Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain

André Mischo,Matthias Görlach,Oliver Ohlenschläger,Peter Hortschansky,Ramadurai Ramachandran,Olivier Kocher

doi:10.1371/journal.pone.0062584

André Mischo, Matthias Görlach + Show 4 more

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https://doi.org/10.1371/journal.pone.0062584

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Abstract

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.

Highlights

Infections are responsible for 17.8% of all cancers [1]
The human papilloma virus (HPV) oncoproteins E6 and E7 of tumor-associated so-called ‘‘high-risk’’ HPVs prevent the differentiation of HPV-infected keratinocytes and immortalize primary, cultured human keratinocytes [5,6,7]
Assessment of solubility of amino-terminally His6-tagged, recombinant E6 constructs derived from the high-risk types 16, 18, 26, 31, 45, 51, 59 and 97 as well as for the non-tumorigenic cutaneous type 1a was performed after bacterial expression in presence of 10 mM of Zn2+ as solubility of E6 constructs is dependent on low mM concentrations of zinc [54]

Summary

Introduction

Infections are responsible for 17.8% of all cancers [1]. Several human papilloma virus (HPV) types are the most frequent viral cancer-causing agents and are linked to well over half a million incidences of cervical cancer each year [1,2,3]. Development of therapies against HPV-caused malignancies requires further mechanistic insight into how oncogenic HPV drive tumor development and maintenance. The HPV oncoproteins E6 and E7 of tumor-associated so-called ‘‘high-risk’’ HPVs prevent the differentiation of HPV-infected keratinocytes and immortalize primary, cultured human keratinocytes [5,6,7]. Primary human cervical carcinoma cells require E6 and E7 expression for proliferation [8]. Upon depletion of E6 and/or E7, HPV-transformed cervical cancer derived cell lines undergo apoptosis or senescence [9,10,11,12,13,14]. Apoptotic HPV positive cancer cells may transform human primary fibroblasts by horizontal gene transfer of the E6/E7 ORF [15]

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