Design and characterization of helical peptides that inhibit the E6 protein of papillomavirus

The E6 protein from HPV type 16 binds proteins containing a seven-residue leucine-containing motif. Previous work demonstrated that peptides containing the consensus sequence are a mixture of alpha-helix and unstructured conformations. To design monomeric E6-binding peptides that are stable in aqueous solution, we used a protein grafting approach where the critical residues of the E6-binding motif of E6-associated protein, E6AP, LQELLGE, were incorporated into exposed helices of two stably folded peptide scaffolds. One series was built using the third zinc finger of the Sp1 protein, which contains a C-terminal helix. A second series was built using a Trp-cage scaffold, which contains an N-terminal helix. The chimeric peptides had very different activities in out-competing the E6-E6AP interaction. We characterized the peptides by circular dichroism spectroscopy and determined high-resolution structures by NMR methods. The E6-binding consensus motif was found to be helical in the high-quality structures, which had backbone root-mean-square deviations of less than 0.4 A. We have successfully grafted the E6-binding motif into two parent peptides to create ligands that have biological activity while preserving the stable, native fold of their scaffolds. The data also indicate that conformational change is common in E6-binding proteins during the formation of the complex with the viral E6 protein.     

Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance

Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation. E6AP, like other E6 binding proteins such as E6BP, IRF-3 and paxillin, interacts with E6 via a consensus leucine-charged motif. Here we have investigated the kinetics of the interactions of a 15-mer peptide containing the LxxvarphiLsh motif of E6AP with E6. For this we have developed a Biacore assay based on antibody-capture on the sensor surface of GST- and/or MBP-E6AP peptide constructs followed by E6 protein injection. Our experiments show that E6 oncoproteins from four major high-risk (16, 18, 33 and 58) HPV types bind to E6AP with equilibrium dissociation constants in the low micromolar range. The kinetic dissociation parameters of these interactions are remarkably similar. On the other hand, low-risk HPV 11 E6 does not interact with E6AP even at relatively high concentrations. We also show that the two zinc-binding domains of E6 are required for E6AP recognition. Finally, we have analysed the binding properties of site-directed mutants of the E6AP-derived peptide. We demonstrate the importance for binding of conserved aliphatic side-chains and the moderate role of the global negative charge of the peptide. This work provides the first quantitative data on an HPV E6-mediated interaction, which support the current models of E6AP-mediated degradation.     

PATJ, a tight junction-associated PDZ protein, is a novel degradation target of high-risk human papillomavirus E6 and the alternatively spliced isoform 18 E6

The E6 protein from high-risk human papillomavirus types interacts with and degrades several PDZ domain-containing proteins that localize to adherens junctions or tight junctions in polarized epithelial cells. We have identified the tight junction-associated multi-PDZ protein PATJ (PALS1-associated TJ protein) as a novel binding partner and degradation target of high-risk types 16 and 18 E6. PATJ functions in the assembly of the evolutionarily conserved CRB-PALS1-PATJ and Par6-aPKC-Par3 complexes and is critical for the formation of tight junctions in polarized cells. The ability of type 18 E6 full-length to bind to, and the subsequent degradation of, PATJ is dependent on its C-terminal PDZ binding motif. We demonstrate that the spliced 18 E6* protein, which lacks a C-terminal PDZ binding motif, associates with and degrades PATJ independently of full-length 18 E6. Thus, PATJ is the first binding partner that is degraded in response to both isoforms of 18 E6. The ability of E6 to utilize a non-E6AP ubiquitin ligase for the degradation of several PDZ binding partners has been suggested. We also demonstrate that 18 E6-mediated degradation of PATJ is not inhibited in cells where E6AP is silenced by shRNA. This suggests that the E6-E6AP complex is not required for the degradation of this protein target.     

Recognition of accessory protein motifs by the gamma-adaptin ear domain of GGA3

Adaptor proteins load transmembrane protein cargo into transport vesicles and serve as nexuses for the formation of large multiprotein complexes on the nascent vesicles. The gamma-adaptin ear (GAE) domains of the AP-1 adaptor protein complex and the GGA adaptor proteins recruit accessory proteins to these multiprotein complexes by binding to a hydrophobic motif. We determined the structure of the GAE domain of human GGA3 in complex with a peptide based on the DFGPLV sequence of the accessory protein Rabaptin-5 and refined it at a resolution of 2.2 A. The leucine and valine residues of the peptide are partly buried in two contiguous shallow, hydrophobic depressions. The anchoring phenylalanine is buried in a deep pocket formed by the aliphatic portions of two conserved arginine residues, along with an alanine and a proline, illustrating the unusual function of a cluster of basic residues in binding a hydrophobic motif.     

