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 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.
Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg (IREBS), 67412, Illkirch, France.
Katia
K
Zanier
Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany.
Christina
C
Stutz
Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany.
Susanne
S
Kintscher
Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany.
Eileen
E
Reinz
EMBL-DKFZ Chemical Biology Core Facility, European Molecular Biology Laboratory (EMBL), 69117, Heidelberg, Germany.
Peter
P
Sehr
Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany.
Julia
J
Bulkescher
Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany.
Karin
K
Hoppe-Seyler
Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg (IREBS), 67412, Illkirch, France.
Gilles
G
Travé
Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany.
Felix
F
Hoppe-Seyler
D000595:Amino Acid Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D015195:Drug Design; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009856:Oncogene Proteins, Viral / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D010455:Peptides; D012097:Repressor Proteins; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases