Targeting mechanism of the retinoblastoma tumor suppressor by a prototypical viral oncoprotein Structural modularity, intrinsic disorder and phosphorylation of human papillomavirus E7 Lucı ´ a B. Chemes, Ignacio E. Sa ´ nchez, Clara Smal and Gonzalo de Prat-Gay Protein Structure-Function and Engineering Laboratory, Fundacio ´ n Instituto Leloir and IIBBA-CONICET, Buenos Aires, Argentina Keywords LxCxE motif; natively unfolded proteins; phosphorylation; retinoblastoma protein; viral oncoprotein Correspondence Gonzalo de Prat-Gay, Protein Structure- Function and Engineering Laboratory, Fundacio ´ n Instituto Leloir and IIBBA-CONICET, Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina Fax: +54 11 5238 7501 Tel: +54 11 5238 7500 ext. 3209 E-mail: gpg@leloir.org.ar (Received 16 November 2009, revised 4 December 2009, accepted 7 December 2009) doi:10.1111/j.1742-4658.2009.07540.x DNA tumor viruses ensure genome amplification by hijacking the cellular replication machinery and forcing infected cells to enter the S phase. The retinoblastoma (Rb) protein controls the G1 ⁄ S checkpoint, and is targeted by several viral oncoproteins, among these the E7 protein from human papillomaviruses (HPVs). A quantitative investigation of the interaction mechanism between the HPV16 E7 protein and the RbAB domain in solu- tion revealed that 90% of the binding energy is determined by the LxCxE motif, with an additional binding determinant (1.0 kcalÆmol )1 ) located in the C-terminal domain of E7, establishing a dual-contact mode. The stoichiometry and subnanomolar affinity of E7 indicated that it can bind RbAB as a monomer. The low-risk HPV11 E7 protein bound 2.0 kcalÆmol )1 more weakly than the high-risk HPV16 and HPV18 type counterparts, but the modularity and binding mode were conserved. Phos- phorylation at a conserved casein kinase II site in the natively unfolded N-terminal domain of E7 affected the local conformation by increasing the polyproline II content and stabilizing an extended conformation, which allowed for a tighter interaction with the Rb protein. Thus, the E7–RbAB interaction involves multiple motifs within the N-terminal domain of E7 and at least two conserved interaction surfaces in RbAB. We discussed a mechanistic model of the interaction of the Rb protein with a viral target in solution, integrated with structural data and the analysis of other cellu- lar and viral proteins, which provided information about the balance of interactions involving the Rb protein and how these determine the progres- sion into either the normal cell cycle or transformation. Structured digital abstract l MINT-7383794, MINT-7383812, MINT-7383830, MINT-7383868, MINT-7383891, MINT- 7384056: E7 (uniprotkb:P03129) and Rb (uniprotkb:P06400) bind (MI:0407)byfluorescence technologies ( MI:0051) l MINT-7383923: E7 (uniprotkb:P04020) and Rb (uniprotkb:P06400) bind (MI:0407)bycom- petition binding ( MI:0405) Abbreviations AdE1A, adenovirus E1A; BPVN, N-terminal fragment of the BPV1 E7 protein; CKII, casein kinase II; CR1, conserved region 1; CR2, conserved region 2; CtIP, transcriptional corepressor CtBP-interacting protein; E7(16-40)PP, a synthetic E7(16-40) peptide phosphorylated at serine residues 31 and 32; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; HDAC, histone deacetylase; HPV, human papillomavirus; IPTG, isopropyl thio-b- D-galactoside; MBP, maltose-binding protein; PII, polyproline type II; Rb, retinoblastoma; SV40LT, SV40 large T antigen; TA, transactivation region; TFE, 1,1,1, trifluoroethanol. FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS 973 Introduction The retinoblastoma tumor suppressor gene (RB1) was first identified as the causative agent whose loss resulted in retinoblastoma, a heritable disease of pedi- atric relevance [1]. To date, over 500 distinct muta- tions in the RB1 gene have been identified in retinoblastoma tumors, 50 of which are missense mutations [2,3]. The tumor suppressor function of the Rb protein is underscored by its mutation in a broad range of human tumors [4]. The most extensively studied function of the Rb protein is in the control of cell cycle progression at the G1 ⁄ S boundary, medi- ated through its interaction with the E2F family of transcription factors [5]. The Rb protein also plays important roles in chromatin remodeling, develop- ment, differentiation and apoptosis [6]. These multiple functions are mediated by over 100 interactions with different protein partners that are dependent on the cell type, and on the developmental and cell cycle stages [7]. The Rb protein has a molecular mass of 105 kDa and is composed of three domains. Both the N-terminal and the AB (RbAB) domains consist of a double cyclin fold [8,9], while the C-terminal domain (RbC) appears to be natively unfolded [10]. The function of the N-ter- minal domain is still poorly defined. The RbAB domain mediates transcriptional repression and, together with the C-terminal domain (RbC), promotes growth arrest [11,12]. Most interacting partners contact more than one structural domain in the Rb protein [13–15]. For example, the ‘transactivation’ domain of E2F (E2F-TA) binds to RbAB, whereas the ‘marked box’ domain (E2F-MB) binds to RbC [10,16]. Moreover, there are at least two distinct highly conserved ligand-binding sites within the RbAB domain [8] (Figs 1 and S1). Cellular proteins containing an LxCxE motif interact with a site located on the B subdomain of RbAB [8,17] (Fig. 1A,B). The E2F-TA domains bind to a site located at the cleft between the A and B subdomains on the opposite side of RbAB [16,18] (Fig. 1C,D). Early evidence for the tumor suppressor role of the Rb protein came from the mechanism of action of the human papillomavirus (HPV) E7 major transforming protein [19]. The interaction between E7 and the Rb protein is required for the induction and maintenance of the transformed state of human keratinocytes [20]. Deregulation of E7 expression upon integration of the HPV genome is believed to play a role in HPV-medi- ated oncogenesis. The DNA tumor virus proteins SV40 large T antigen (SV40LT) and adenovirus E1A (AdE1A) also target the Rb protein and share sequence and functional conservation with the HPV E7 protein [21,22]. E7, AdE1A and SV40LT each con- tain several functional and structural domains, each of which mediates interactions with different cellular tar- gets. The three transforming proteins share conserved region 2 (CR2); E7 and AdE1A also share conserved region 1 (CR1). E7 is a small ($ 100 amino acids) protein composed of two structural domains. We have previously deter- mined that the N-terminal domain (E7N) is natively unfolded [23,24], includes CR1 and CR2, and contains dynamic elements of helical and polyproline type II (PII) secondary structure [23]. The globular C-terminal domain (E7C) constitutes conserved region 3 (CR3) and is responsible for protein dimerization and zinc binding [24,25] (Fig. 2A). While the CR1 and CR2 domains are required for Rb protein degradation, all conserved E7 regions participate in transformation [26,27]. E7 can also oligomerize in vitro and in vivo [28–30]. The conformational diversity of E7 may be an evolved trait that allows for multiple modes of pro- tein–protein interaction [31,32]. E7 binds to two structural domains in the Rb protein, namely the RbAB and RbC domains. Binding to both domains is required for E2F displacement [33]. The LxCxE motif within the CR2 region of E7 mediates high-affinity binding to the RbAB domain [8,34] (Fig. 1A), while the isolated E7C binds to the RbC domain with micromolar affinity [25,35]. The crystal structure of the LxCxE–RbAB complex reveals that the motif binds to a conserved shallow groove of the B subdomain in an extended conformation (Fig. 1A). The LxCxE motif is followed in E7 CR2 by two conserved serine residues (S31 and S32) and by a l MINT-7383777, MINT-7384078, MINT-7383848, MINT-7384113, MINT-7384096: Rb (uni- protkb: P06400) and E7 (uniprotkb:P03129) bind (MI:0407)bycompetition binding (MI:0405) l MINT-7383963: Rb (uniprotkb:P06400) and E7 (uniprotkb:P06788) bind (MI:0407)bycom- petition binding ( MI:0405) l MINT-7384022, MINT-7384040: E7 (uniprotkb:P03129) and Rb (uniprotkb:P06400) bind ( MI:0407)bycomigration in non denaturing gel electrophoresis (MI:0404) l MINT-7384004, MINT-7383984: Rb (uniprotkb:P06400) binds (MI:0407)toE7 (uni- protkb: P03129)bypull down (MI:0096) Viral targeting of the retinoblastoma protein L. B. Chemes et al. 974 FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS stretch of acidic amino acids, and HPV16 E7 is phosphorylated at S31 and S32 by casein kinase II (CKII) in vitro and in vivo [36,37]. Phosphorylation is required