Identification of a second transforming function in bovine papillomavirus type 1 E6 and the role of E6 interactions with paxillin, E6BP, and E6AP

Papillomavirus E6 oncoproteins transform mammalian cells through interaction with cellular proteins. Bovine papillomavirus type 1 E6 (BE6) interacts with three previously described cellular targets: the E6AP E3 ubiquitin ligase, the calcium-binding protein E6BP (also known as ERC-55), and paxillin, which is a focal adhesion adapter protein. BE6 interacts strongly with each of these proteins in vitro, binding to similar peptide sequences found in E6AP, E6BP, and paxillin. To determine which BE6 interactions are necessary for transformation by BE6, we used a novel selection strategy for temperature-sensitive BE6 mutants in yeast that could discriminate in their interaction between E6AP, E6BP, and paxillin. All BE6 mutants that retained transforming ability retained association with paxillin, while some mutants that were transformation positive failed to interact with E6AP or E6BP. This study demonstrates that oncogene mutants that are temperature sensitive for transformation can be selected in yeast and that the induction of anchorage-independent cell proliferation by BE6 does not require strong association of BE6 with either E6AP or E6BP. Of particular interest is the identification of a BE6 mutant that interacts strongly with the acidic charged leucine motifs of E6AP, E6BP, and paxillin but is devoid of transformation activity, thereby genetically identifying a second essential transformation function in BE6 that is independent of interaction with acidic charged leucine motifs.     

The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

Employing ¹²⁵I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508 Ų and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe⁷²⁷ and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases k_cat 62-fold but fails to affect E2∼ubiquitin binding or subsequent formation of the Hect domain Cys⁸²⁰∼ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe⁷²⁷-dependent trimer formation and catalytic activity (K_i = 12 mm), as does a conserved α-helical peptide corresponding to residues 474–490 of E6AP isoform 1 (K_i = 22 μm) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe⁷²⁷ intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (K_activation = 1.5 nm) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the k_cat defect of the Phe⁷²⁷ mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.     

Molecular Determinants of PAM2 Recognition by the MLLE Domain of Poly(A)-Binding Protein

MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.     

Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin

Ubiquitin is an important cellular signal that targets proteins for degradation or regulates their functions. The previously identified BMSC-UbP protein derived from bone marrow stromal cells contains a ubiquitin-associated (UBA) domain at the C terminus that has been implicated in linking cellular processes and the ubiquitin system. Here, we report the solution NMR structure of the UBA domain of human BMSC-UbP protein and its complex with ubiquitin. The structure determination was facilitated by using a solubility-enhancement tag (SET) GB1, immunoglobulin G binding domain 1 of Streptococcal protein G. The results show that BMSC-UbP UBA domain is primarily comprised of three alpha-helices with a hydrophobic patch defined by residues within the C terminus of helix-1, loop-1, and helix-3. The M-G-I motif is similar to the M/L-G-F/Y motifs conserved in most UBA domains. Chemical shift perturbation study revealed that the UBA domain binds with the conserved five-stranded beta-sheet of ubiquitin via hydrophobic interactions with the dissociation constant (KD) of approximately 17 microM. The structural model of BMSC-UbP UBA domain complexed with ubiquitin was constructed by chemical shift mapping combined with the program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, three residues (Met76, Ile78, and Leu99) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin defined by residues Leu8, Ile44, His68, and Val70. This complex structure may provide clues for BMSC-UbP functions and structural insights into the UBA domains of other ubiquitin-associated proteins that share high sequence homology with BMSC-UbP UBA domain.     

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

Evaluation of different glutathione S-transferase-tagged protein captures for screening E6/E6AP interaction inhibitors using AlphaScreen

Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins.     

