Structures of a Human Papillomavirus (HPV) E6 Polypeptide Bound to MAGUK Proteins: Mechanisms of Targeting Tumor Suppressors by a High-Risk HPV Oncoprotein

Abstract

Human papillomavirus (HPV) E6 oncoprotein targets certain tumor suppressors such as MAGI-1 and SAP97/ hDlg for degradation. A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of these tumor suppressors, which is a property unique to high-risk HPVs that are associated with cervical cancer. The detailed recognition mechanisms between HPV E6 and PDZ proteins are unclear. To understand the specific binding of cellular PDZ substrates by HPV E6, we have solved the crystal structures of the complexes containing a peptide from HPV18 E6 bound to three PDZ domains from MAGI-1 and SAP97/Dlg. The complex crystal structures reveal novel features of PDZ peptide recognition that explain why high-risk HPV E6 can specifically target these cellular tumor suppressors for destruction. Moreover, a new peptide-binding loop on these PDZs is identified as interacting with the E6 peptide. Furthermore, we have identified an arginine residue, unique to high-risk HPV E6 but outside the canonical core PDZ recognition motif, that plays an important role in the binding of the PDZs of both MAGI-I and SAP97/Dlg, the mutation of which abolishes E6's ability to degrade the two proteins. Finally, we have identified a dimer form of MAGI-1 PDZ domain 1 in the cocrystal structure with E6 peptide, which may have functional relevance for MAGI-1 activity. In addition to its novel insights into the biochemistry of PDZ interactions, this study is important for understanding HPV-induced oncogenesis; this could provide a basis for developing antiviral and anticancer compounds.Human papillomaviruses (HPVs) are small double-stranded DNA tumor viruses that induce hyperproliferative lesions in epithelial tissues (28). A subset of HPV types that act as the etiological agent of cervical cancers are called "high-risk" types; these include HPV16, HPV18, HPV31, and HPV45 among others (26,27,29,45). The oncogenic potential of the high-risk types is dependent on the cooperative action of the two early viral gene products, E6 and E7, which bind and alter the activity of cell cycle regulatory proteins.The targeting of p53 for degradation by E6 has extensively been studied, and this is an important feature for the oncogenic activity of the high-risk papillomaviruses that cause cancer (19,36,37). However, a large amount of evidence suggests the existence of p53-independent functions of E6 that are also necessary and important for transformation. Support for this idea comes from the observation that the transformation of cells or the induction of epithelial hyperproliferation in transgenic animals by E6 does not always correlate with its ability to degrade p53 (22,33).Numerous cellular proteins have been identified as targets of HPV E6 in cell transformation. These proteins are involved in a variety of cellular processes, such as calcium signaling (4), cell adhesion (41), transcriptional control (5), DNA synthesis (25), apoptosis (8), cell cycle control (10), DNA repair (20), and small G-protein signaling (11). One group of these targets of...

Human papillomaviruses, cervical cancer and cell polarity

“…They have been shown to be targets for virus oncoproteins, such as adenovirus type 9 E4ORF1 protein and the HTLV-1 Tax protein Ohashi et al, 2004;reviewed in Javier (2008), this issue), and are targeted by the HPV-16 and HPV-18 E6 proteins for proteasome-mediated degradation Thomas et al, 2001Thomas et al, , 2002. Indeed, biochemically they appear to be the most avidly bound PDZ targets of E6 (Zhang et al, 2007b) and the most susceptible to degradation (M Thomas, personal observation).…”

“…Conversely, hScrib is bound more strongly by HPV-16 E6 than by HPV-18 E6, again dependent upon the carboxy terminal amino-acid residue, and, equally, it is degraded more effectively in the presence of HPV-16 E6 (Thomas et al, 2005), thus showing that the exact sequence of the PDZ-binding motif is instrumental in substrate selection. An extra dimension was added to this with the description of the crystal structures of the MAGI-1 PDZ1 and the Dlg1 PDZ2 and PDZ3 domains, each bound to a peptide homologous to the HPV-18 E6 C terminus (Zhang et al, 2007b). The Dlg PDZ3 is not an in vivo target of HPV-18 E6, and analysis showed that it made a minimum of binding contacts with the peptide; in contrast, the MAGI-1 PDZ1 and Dlg PDZ2 domains, both of which are in vivo targets of HPV-18 E6, each made several additional contacts with the peptide, including those with aminoacid residues outside the canonical PDZ-binding motif.…”

A conserved C-terminal sequence of high-risk cutaneous beta-human papillomavirus E6 proteins alters localization and signalling of β1-integrin to promote cell migration

“…Consequently, these E6 proteins bind and target PDZ domain proteins that have differing functions with different affinities (Gardiol et al, 1999; Kiyono et al, 1997;Pim et al, 2000;Thomas et al, 2005). Generally, the ETQV/L motif inserts into a peptidebinding loop within the PDZ domain, formed by the strand bB and the helix ab (Zhang et al, 2007). It is therefore interesting that there is no variation between the HPV5 and -8 E6 proteins, suggesting a strict conservation of function.…”

“…It is therefore interesting that there is no variation between the HPV5 and -8 E6 proteins, suggesting a strict conservation of function. Studies have shown that phosphorylation of the ETQV/L motif of the high-risk HPV types by protein kinase A inhibits the ability of the protein to bind PDZ motifs (Kühne et al, 2000;Zhang et al, 2007), and mutation of this region impacts on the phenotype of E6-expressing cells (Watson et al, 2003). However, interrogation of the PHOSIDA and NetPhos2.0 prediction tools suggests that the YHDW motif is not likely to be a phosphorylation site, implying that the regulation of function of the different C-terminal motifs is not conserved.…”

Molecular Bases of Human Papillomavirus Pathogenesis in the Development of Cervical Cancer

“…The PDZ proteins known to bind E6 are hDlg1 and 4, tumor suppressor proteins (Kiyono et al, 1997; S.S. Lee et al, 1997), MAGI-1, -2 and -3, Membrane Associated Guanylate kinase homolog proteins (Thomas et al, 2001), MUPP-1 a multi PDZ protein (S.S. Lee et al, 2000), hScrib (Nakagawa & Huibregtse 2000) and PTPN3, both tyrosine phosphatase proteins (Jing et al, 2007). These proteins bind to E6 by the PDZ domain (aa 141-151) (Pim et al, 2009;Storrs & Silverstein, 2007;Y. Zhang et al, 2007), acting as an adaptor to link the ubiquitin ligase to the target for ubiquitination by means of its C-terminal region (Pim et al, 2009).…”