Solution Structure Analysis of the HPV16 E6 Oncoprotein Reveals a Self-Association Mechanism Required for E6-Mediated Degradation of p53

Zanier, Katia; Sidi, Abdellahi Ould M'hamed Ould; Boulade-Ladame, Charlotte; Rybin, Vladimir; Chappelle, A.; Atkinson, R. Andrew; Kieffer, Bruno; Travé, Gilles

doi:10.1016/j.str.2012.02.001

Cited by 111 publications

(162 citation statements)

References 60 publications

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“…We employed a solubility-optimized mutant of HPV16 E6, named E6 F47R 4C/4S, which harbors the F47R mutation preventing dimerization of the E6N domain and four cysteine to serine substitutions in the E6C domain for suppression of disulfide cross-bridging [19]. Biotinylated peptides comprising either the pep11** sequence or E6APpep were captured on a streptavidin-coated surface.…”

Section: Resultsmentioning

confidence: 99%

“…These experiments were based on the analysis of backbone amide groups, since the 2D 1 H- 15 N correlation spectrum of the monomeric E6 construct used is relatively well dispersed and could be assigned to 92% of completeness [19]. After optimization of the experimental setup, we were able to record NMR spectra of E6 bound to pep11** or E6APpep at conditions of minimal E6 aggregation.…”

Section: Discussionmentioning

confidence: 99%

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The E6AP Binding Pocket of the HPV16 E6 Oncoprotein Provides a Docking Site for a Small Inhibitory Peptide Unrelated to E6AP, Indicating Druggability of E6

et al. 2014

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The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The E6AP Binding Pocket of the HPV16 E6 Oncoprotein Provides a Docking Site for a Small Inhibitory Peptide Unrelated to E6AP, Indicating Druggability of E6

et al. 2014

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“…In this way we created a soluble construct of HPV16 E6 comprising 5 point mutations, which did not alter E6 structure. 4 These findings, together with strategies based on fusion carrier proteins, opened the way to structural studies of complexes of HPV E6.…”

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confidence: 99%

HPV-mediated inactivation of tumor suppressor p53

Travé

Zanier

2016

Cell Cycle

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“…It is unclear if this is due to an unmasking of an intact p53-interacting site on 16E6 upon binding LXXLL or if the interaction of 16E6 with LXXLL peptides causes a de novo p53 interaction site to be formed by allosteric modification of 16E6. A recent nuclear magnetic resonance (NMR) solution structure of 16E6 shows strong conservation of the N-terminal (E6N) and C-terminal (E6C) zinc structured domains and poor resolution of the interdomain connecting region in the absence of bound LXXLL peptide (27). Since this solution structure poorly resolved the linker region of E6, it remains unclear how the structure of E6 is altered upon peptide binding or how peptide binding could alter the fold of either the E6N or E6C domain.…”

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confidence: 99%

Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

Ansari

Brimer

Pol

2012

J Virol

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). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule. P apillomavirus E6 oncoproteins interact with target cellular proteins through docking on short peptides often containing the sequence LXXLL (4, 6, 26). The cancer-associated human papillomavirus type 16 (HPV-16) protein E6 (16E6) binds to an LXXLL motif (LQELL) on the cellular E3 ubiquitin ligase E6AP (10); 16E6-E6AP recruits and ubiquitinates p53. Neither 16E6 nor E6AP interacts alone with p53 (8-10, 17, 18) (20). A form of E6AP with a C833A mutation still binds E6 and p53 but fails to ubiquitinate or degrade p53 (19).The necessity of E6AP for p53 degradation by 16E6 is controversial. While E6AP is required for in vitro degradation systems (9) and immortalized mouse fibroblasts (5), E6AP-null mice that express 16E6 in the skin do not accumulate p53 when irradiated, and mouse embryo kidney cells from those E6AP-null mice are reported to lose p53 upon overexpression of 16E6 (12,22). Further, ubiquitin-independent degradation of p53 by E6 has been described (2). These disparate observations raise the questions of whether of E6AP is necessary for degradation and how the specificity of 16E6 for p53 is determined.16E6 binding to the LQELL peptide alone recruits p53 to 16E6 in the absence of full-length E6AP. Our previous studies in yeast (Saccharomyces cerevisiae) had shown that p53 was very weakly recruited by LexA fusions to 16E6, but coexpression of LexA_16E6 with the E6AP C833A mutant (here termed E6AP-Ub Ϫ , for ubiquitin ligase negative) strongly recruited p53 to 16E6 (5). We reasoned that the binding of 16E6 solely to the minimal LQELL peptide of E6AP or similar LXXLL peptides might be sufficient to induce a p53-binding conformation of 16E6. To test this, we fused the 10-residue E6AP LQELL peptide between LexA and 16E6 to provide 16E6 with an LQELL docking site in cis; we similarly fused a mutated LXXLL peptide (LQEAS) between LexA and 16E6 (Fig. 1A). We tested interactions of these LexA fusions by yeast two-hybrid assays with a B42 transactivator-tagged LQELL peptide, B42_E6AP-Ub Ϫ , or with B42_E6AP-Ub Ϫ molecules where LQELL was progressively mutated from LQELL to LQELS or LQEAS. Fusion of the LQELL peptide in cis to 16E6 blocked interaction with B42_LQELL peptide in trans as expected, and mutation of LexA_LQELL_16E6 to LexA_LQEAS_16E6 restored interactions in trans (Fig. 1B, spots 4C and 4D). This is consistent with the LQELL peptide binding in cis to the 16E6 portion of the fusion protein and blocking trans interaction with the B42-fused peptide. Interaction of LQELL_16E6 with B42_E6AP-Ub Ϫ in trans was not blocked, indicating that the interaction of 16E6 with the full E6AP protein is more robust than the interaction with the free LQELL peptide but is still dependent upon an in...

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Solution Structure Analysis of the HPV16 E6 Oncoprotein Reveals a Self-Association Mechanism Required for E6-Mediated Degradation of p53

Cited by 111 publications

References 60 publications

The E6AP Binding Pocket of the HPV16 E6 Oncoprotein Provides a Docking Site for a Small Inhibitory Peptide Unrelated to E6AP, Indicating Druggability of E6

The E6AP Binding Pocket of the HPV16 E6 Oncoprotein Provides a Docking Site for a Small Inhibitory Peptide Unrelated to E6AP, Indicating Druggability of E6

HPV-mediated inactivation of tumor suppressor p53

Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

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