Structural basis of enhanced binding of extended and helically constrained peptide epitopes of the broadly neutralizing HIV-1 antibody 4E10

Potent, broadly HIV-1 neutralizing antibodies (nAbs) may be invaluable for the design of an AIDS vaccine. 4E10 is the broadest HIV-1 nAb known to date and recognizes a contiguous and highly conserved helical epitope in the membrane-proximal region of gp41. The 4E10 epitope is thus an excellent target for vaccine design as it is also highly amenable to peptide engineering to enhance its helical character. To investigate the structural effect of both increasing the peptide length and of introducing helix-promoting constraints in the 4E10 epitope, we have determined crystal structures of Fab 4E10 bound to an optimized peptide epitope (NWFDITNWLWYIKKKK-NH(2)), an Aib-constrained peptide epitope (NWFDITNAibLWRR-NH(2)), and a thioether-linked peptide (NWFCITOWLWKKKK-NH(2)) to resolutions of 1.7 A, 2.1 A, and 2.2 A, respectively. The thioether-linked peptide is the first reported structure of a cyclic tethered helical peptide bound to an antibody. The introduced helix constraints limit the conformational flexibility of the peptides without affecting interactions with 4E10. The substantial increase in affinity (10 nM versus 10(4) nM of the IC(50) of the original KGND peptide template) is largely realized by 4E10 interaction with an additional helical turn at the peptide C terminus that includes Leu679 and Trp680. Thus, the core 4E10 epitope was extended and modified to a WFX(I/L)(T/S)XX(L/I)W motif, where X does not play a major role in 4E10 binding and can be used to introduce helical-promoting constraints in the peptide epitope.     

Strategies for bacterial expression of protein-peptide complexes: application to solubilization of papillomavirus E6

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif.     

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex

In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Herein, we performed extensive molecular dynamics simulations (totally ～13 μs) on both heterodimer and heterotrimer to elucidate at an atomic level how each p53-degradation-defective HPV16 E6 mutant reduces the structural stabilities of the two complexes. Our simulations reveal that the three E6 mutations destabilize the structure of E6/E6AP/p53 complex through distinct mechanisms. Although F47R^E6 mutation has no effect on the structure of E6/E6AP heterodimer, it results in an electrostatic repulsion between R47^E6 and R290^p53, which is unfavorable for E6-p53 binding. R102A^E6 mutation destabilizes the structure of E6/E6AP heterodimer and significantly disrupts hydrophobic and cation-π interactions between F47^E6 and E286^p53/L298^p53/R290^p53. L50E^E6 mutation impairs both E6 interdomain interactions (especially F47-K108 cation-π interaction) and E6-E6AP intermolecular interactions important for the stabilization of E6/E6AP heterodimer. This study identifies the intra- and intermolecular interactions crucial for the complex stability, which may provide mechanistic insights into the inhibition of complex formation by the three HPV16 E6 mutations.     

Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase

"Charged-to alanine" scanning mutagenesis of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase (C1) identified three glutamate residues, E171, E214, and E274, that are involved in the recognition of a peptide substrate, kemptide (Leu1Arg2Arg3Ala4Ser5Leu6Gly7). These glutamate residues are conserved or conservatively substituted with asparate in the serine/threonine protein kinases that have a requirement for basic residues on the N-terminal side of their phosphorylation sites. Alanine replacement mutants in C1 were subjected to kinetic analysis using alanine-substituted peptides as substrates. The additivity or nonadditivity of the effects of the alanine substitutions on the catalytic efficiency (kcat/Km) was analyzed. This allowed the identification of electrostatic interactions between the three glutamate residues in the enzyme and the two arginine residues present in the peptide substrate. The data suggest that E171 interacts with Arg2 in the substrate and that E214 and E274 both interact with Arg3. This may be a general method for identifying simple intermolecular interactions involving proteins when there is no three-dimensional structure available of the complex of interacting species. The identification of these interactions provides the potential for rational protein engineering of enzymes with alternative specificities.     

Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity

The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F(1). Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.     

