), B1a(1–2), B1b(1–2), B2, and E1. Each green patch represents a species that includes several HPV

types, each indicated by its number. Homology models of E6C domains were produced for one representative type of each HPV species by using

the structured part of HPV16 E6C (residues 80–140) as a template, and surface charge potentials were computed as in Figure 1. Views corre-

spond to those in Figure 1D. The isoelectric points and the net charges of each modeled domain are also indicated.

As expected for a nucleic acid binding domain, HPV16E6C displays a strongly positively charged surface (Fig-ure 1D). We generated homology models for all E6Csequences aligned above and compared their electro-static surface potentials in the context of HPV phylogeny(Figure 5B). The isoelectric points of the domains covera wide range, from highly basic (pI = 10.61 for HPV 16) toneutral (pI = 7.28 for HPV 27). The E6C domains with themost positive surface potential are those of the twohighest-risk genital HPV species A9 and A7.

Screening of E6 Surface Residues Important

for p53 Degradation In VivoStructural information allows us to probe the function ofsurface residues by mutagenesis. We constructed a se-

ries of mutants with modifications at 32 distinct residueson the surface of E6 (Figure 6A) with the aim of testingthe effect of these modifications on the p53 degradationactivity of E6 in human cells. A subset of these mutantswas designed to alter all surface residues comprised be-tween L19 and C33 and between K121 and I128, two re-gions previously suggested to be involved in E6AP bind-ing (Liu et al., 1999; Pim and Banks, 1999). Additionalmutants altered on other regions originate from a previ-ous in vitro study (Ristriani et al., 2002).

All surface mutants were transfected in human C33Acells, and their localization was analyzed using anti-E6monoclonal antibodies as published before (Massonet al., 2003). Most retained the nuclear localizationnormally observed for wild-type E6 (Figure 6B and

Molecular Cell672

Figure 6. A Screening of p53 Degradation-Defective E6 Surface Mutants

(A) E6 positions mutated in this study, indicated on E6 sequence, on ribbon plots and on van der Waals surface plots of the model of E6.

Structure-Function Analysis of HPV E6 Oncoprotein673

Figure S4). This indicates that these mutants were prop-erly folded, since misfolded E6 forms aggregates (Ris-triani et al., 2002) that should hinder nuclear localization.E6 6C/6S K121A/K122A/Q123A/R124A showed an al-tered localization pattern that may confirm the proposedrole of the 121KKQR124 sequence in nuclear localizationof E6 (Le Roux and Moroianu, 2003) but may also bedue to misfolding problems. This mutant is indeedknown to aggregate upon bacterial overexpression (Ris-triani et al., 2002).

Analysis of E6-transfected human cells using a doubleimmunofluorescence assay with anti-E6 and anti-p53antibodies provides a straightforward test of the p53degradation activity of E6 in vivo: in E6-positive cells,the p53 signal is dramatically reduced compared tothat observed in E6-negative cells (Figure 6B). In this as-say, the E6 6C/6S mutant was indistinguishable fromwild-type E6 (Figure 6B), confirming previous in vitro ob-servations that this mutant conserved full wild-type E6activities (Nomine et al., 2001b). Almost all E6 mutantsretained p53 degradation activity in vivo (Figure 6Cand Figure S4), but several mutants that fully degradedp53 in vivo could not degrade it in vitro in the contextof rabbit reticulocyte extracts (Figure 6C and Figure S4).Notably, the F47R mutation altering helix H1 in the E6Ndomain of E6 6C/6S was the only one to inactivate p53degradation both in vitro and in vivo (Figures 6B and6C and Figures S4 and S5).

Remarkably, all the point mutants directed at the pu-tative E6AP binding sites L19-C33 and K121-I128 effi-ciently degraded p53 in human cells (Figure 6C) andalso retained coprecipitation with the minimal E6 bind-ing site of E6AP (residues 403–417) in a GST-pull-downassay (data not shown). Therefore, none of these two re-gions seems involved in binding to E6AP.

E6 6C/6S F47R Is Defective in Recruiting p53to E6AP and Acts as a Dominant Negative versus

HPV18E6-Mediated p53 Degradation in HeLa CellsE6 6C/6S F47R was transfected into cervical cancer-derived HeLa cells, which constitutively express HPV18E6 and are therefore characterized by low steady-statelevels of p53. Interestingly, endogeneous p53 levelswere strongly increased in E6 6C/6S F47R-transfectedcells (Figure 7A). A comparable increase in p53 levelswas observed when cells were transfected by an anti-HPV18 E6 synthetic siRNA (Figure 7A). E6 6C/6S F47Rtherefore exhibits a strong dominant-negative effectover the p53 degradation activity of endogeneousHPV18 E6.

