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* This work was supported by funds from the Swedish Research Council (to P. J. and P. L.), Jeansson's Foundation (to P. J.), Clas Groschinskys Minnesfond (to P. J.), Science Foundation Ireland Awards 07/SK/B1224and 09/YI/B1682 (to N. F.), and a start-up award from University College Dublin (to N. F.). The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Fig. S1. ♦ This article was selected as a Paper of the Week.
The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ2 domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.
), and HPV16 and 18 are the two most common high risk types of HPV. One of the hallmarks of HPV pathogenesis is the E6-mediated inactivation of the p53 tumor suppressor protein. However, a number of studies have shown the existence of a p53-independent mechanism that leads to uncontrolled cell growth (
). For example, the E6 protein of the high risk (but not the low risk type) HPV interacts with the PDZ (PSD-95/Discs large/ZO-1) domains of several proteins such as SAP97 (synapse-associated protein 97), hScrib, MAGI, and MUPP1, through a conserved C-terminal motif (
). The tumor suppressor protein SAP97 contains a series of three consecutive PDZ domains (PDZ1, PDZ2, and PDZ3), one Src homology 3 domain, and one guanylate kinase-like domain. The mechanism by which the E6 protein interacts with these three PDZ domains is not well understood. It has been shown through cell pulldown assays that only the PDZ2 domain of SAP97 interacts with the E6 protein of HPV16, whereas the E6 protein of HPV18 interacts with all three PDZ domains (
). Biochemical characterization of the PDZ-E6 interaction have shown that discrimination between the E6 proteins of HPV16 and HPV18 is due to the C-terminal amino acid, which is Leu-151 in HPV16 and Val-158 in HPV18 (
), this poses the question: how do such repeats affect the binding to the HPV E6 protein, if at all? For example, steric occlusion, allostery, or cooperativity in binding could regulate SAP97-E6 interactions and further contribute to differential specificity toward HPV E6 variants.
To dissect these issues, we analyzed the interaction between HPV E6 and six SAP97 PDZ domain constructs: PDZ1; PDZ2; PDZ3; the tandem constructs PDZ12 and PDZ23; and PDZ123, which contains all three concatenated PDZ domains. We used the complete C-terminal domain of E6 and the entire PDZ region of SAP97 and combined both equilibrium and kinetic experiments in solution (
Cervical cancer is caused by certain strains of HPV through expression of the oncogenic proteins E6 and E7. The E6 protein has evolved to bind to several important host regulatory proteins, such as the tumor suppressor proteins p53 and SAP97, and thus mark these proteins for destruction to keep HPV-infected cells alive. The exact mechanisms by which host proteins are targeted by the viral proteins are very complex (
). Here we have looked at mechanistic aspects of the complex formation between E6 and the PDZ domains of SAP97 and revealed several novel facets of this interaction that are recapitulated in Fig. 4.
One major finding is that the E6 proteins of both HPV16 and HPV18 bind to all three PDZ domains of SAP97 in vitro. By using a cell pulldown assay, it was previously suggested that only the PDZ2 domain of SAP97 could bind the E6 protein of HPV16 (
). In this study we have measured the affinities of the respective PDZ domains with the E6 from HPV16 and a “pseudo” HPV18 E6 protein (E6L151V). All three PDZ domains have similar association or on rate constants for the two different E6 constructs. However, the dissociation (off rate) constants differed and may explain the observed difference in virulence between the two HPV types (
). Although Val and Leu residues both have a hydrophobic side chain, the Val residue of HPV18 E6 is smaller than the Leu of HPV16 E6, which may allow for a more snug fit in the hydrophobic binding pockets of the PDZ domains.
Contrary to the previous results, we find that PDZ1 displays the highest affinity for HPV16 E6, whereas PDZ3 binds strongest to E6L151V (i.e. to HPV18 E6). However, the affinities are within the same order of magnitude (TABLE 1, TABLE 3), suggesting a high degree of promiscuity in E6-PDZ interactions. Furthermore, SEC-MALS, ITC, and kinetic studies show that all three putative binding sites in SAP97 are occupied by E6 proteins in a quaternary complex. Previous experiments using electron microscopy on full-length SAP97 showed that the molecule is present in a monomer-dimer equilibrium and that the dimerization occurs via its N-terminal L27 domain (
). Furthermore, it was demonstrated that monomeric SAP97 is a relatively dynamic protein that exists either in an extended conformation or as a more compact ring-like structure. In another study on the PDZ region of SAP97, it was demonstrated by small angle x-ray scattering that PDZ123 is indeed flexible, in particular the region between PDZ2 and PDZ3 (
). It was further shown that the PDZ12 part was more conformationally restricted and displayed a dumbbell-like shape, in agreement with our kinetic experiments where the on rate constant for PDZ12 is not the sum of those for PDZ1 and PDZ2. The SEC-MALS experiments presented here are consistent with the PDZ region of SAP97 undergoing a structural change, perhaps a collapse or a compaction, upon interaction with the E6 proteins. These data are supported by rate constants of dissociation from stopped flow fluorescence, in particular the slower dissociations from PDZ12, PDZ23, and PDZ123, as compared with those of the single domains (Fig. 2, c and d, and Table 3). Whether the quaternary complex occurs in vivo depends on the expression levels of the E6 protein and of SAP97. Likewise, the reported oligomerization of E6 (
), but actual concentrations have not been reported and are very difficult to estimate. Expression of E6 may also vary both temporally and spatially, and the effects of oligomerization and dissociation of E6 in the infected cell remain to be investigated.
PDZ domains bind ligands like the E6 protein through the C terminus of the ligand, which becomes a strand in an extended β-sheet (
). Here, by NMR experiments, we found residues in PDZ2 that are not part of the peptide-binding pocket but nevertheless experience chemical shift changes upon binding of E6. Importantly, four of these residues (Fig. 3) were unaffected by binding of an E6 C-terminal peptide (
) or by direct interactions between other parts of the E6 than its C terminus. In either case these noncanonical interactions may be employed by the E6 protein to increase affinity or even modulate binding to a third partner. Low affinity nonspecific interactions between E6 and PDZ2 are less likely to cause these changes in chemical shifts because these four residues saturate at similar concentration as residues in the canonical binding pocket, upon titration with E6.
In conclusion, we report affinities, stoichiometries, rate constants, and conformational change(s) for the complex between E6 and SAP97. As a step toward better understanding of E6-mediated oncogenesis, our results highlight the dynamic nature of the E6-SAP97 binding and reveal mechanistic and molecular details of the interaction.
The human papillomavirus (HPV) E6 protein plays an important role in the development of cancer associated with certain high risk strains. In addition to binding to p53, E6 also binds to the PDZ domain of several proteins that participate in cell adherence and polarity. The mechanism by which E6 interacts with PDZ is not well understood, but in this Paper of the Week, Celestine Chi and colleagues employed multiple biophysical techniques to explore E6 binding to the PDZ domains of the tumor suppressor protein SAP97; they developed various constructs that consisted of one, two, or all three PDZ domains.