Sequence analysis of Arabidopsis thaliana E/ANTH-domain-containing proteins: membrane tethers of the clathrin-dependent vesicle budding machinery

Summary. The epsin N-terminal homology (ENTH) domain is a conserved protein module present in cytosolic proteins which are required in clathrin-mediated vesicle budding processes. A highly similar, yet unique module is the AP180 N-terminal homology (ANTH) domain, which is present in a set of proteins that also support clathrin-dependent endocytosis. Both ENTH and ANTH (E/ANTH) domains bind to phospholipids and proteins, in order to support the nucleation of clathrin coats on the plasma membrane or the trans-Golgi-network membrane. Therefore, E/ANTH proteins might be considered as universal tethering components of the clathrin-mediated vesicle budding machinery. Since the E/ANTH protein family appears to be crucial in the first steps of clathrin-coated vesicle budding, we performed data base searches of the Arabidopsis thaliana genome. Sequence analysis revealed three proteins containing the ENTH signature motif and eight proteins containing the ANTH signature motif. Another six proteins were found that do not contain either motif but seem to have the same domain structure and might therefore be seen as VHS-domain-containing plant proteins. Functional analysis of plant E/ANTH proteins are rather scarce, since only one ANTH homolog from A. thaliana, At-AP180, has been characterized so far. At-AP180 displays conserved functions as a clathrin assembly protein and as an α-adaptin binding partner, and in addition shows features at the molecular level that seem to be plant-specific. Correspondence and reprints: Cell Biology, Heidelberg Institute for Plant Sciences, Im Neuenheimer Feld 230, 69120 Heidelberg, Federal Republic of Germany.     

Solution structure of apo CopZ from Bacillus subtilis: further analysis of the changes associated with the presence of copper

The solution structure of apo CopZ from Bacillus subtilis has been determined with the aim of investigating the changes in the hydrophobic interactions around the M-X-C-X-X-C copper(I) binding motif upon metal binding. The methionine of this motif (Met 11 in CopZ) points toward the solvent in apo CopZ, whereas its sulfur atom is close to the metal ion in the metal-loaded protein, though probably not at binding distance. This change is associated with the weakening of the interaction between Leu 37 and Cys 16, present in the apo form, and the formation of an interaction between Met 11 and Tyr 65. Loops 1, 3, and 5 are affected by metal binding. Comparison with the structure of other homologous proteins confirms that often metal binding affects a hydrophobic patch around the metal site, possibly for optimizing and tuning the hydrophobic interactions with the partners. It is also shown that copper(I) exchanges among apo CopZ molecules in slow exchange on the NMR time scale, whereas it is known that such exchange between partner molecules (i.e., metallochaperones and metal pumps) is fast.     

Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding

Monoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins.     

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.     

Transforming properties of the cottontail rabbit papillomavirus oncoproteins Le6 and SE6 and of the E8 protein.

Cottontail rabbit papillomavirus induces on cottontail and domestic rabbits papillomas which progress at a high frequency to carcinoma. The virus encodes three transforming proteins; one is translated from open reading frame (ORF) E7 and binds the retinoblastoma protein, and two, LE6 and SE6, are translated from the first and second ATGs of ORF E6, respectively. Here we show that neither of the E6 proteins coprecipitated with p53 in vitro, nor did they bind to a recently identified E6-binding protein (J. J. Chen, C. E. Reid, V. Band, and E. Androphy, Science 269:529-531, 1995). This protein was shown to bind to the E6 proteins of the high-risk human papillomairus types 16 and 18 but not to the low-risk human papillomavirus types VI and II. In-frame deletions cloned into the pZipNeo vector were used to identify structural features of SE6 and LE6 important for transformation of NIH 3T3 cells. Three deletions covering the amino-terminal half of SE6 did not transform cells. In two of the three deletions, two Cys-X-X-Cys motifs were deleted, each deletion preventing the formation of one of the potential small Zn fingers of SE6. Among the LE6 deletions, only one had a reduced transformation efficiency, while seven transformed cells at least as efficiently as wild-type LE6. In each of three of these seven mutants, two Cys-X-X-Cys motifs were deleted. None of the three amino acid deletions which abolished transformation by SE6 reduced transformation by LE6. Furthermore, transformation did not correlate with the level of SE6 or LE6 proteins detectable. ORF E8 colinear with ORF E6, which could generate a 50-amino-acid protein with a hydrophobic segment, did not transform cells when cloned into the pZipNeo vector. However, mutation of the E8 ATG, which did not alter the amino acid sequence of LE6, increased transformation by LE6 without affecting the level of LE6 expression. The data suggest that transformation by the E6 proteins is not mediated by interfering with p53 function or through binding to the E6-binding protein. Furthermore, different structural features are important to maintain transformation functions and protein stability of LE6 and SE6. Finally, E8 seems not to be a transforming protein but rather appears to modulate transformation bv LE6.