We designed an in vivo GST pull-down assay to ana-lyze the E6AP and p53 binding properties of E6 6C/6SF47R directly in human cells. A fusion of GST to E6APC843A (a E6AP mutant that binds to E6 but is inactiveas a ubiquitin ligase [Talis et al., 1998]) was expressedin C33A cells together with either E6 wt, E6 6C/6S, orE6 6C/6S F47R. Following lysis of the transfected cells,the three E6 constructs coprecipitated with GST-E6APC843A in equal amounts (Figure 7B, lanes 14–16). This

is coherent with in vitro BIAcore measurements showingthat the F47R mutation does not decrease the affinity ofE6 for the 15 residue minimal E6 binding region of E6AP(residues 403–417) (Zanier et al., 2005). However, in con-trast to E6 wild-type and E6 6C/6S, E6 6C/6S F47R wasunable to recruit p53 to the GST-E6AP C843A fusion(Figure 7B, compare lane 24 to lanes 22 and 23), despitethe higher levels of p53 in cells expressing E6 6C/6SF47R (Figure 7C).

In the above experiments, we expected that ubiquiti-nation-deficient GST-E6AP C843A:E6 complexes wouldrescue large amounts of p53 by competing with ubiqui-tination-proficient endogeneous E6AP:E6 complexes,thus maximizing the amounts of pulled down p53. Tothis end, the GST-E6AP C843A-encoding plasmid wastransfected in higher amounts (5- to 7-fold) than theE6-encoding plasmids. Under such conditions, bothE6 wt and E6 6C/6S F47R were relocalized from nucleusto cytoplasm (compare Figure 7C to Figure 6B), sug-gesting that overexpressed GST-E6AP C843A capturesE6 in the cytoplasm. Surprisingly, in cells transfectedwith E6 wt, p53 levels are not increased by the presenceof excess GST-E6AP C843A (compare Figures 6A and7C), although coprecipitation results clearly indicatethat p53 is efficiently recruited by the resulting GST-E6AP C843A:E6 wt complexes (Figure 7B). This sug-gests that these ubiquitination-deficient complexesmay recruit additional cellular factors that promote effi-cient p53 degradation. By contrast, in cells transfectedwith E6 6C/6S F47R, the corresponding complexes donot drive p53 degradation (Figure 7C), in agreementwith the defect in p53 recruitment observed for GST-E6AP C843A:E6 6C/6S F47R complexes (Figure 7B).

Discussion

The difficult biochemical behavior of E6 proteins hadlong hindered their structural analysis. We have deter-mined the solution structure of HPV16 E6C domain, re-vealing a novel zinc binding fold. In addition, we havebuilt a model of full-length E6 based on the sequencehomology between E6C and E6N and on the assumptionthat interaction between the two domains should burytheir conserved hydrophobic patches. We found thatonly the proposed pseudodimeric arrangement satisfiesthis assumption while providing a proper fit of second-ary structure elements. Intriguingly, the model suggeststhat a single-domain protein possessing the same foldand forming a real symmetric homodimer might haveonce existed. This is in agreement with previous phylo-genetic analysis indicating that E6 evolved from a sin-gle-domain protein through gene duplication (Cole andDanos, 1987). The pseudodimeric model also shedsnew light on previous work showing that E6*, a splicingmutant encompassing residues 1–43 of HPV18 E6(equivalent to 1–41 in HPV16 E6), binds to full-lengthE6 from both HPV18 and HPV16 (Pim and Banks,1999). Remarkably, E6* includes the essential elementsof the E6N/E6C interface in the model of E6: strand 1,

(B) Double immunofluorescence detection of E6 and p53 in C33A cells 24 hr after transient transfection with p513 vector expressing wild-type E6,

E6 6C/6S, or E6 6C/6S F47R. Note that p53 signal selectively disappears in wild-type E6- and E6 6C/6S-transfected cells but does not decrease in

E6 6C/6S F47R-transfected cells.

(C) Summary of p53 degradation activities for all E6 mutants evaluated either in C33A cells or in rabbit reticulocyte extracts.

Molecular Cell674

Figure 7. E6 6C/6S F47R Mutant Acts as a Dominant-Negative Mutant in HeLa Cells and Is Impaired in the Recruitment of p53

(A) Double immunofluorescence detection of E6 and p53 in HeLa cells 30 hr after transient transfection with pXJ vector expressing wild-type E6

or E6 6C/6S F47R. Note the strong increase in p53 signal in E6 6C/6S F47R-transfected cells. As a positive control, HeLa cells were transfected

with a double-stranded siRNA against HPV18 E6 mRNA (Butz et al., 2003) and as a negative control with a scrambled siRNA. p53 expression was

visualized after 48 hr by immunofluorescence.

(B) C33A cells were transiently cotransfected by pBC vectors expressing either GST or GST-E6AP C843A and pXJ vectors expressing either wild-

type E6, E6 6C/6S, or E6 6C/6S F47R. Cell extracts were incubated with glutathione Sepharose beads and resolved by SDS-PAGE. Western blot-

ting was performed using anti-GST-E6AP polyclonal antibody Ab-1 (lanes 1–8), anti-E6 monoclonal antibody mAb 6F4 (lanes 9–16), and anti-p53

monoclonal antibody pAb 1801 (lanes 17–24).

(C) C33A cells were transiently cotransfected with pXJ vector expressing wild-type E6 or E6 6C/6S F47R and an excess of pBC vector expressing

GST-E6AP C843A. Expression of E6 and p53 was visualized by double immunofluorescence using anti-E6 monoclonal antibody mAb 6F4 and

anti-p53 polyclonal antibody FL393. Note that, upon coexpression of GST-E6AP C843A, E6 is relocalized in the cytoplasm, whereas it is normally

nuclear (see Figures 7A and 6B).

loop 1, helix 1, and the start of loop 2 (Figure 3C and Fig-ure S2D). We hypothesize that E6* binds to full-length E6by competing with intramolecular E6N/E6C association.

The DNA binding surface of E6 coincides with a strongpositive potential conserved in high-risk genital HPVspecies A9 and A7. This confirms our previous observa-tion that structure-dependent DNA binding is a special-ized activity of high-risk genital E6 (Ristriani et al., 2000,2001). This activity points to a role of E6 in HPV DNA rep-lication, a process during which intermediate DNA struc-tures resembling four-way junctions may be formed. In-deed, E6 from HPV 16 and 18 strongly inhibits HPV DNAreplication in vivo (Grm et al., 2005). E6 might also recog-nize other nucleic acids presenting comparable struc-tural features. Bacterially expressed E6 and E6C fromhigh-risk genital HPV (but not from other HPV types)copurify with RNA molecules, and these are only re-leased at high ionic strength upon gel filtration (G.T., un-published data), suggesting that E6 contains an RNA

recognition site that may overlap with its DNA bindingsite. Accordingly, in HPV-transformed cells, some E6molecules colocalize with nuclear ribonucleoproteic ul-trastructures and with ribosomal areas of the cytoplasm(Masson et al., 2003).

The structural data indicate that many mutations pre-viously performed on E6 might have indirectly altered itsfunction through structure destabilization, leading to in-accurate maps of E6 functional regions. Short deletions(Crook et al., 1991; Foster et al., 1994; Pim et al., 1994)cut through essential secondary structure elements.Point mutations (Cooper et al., 2003; Dalal et al., 1996;Foster et al., 1994; Gao et al., 2001; Nakagawa et al.,1995; Ristriani et al., 2002; Slebos et al., 1995) often tar-geted key buried positions of E6N or E6C (highlighted inFigures 2B and 2C). However, several mutations proneto destabilize the fold were found to retain some func-tion. For instance, a mutation targeting the central coreresidue of E6C (W132) did not suppress the capability

Structure-Function Analysis of HPV E6 Oncoprotein675

of E6 to immortalize mammary epithelial cells (Dalalet al., 1996); two mutations targeting the central coreresidue of E6N (Y54) retained the p53 degradation activ-ity of E6 in certain conditions (Liu et al., 1999), and a frag-ment of E6C missing strand S1 and helix H1 retained thecapacity to recognize DNA junctions with high affinityand selectivity (Ristriani et al., 2001). This indicatesthat, in the case of E6 and its domains, there is not al-ways a straight correlation between structure destabili-zation and loss of function. Destabilized mutant E6 con-structs might recover their active conformation uponbinding to some of their targets, as has been describedbefore for naturally disordered proteins (Demarest et al.,2002).

To limit destabilizing effects, the structural informa-tion can be used to design mutations altering only thesurface of E6. Using such an approach, we have demon-strated that a large part of E6 surface is not important forp53 degradation in human cells. In particular, we havemutated all surface residues within two regions of E6formerly proposed to participate in E6AP binding (Liuet al., 1999; Pim and Banks, 1999), and our resultsstrongly suggest that these regions are not involved inthe E6AP binding site. In a recent BIAcore kinetic study(Zanier et al., 2005), now confirmed by pull-down andNMR mapping experiments (data not shown), we actu-ally showed that neither E6N nor E6C constructs re-tained detectable binding to E6AP(403-417). Thus, theE6AP binding surface of E6 appears to consist of anas-yet-undefined set of residues from both E6N andE6C, probably situated at the periphery of the domains’interface. Our surface mutagenesis also provides insighton the E6AP-dependent p53 binding site of high-riskgenital E6 postulated by Li et al. (Li and Coffino, 1996).Mutation F47R preserved binding of E6 to E6AP but in-activated E6-mediated recruitment of p53 to E6AP(Figure 7B). Comparable defects in the recruitment ofp53 to E6AP were observed for point mutations in theN-terminal tail of E6 (Cooper et al., 2003), and an anti-E6 antibody recognizing the N-terminal tail inhibitedE6-p53 binding but not E6-E6AP binding (Lagrangeet al., 2005). Within full-length E6, F47 might cooperatewith the N-terminal tail to build up the E6AP-dependentp53 binding site. Mutant E6 6C/6S F47R was also foundto exert a strong dominant-negative effect on the p53degradation activity of endogeneous HPV18 E6 inHeLa cells. The mutant may act either by titrating the en-tire cellular pool of E6AP or by binding directly to HPV18E6. The latter mechanism was proposed to account forthe inhibition of E6-mediated degradation of p53 bythe HPV18 E6* splicing mutant (Pim and Banks, 1999).Further characterization of the F47R mutation and inves-tigation of its ability to induce apoptosis in HPV-trans-formed cells may suggest approaches to inactivatingE6-mediated degradation of p53 in cervical tumors.

E6 mutagenesis altered p53 degradation differentiallydepending on whether it was evaluated in vitro or in vivo,as has been observed previously (Cooper et al., 2003;Foster et al., 1994; Gardiol and Banks, 1998; Liu et al.,1999). In rabbit reticulocyte extracts, the trimeric inter-action between E6, E6AP, and p53 leads to degradationof p53 via a process requiring E6AP molecules proficientfor ubiquitin ligation (Scheffner et al., 1993). By contrast,in human cells, our data indicate that E6/GST-E6AP

C843A complexes deficient for ubiquitin ligation remainproficient for p53 degradation, suggesting that thesecomplexes may recruit additional cellular factors thatpromote the degradation of p53. One such factor mightbe endogeneous E6AP, requiring that several E6AP mol-ecules be recruited to a single complex. Alternatively,E6/GST-E6AP C843A complexes may recruit ubiquitin li-gases other than E6AP. Indeed, E6 is known to bind tosuch factors, leading either to its own degradation(Stewart et al., 2004) or to the degradation of targetsother than p53 (Grm and Banks, 2004). We hypothesizethat, in human cells, E6AP is required only to bring E6into contact with p53 and that its ubiquitin ligase activityis dispensable as other ubiquitin ligases are recruited tothe trimeric complexes and catalyze polyubiquitinationof p53. These other ligases may be absent in rabbit retic-ulocyte extracts, explaining the strict requirement forthe ubiquitin ligase activity of E6AP for efficient E6-mediated p53 degradation in vitro. In such a model, dif-ferences in in vitro and in vivo p53 degradation may re-sult if, for instance, E6 mutants retain the ability toform trimeric complexes with E6AP and p53 but abolishthe ability of E6AP to polyubiquitinylate p53 in thesecomplexes.

Surface analysis of E6 models revealed residues pos-sibly involved in generic activities common to all HPVspecies (Figure 3E) or in specialized activities of particu-lar species (Figure 4). Until now, generic E6 activitiescould not be clearly defined, since E6 proteins frommost HPV species have not been characterized. It isnow possible to investigate these activities by rationalmutagenesis of generic surface positions of a subsetof E6 proteins from distinct species. On the other hand,many specialized E6 activities have been described, al-most exclusively for E6 of high-risk genital HPV species(reviewed in Chakrabarti and Krishna [2003]). It will be in-teresting to identify specialized surface positions in-volved in these activities, then to examine the role of cor-responding positions in other species. For example, the‘‘ETQV’’ C-terminal PDZ binding motif of high-risk genitalE6s (Thomas et al., 2002) is replaced in groups A10 andB1a1 by distinct consensus sequences (Figure 4C) thatmay recognize targets yet to be identified. By extension,the highly variable surfaces of E6 (Figure 4A) may recog-nize a plethora of specialized targets mostly unknown forall but high-risk genital HPV species. The structural datapresented in this work should give an impulse to thesearch of such novel functions. The data are also the firststep toward the atomic description of the most studiedE6 activities. Finally, it should provide a consistentframework to assist the rational design of prophylacticor therapeutic tools against HPV-driven pathogenesis.

Experimental Procedures

Structure Calculation

Sample preparation and details of NMR data acquisitions have been

published elsewhere (Nomine et al., 2005). NOE crosspeaks from ho-

monuclear and 15N-edited NOESY spectra recorded at 288 K and

600 Mhz (1H frequency) with a mixing time of 200 ms were converted

into upper-limit distance restraints of 2.5, 3.7, and 5.0 A, according

to their intensities. Dihedral f angle target values of 260º 6 30º

and 2120º 6 30º were assigned for 3JHN-HA coupling constants

less than 6 Hz and greater than 8 Hz, respectively. Structure calcu-

lations started from an extended initial structure including the zinc

Molecular Cell676

atom and used a standard simulated annealing protocol in cartesian

coordinates space with the parallhdg force field (Nilges et al., 1988)

in XPLOR-NIH (Schwieters et al., 2003). Tetrahedral zinc coordina-

tion geometry was assumed with Zn-Sg bond lengths set to 2.3 A

and Cb-Sg-Zn and Sg-Zn-Sg angles set to 109.5º. Floating chirality

assignment was used for all methylene and isopropyl groups. A first

set of structures was calculated with a soft potential for NOE dis-

tance restraints and incorporation of dihedral f and c angle re-

straints for regions of unambigously identified elements of second-

ary structure. In iterative rounds of calculations, initially ambiguous

distance restraints were introduced progressively until complete in-

terpretation of the NOE data set was attained. From the resulting set

of 100 calculated structures, the 20 structures with the lowest en-

ergy were subsequently refined using only experimental dihedral f

angle restraints, a square-well potential for distance restraints,

and a protocol that included a torsion angle database potential (Kus-

zewski et al., 1996).Upper-limit distance restraints were increased

by 1.0 A for NOEs involving protons of residues affected by con-

formational exchange, as detected by CPMG-HSQC experiments

(Nomine et al., 2005). The final ensemble of 10 conformations has

no NOE violation greater than 0.6 A and no single dihedral angle

violation greater than 8º. This ensemble of conformations was visu-

alized with MOLMOL (Koradi et al., 1996).

Plasmids and Synthetic siRNAs

The E6AP C843A mutant (including a 45 residue N-terminal deletion

inocute for E6-induced p53 degradation [Huibregtse et al., 1993])

was cloned in the pBC vector, which allows cellular expression of

GST fusions (Chatton et al., 1995). Note that E6AP residue number-

ing is according to the 875 residue-long isoform II of E6AP (Yama-

moto et al., 1997). The coding sequence of the HPV16 E6 gene

was inserted into pXJ40 or p513 vectors allowing E6 expression

controlled by CMV or SV40 promoters, respectively. All E6 mutants

were generated by PCR and checked by sequencing.

Synthetic siRNA against mRNA encoding HPV18 E6 and scram-

bled siRNA were obtained from Ambion, Inc. The target sequence

for HPV18 E6 was 50-CUAACUAACACUGGGUUAU (nucleotides

381–399) (Butz et al., 2003).

In Vitro Transcription/Translation and Degradation Assays

E6 and p53 proteins were translated in vitro at 30ºC for 2 hr using

TNT T7 coupled rabbit reticulocyte system (Promega). E6-mediated

p53 degradation was performed by incubating 5 ml and 2 ml of E6 and

p53 translation products, respectively, in 25 mM Tris HCl (pH 7.5),

100 mM NaCl, 2 mM DTT (total volume 10 ml) at 28ºC for 2 hr. Reac-

tions were stopped by adding 23 SDS loading buffer and analyzed

by SDS-PAGE and autoradiography.

Cell Lines and Transfections

HPV-negative cervical carcinoma C33A cells and HPV18-positive

HeLa cervical carcinoma cells were grown in DMEM medium sup-

plemented with 10% fetal calf serum (FCS) (GIBCO-BRL) at 37ºC

in 10% CO2. C33A and HeLa cells were transfected using Exgen

(Eurogentec) and transfectin (Bio-Rad), respectively. Synthetic

siRNAs (50 nM) were transfected in HeLa cells using jetSi (Polyplus

Transfection, Illkirch, France) according to the manufacturer’s in-

structions.

Indirect Immunofluorescence Analysis

C33A and HeLa cells grown on coverslips were transfected with

pXJ-E6 alone or in the presence of pBC-E6AP C843A. 24 hr later,

cells were rinsed with PBS, fixed for 20 min at 20ºC in 4% parafor-

maldehyde, and then permeabilized for 5 min in 0.1% Triton X-100.

Coverslips were incubated for 30 min in blocking buffer (DMEM,

10% FCS). For simultaneous observation of p53 and E6, cells were

incubated with polyclonal anti-p53 antibody (FL393, Santa Cruz Bio-

technology) diluted 1/200 in blocking buffer, washed with PBS, incu-

bated with monoclonal anti-E6 antibody 6F4 (Masson et al., 2003)

(diluted 1/3000) and a goat anti-rabbit IgG antibody conjugated to

Alexa Fluor 568 (Molecular Probes, diluted 1/2000), washed with

PBS, incubated with goat anti-mouse IgG antibody conjugated to

Alexa Fluor 488 (Molecular Probes, diluted 1/2000), washed with

PBS, and stained for DNA with Hoechst. Coverslips were mounted

onto slides and visualized with a Zeiss fluorescence microscope.

In Vivo GST Pull-Down

C33A cells (60 mm diameter tissue culture plates) were cotrans-

fected with pXJE6 and pBC-E6AP C843A or pBC alone. Thirty hours

later, cells were rinsed with PBS and then lysed for 20 min on ice with

lysis buffer (20 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP40, 2 mM

dithiothreitol and antiprotease cocktail [Roche]). The cell lysate

was cleared by centrifugation for 20 min at 13,000 rpm, then incu-

bated for 2 hr at 4ºC with 40 ml of a 50% slurry of glutathione

(GSH)-agarose beads in 1 ml of binding buffer (as lysis buffer, but

with 0.1% NP40). Beads were washed three times with 1 ml of bind-

ing buffer, boiled for 5 min in 20 ml of 23 SDS-loading buffer, and run

on SDS-PAGE. Proteins were electrotransferred onto nitrocellulose

filters and then analyzed by Western blotting using either monoclo-

nal anti-E6 antibody (6F4), monoclonal anti-p53 antibody (pAb 1801,

Santa Cruz Biotechnology), or anti-GST-E6AP polyclonal antibodies

(Ab-1, Calbiochem).

Supplemental Data

Supplemental Data include five figures and Supplemental Refer-

ences and can be found with this article online at http://www.

molecule.org/cgi/content/full/21/5/665/DC1/.

Acknowledgments

This paper is dedicated to the memory of Jean-Francois Lefevre,

who promoted initiation of the work and constantly supported it in

the most critical phases. G.T. thanks Peter Howley and Gerhard

Wagner for meetings and discussions in the early phase of the struc-

tural work. This work was sponsored by grants from ARC, Ligue Na-

tionale contre le Cancer, and CNRS. T.R. and S.C. were sponsored

by grants from Ligue Nationale contre le Cancer and K.Z. by a grant

from Universite Louis Pasteur. The NMR platform was supported by

the European program SPINE QLG2-CT-2002-00988.

Received: July 2, 2005

Revised: October 26, 2005

Accepted: January 19, 2006

Published: March 2, 2006

References

Bonnefoy, E., Takahashi, M., and Yaniv, J.R. (1994). DNA-binding

parameters of the HU protein of Escherichia coli to cruciform DNA.

J. Mol. Biol. 242, 116–129.

Butz, K., Ristriani, T., Hengstermann, A., Denk, C., Scheffner, M., and

Hoppe-Seyler, F. (2003). siRNA targeting of the viral E6 oncogene ef-

ficiently kills human papillomavirus-positive cancer cells. Oncogene

22, 5938–5945.

Chakrabarti, O., and Krishna, S. (2003). Molecular interactions of

‘high risk’ human papillomaviruses E6 and E7 oncoproteins: implica-

tions for tumour progression. J. Biosci. 28, 337–348.

Chan, S.Y., Delius, H., Halpern, A.L., and Bernard, H.U. (1995). Anal-

ysis of genomic sequences of 95 papillomavirus types: uniting typ-

ing, phylogeny, and taxonomy. J. Virol. 69, 3074–3083.

Chatton, B., Bahr, A., Acker, J., and Kedinger, C. (1995). Eukaryotic

GST fusion vector for the study of protein-protein associations in

vivo: application to interaction of ATFa with Jun and Fos. Biotechni-

ques 18, 142–145.

Chothia, C. (1984). Principles that determine the structure of pro-

teins. Annu. Rev. Biochem. 53, 537–572.

Cole, S.T., and Danos, O. (1987). Nucleotide sequence and compar-

ative analysis of the human papillomavirus type 18 genome. Phylog-

eny of papillomaviruses and repeated structure of the E6 and E7

gene products. J. Mol. Biol. 193, 599–608.

Cooper, B., Schneider, S., Bohl, J., Jiang, Y., Beaudet, A., and Vande Pol,

S. (2003). Requirement of E6AP and the features of human papillomavi-

rus E6 necessary to support degradation of p53. Virology 306, 87–99.

Crook, T., Tidy, J.A., and Vousden, K.H. (1991). Degradation of p53

can be targeted by HPV E6 sequences distinct from those required

for p53 binding and trans-activation. Cell 67, 547–556.

Dalal, S., Gao, Q., Androphy, E.J., and Band, V. (1996). Mutational

analysis of human papillomavirus type 16 E6 demonstrates that

Structure-Function Analysis of HPV E6 Oncoprotein677

p53 degradation is necessary for immortalization of mammary epi-

thelial cells. J. Virol. 70, 683–688.

Dell, G., and Gaston, K. (2001). Human papillomaviruses and their

role in cervical cancer. Cell. Mol. Life Sci. 58, 1923–1942.

Demarest, S., Martinez-Yamout, M., Chung, J., Chen, H., Xu, W.,

Dyson, H., Evans, R., and Wright, P. (2002). Mutual synergistic fold-

ing in recruitment of CBP/p300 by p160 nuclear receptor coactiva-

tors. Nature 415, 549–553.

Foster, S.A., Demers, G.W., Etscheid, B.G., and Galloway, D.A.

(1994). The ability of human papillomavirus E6 proteins to target

p53 for degradation in vivo correlates with their ability to abrogate

actinomycin D-induced growth arrest. J. Virol. 68, 5698–5705.

Gao, Q., Singh, L., Kumar, A., Srinivasan, S., Wazer, D.E., and Band,

V. (2001). Human papillomavirus type 16 E6-induced degradation of

E6TP1 correlates with its ability to immortalize human mammary ep-

ithelial cells. J. Virol. 75, 4459–4466.

Gardiol, D., and Banks, L. (1998). Comparison of human papilloma-

virus type 18 (HPV-18) E6-mediated degradation of p53 in vitro and

in vivo reveals significant differences based on p53 structure and

cell type but little difference with respect to mutants of HPV-18 E6.

J. Gen. Virol. 79, 1963–1970.

Gewin, L., Myers, H., Kiyono, T., and Galloway, D. (2004). Identifica-

tion of a novel telomerase repressor that interacts with the human

papillomavirus type-16 E6/E6-AP complex. Genes Dev. 18, 2269–

2282.

Grm, H., and Banks, L. (2004). Degradation of hDlg and MAGIs by hu-

man papillomavirus E6 is E6-AP-independent. J. Gen. Virol. 85,

2815–2819.

Grm, H., Massimi, P., Gammoh, N., and Banks, L. (2005). Crosstalk

between the human papillomavirus E2 transcriptional activator

and the E6 oncoprotein. Oncogene 24, 5149–5164.

Huibregtse, J.M., Scheffner, M., and Howley, P.M. (1993). Localiza-

tion of the E6-AP regions that direct human papillomavirus E6 bind-

ing, association with p53, and ubiquitination of associated proteins.

Mol. Cell. Biol. 13, 4918–4927.

Klingelhutz, A.J., Oster, S.A., and Mc.Dougall, J.K. (1996). Telome-

rase activation by the E6 gene product of human papillomaviruses

type 16. Nature 380, 79–82.

Koradi, R., Billeter, M., and Wuthrich, K. (1996). MOLMOL: a program

for display and analysis of macromolecular structures. J. Mol.

Graph. 14, 29–32.

Kuszewski, J., Gronenborn, A.M., and Clore, G.M. (1996). Improving

the quality of NMR and crystallographic protein structures by means

of a conformational database potential derived from structure data-

bases. Protein Sci. 5, 1067–1080.

Lagrange, M., Charbonnier, S., Orfanoudakis, G., Robinson, P.,

Zanier, K., Masson, M., Lutz, Y., Trave, G., Weiss, E., and Deryckere,

F. (2005). Binding of human papillomavirus 16 E6 to p53 and E6AP is

impaired by monoclonal antibodies directed against the second

zinc-binding domain of E6. J. Gen. Virol. 86, 1001–1007.

Lepik, D., Ilves, I., Kristjuhan, A., Maimets, T., and Ustav, M. (1998).

p53 protein is a suppressor of papillomavirus DNA amplificational

replication. J. Virol. 72, 6822–6831.

Le Roux, L., and Moroianu, J. (2003). Nuclear entry of high-risk hu-

man papillomavirus type 16 E6 oncoprotein occurs via several path-

ways. J. Virol. 77, 2330–2337.

Li, X., and Coffino, P. (1996). High-risk human papillomavirus E6 pro-

tein has two distinct binding sites within p53, of which one deter-

mines degradation. J. Virol. 70, 4509–4516.

Lipari, F., McGibbon, G.A., Wardrop, E., and Cordingley, M.G. (2001).

Purification and biophysical characterization of a minimal functional

domain and of an N-terminal Zn2+-binding fragment from the human

papillomavirus type 16 E6 protein. Biochemistry 40, 1196–1204.

Liu, Y., Chen, J.J., Gao, Q., Dalal, S., Hong, Y., Mansur, C.P., Band,

V., and Androphy, E.J. (1999). Multiple functions of human papillo-

mavirus type 16 E6 contribute to the immortalization of mammary

epithelial cells. J. Virol. 73, 7297–7307.

Massimi, P., Gammoh, N., Thomas, M., and Banks, L. (2004). HPV E6

specifically targets different cellular pools of its PDZ domain-

containing tumour suppressor substrates for proteasome-mediated

degradation. Oncogene 23, 8033–8039.

Masson, M., Hindelang, C., Sibler, A., Trave, G., and Weiss, E. (2003).

Preferential nuclear sublocalization of HPV16 E6 oncoprotein in cer-

vical carcinoma cells. J. Gen. Virol. 84, 2099–2104.

Nakagawa, S., Watanabe, S., Yoshikawa, H., Taketani, Y., Yoshiike,

K., and Kanda, T. (1995). Mutational analysis of human papilloma-

virus type 16 E6 protein: transforming function for human cells and

degradation of p53 in vitro. Virology 212, 535–542.

Nilges, M., Gronenborn, G.M., and Clore, A.M. (1988). Determination

of three-dimensional stuctures of proteins by hybrid distance geom-

etry-dynamical simulated annealing calculations. FEBS Lett. 229,

317–324.

Nomine, Y., Ristriani, T., Laurent, C., Lefevre, J.F., Weiss, E., and

Trave, G. (2001a). Formation of soluble inclusion bodies by HPV E6

oncoprotein fused to Maltose-binding protein. Protein Expr. Purif.

23, 22–32.

Nomine, Y., Ristriani, T., Laurent, C., Lefevre, J.F., Weiss, E., and

Trave, G. (2001b). A strategy for optimizing the monodispersity of fu-

sion proteins: application to purification of recombinant HPV E6 on-

coprotein. Protein Eng. 14, 297–305.

Nomine, Y., Charbonnier, S., Ristriani, T., Stier, G., Masson, M.,

Cavusoglu, N., Van Dorsselaer, A., Weiss, E., Kieffer, B., and Trave,

G. (2003). Domain substructure of HPV E6 protein: biophysical char-

acterization of E6 C-terminal DNA-binding domain. Biochemistry 42,

4909–4917.

Nomine, Y., Charbonnier, S., Miguet, L., Potier, N., Van Dorsselaer,

A., Trave, G., and Kieffer, B. (2005). 1H and 15N resonance assign-

ment, secondary structure and dynamic behaviour of the C-terminal

domain of human papillomavirus oncoprotein E6. J. Biomol. NMR

31, 129–141.

Pfister, H. (2003). Chapter 8: human papillomavirus and skin cancer.

J. Natl. Cancer Inst. Monogr. 31, 52–56.

Pim, D., and Banks, L. (1999). HPV-18 E6*I protein modulates the

E6-directed degradation of p53 by binding to full-length HPV-18

E6. Oncogene 18, 7403–7408.

Pim, D., Storey, A., Thomas, M., Massimi, P., and Banks, L. (1994).

Mutational analysis of HPV-18 E6 identifies domains required for

p53 degradation in vitro, abolition of p53 transactivation in vivo

and immortalisation of primary BMK cells. Oncogene 9, 1869–1876.

Ristriani, T., Masson, M., Nomine, Y., Laurent, C., Lefevre, J.F.,

Weiss, E., and Trave, G. (2000). HPV oncoprotein E6 is a structure-

dependent DNA-binding protein that recognizes four-way junctions.

J. Mol. Biol. 296, 1189–1203.

Ristriani, T., Nomine, Y., Masson, M., Weiss, E., and Trave, G. (2001).

Specific recognition of four-way DNA junctions by the C-terminal

zinc-binding domain of HPV oncoprotein E6. J. Mol. Biol. 305,

729–739.

Ristriani, T., Nomine, Y., Laurent, C., Weiss, E., and Trave, G. (2002).

Protein mutagenesis with monodispersity-based quality probing:

selective inactivation of p53 degradation and DNA-binding proper-

ties of HPV E6 oncoprotein. Protein Expr. Purif. 26, 357–367.

Scheffner, M., Werness, B.A., Huibregtse, J.M., Levine, A.J., and

Howley, P.M. (1990). The E6 oncoprotein encoded by human papil-

lomavirus types 16 and 18 promotes the degradation of p53. Cell 63,

1129–1136.

Scheffner, M., Huibregtse, J.M., Vierstra, R.D., and Howley, P.M.

(1993). The HPV-16 E6 and E6-AP complex functions as a ubiqui-

tin-protein ligase in the ubiquitination of p53. Cell 75, 495–505.

Schwarz, E., Freese, U.K., Gissmann, L., Mayer, W., Roggenbuck,

B., Stremlau, A., and zur Hausen, H. (1985). Structure and transcrip-

tion of human papillomavirus sequences in cervical carcinoma cells.

Nature 314, 111–114.

Schwieters, C.D., Kuszewski, J.J., Tjandra, N., and Clore, G.M.

(2003). The Xplor-NIH NMR molecular structure determination pack-

age. J. Magn. Reson. 160, 65–73.

Simonson, S., Difilippantonio, M., and Lambert, P. (2005). Two dis-

tinct activities contribute to human papillomavirus 16 E6’s onco-

genic potential. Cancer Res. 65, 8266–8273.

Molecular Cell678

Slebos, R.J., Kessis, T.D., Chen, A.W., Han, S.M., Hedrick, L., and

Cho, K.R. (1995). Functional consequences of directed mutations

in human papillomavirus E6 proteins: abrogation of p53-mediated

cell cycle arrest correlates with p53 binding and degradation in vitro.

Virology 208, 111–120.

Stewart, D., Kazemi, S., Li, S., Massimi, P., Banks, L., Koromilas, A.,

and Matlashewski, G. (2004). Ubiquitination and proteasome degra-

dation of the E6 proteins of human papillomavirus types 11 and 18.

J. Gen. Virol. 85, 1419–1426.

Talis, A., Huibregtse, J., and Howley, P. (1998). The role of E6AP in

the regulation of p53 protein levels in human papillomavirus (HPV)-

positive and HPV-negative cells. J. Biol. Chem. 273, 6439–6445.

Thomas, M., and Chiang, C. (2005). E6 oncoprotein represses

p53-dependent gene activation via inhibition of protein acetylation

independently of inducing p53 degradation. Mol. Cell 17, 251–264.

Thomas, M., Pim, D., and Banks, L. (1999). The role of the E6-p53 in-

teraction in the molecular pathogenesis of HPV. Oncogene 18, 7690–

7700.

Thomas, M., Laura, R., Hepner, K., Guccione, E., Sawyers, C., Lasky,

L., and Banks, L. (2002). Oncogenic human papillomavirus E6 pro-

teins target the MAGI-2 and MAGI-3 proteins for degradation. Onco-

gene 21, 5088–5096.

Vriend, G. (1990). WHATIF: a molecular modeling and drug design

program. J. Mol. Graph. 8, 52–56.

Yamamoto, Y., Huibregtse, J.M., and Howley, P.M. (1997). The hu-

man E6-AP gene (UBE3A) encodes three potential protein isoforms

generated by differential splicing. Genomics 41, 263–266.

Zanier, K., Charbonnier, S., Baltzinger, M., Nomine, Y., Altschuh, D.,

and Trave, G. (2005). Kinetic analysis of the interactions of human

papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using

surface plasmon resonance. J. Mol. Biol. 349, 401–412.

zur Hausen, H. (1991). Human papillomaviruses in the pathogenesis

of anogenital cancer. Virology 184, 9–13.

Accession Numbers

The solution structure of E6C has been deposited in the Protein Data

Bank with the accession number 2FK4.