for E7 function, and cell culture assays have suggested that phosphorylation modulates the strength of the E7–RbAB interaction, but this proposal remains a matter of debate [37–40]. Indirect evidence suggests that other regions in E7 may contribute to binding to the RbAB domain. For example, mutagenesis of a conserved surface patch in the A subdomain of the RbAB domain (Fig. 1A,B, right) produces a protein capable of arresting the cell cycle of HeLa cells, implying that this protein was resis- tant to E7 inactivation [41]. It is currently unclear whether E7 interacts directly with this surface. Similarly, an E7 construct, encompassing the CR2 and CR3 domains of E7, bound to the RbAB domain more tightly than a CR2 construct and was able to debilitate the E2F–RbAB interaction [16]. Finally, E7 CR1 has been shown to contribute to E2F displacement in com- bination with CR2 [27]. This E7 region shares a high degree of sequence similarity to the AdE1A CR1 region and can functionally complement it [42]. The AdE1A CR1 region binds to the RbAB domain at a site that overlaps with the E2F-TA-binding site [43] (Fig. 1D), leading to disruption of the E2F–Rb complex, but an interaction between E7 CR1 and the RbAB domain has not been demonstrated to date. Mechanistic aspects and structure–function relation- ships for the Rb protein remain ill defined [17], in con- trast to those for other well-known tumor suppressors or oncogenes, such as p53 [44] or Ras [45]. A complete understanding of the Rb protein function requires the dissection of all functional surfaces, along with their partners and the strength and mechanism of interac- tion [46]. We have dissected individual contact sites and their energetic contribution to the E7–RbAB com- plex, using solution-based measurements of binding affinity at equilibrium. This mechanistic and thermody- namic picture of the complex formed by RbAB and E7 paves the way for a better understanding of the Rb cellular complexes that control the cell cycle through- out eukaryotes and their deregulation in HPV infection and oncogenesis. Results Quantitative dissection of the E7–RbAB interaction in solution The minimal region required for the interaction between the HPV16 E7 protein and the RbAB pocket has previously been mapped to residues 21-29 of E7, A B C D Fig. 1. Conserved surface features of the RbAB domain. Conserva- tion scores were calculated using the alignment of the RbAB domain from 46 vertebrate species and CONSURF [74], and figures were gen- erated using PYMOL [75]. Structures correspond to the following com- plexes: (A) RbAB ⁄ E7 (PDB ID: 1GUX); (B) RbAB ⁄ SV40-LT (PDB ID: 1GH6); (C) RbAB ⁄ E2F-TA (PDB ID: 1N4M); and (D) RbAB ⁄ E1A-CR1 (PDB ID: 2R7G). Asterisk: H549Y missense mutation [54]. Arrows indicate the rotation of the molecule along the x-axis between two consecutive images. The color scale indicates the residue conserva- tion score, as calculated using the CONSURF algorithm. L. B. Chemes et al. Viral targeting of the retinoblastoma protein FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS 975 containing the LxCxE motif [8]. The dissociation con- stant for this interaction was shown, by isothermal titration calorimetry, to be 190 nm [8,34], but the con- tribution of other regions of the E7 protein to the affinity of the E7–RbAB complex has not been explored in detail. In order to address this issue, we developed a solution-based assay that allowed us to perform quantitative and accurate determinations of stoichiometry and binding affinity at equilibrium, by measuring the fluorescence anisotropy change upon the binding of fluorescein isothiocyanate (FITC)-labeled E7 fragments to RbAB. This assay was used to mea- sure the binding of different fragments of E7 (corre- sponding to well-defined structural and functional domains and to highly conserved sequence motifs) to RbAB. Figure 2A shows the E7 regions tested. A representative example of the assay is presented in Fig. 2B,C, which show the association of E7N [E7(1- 40)] with RbAB. First, the stoichiometry of the reac- tions was determined by performing titrations at a high peptide concentration (Figs 2B and S2). The anisotropy signal increased linearly up to a 1 : 1 molar ratio, where it reached a constant value indicating the saturation of all binding sites. This implies that there is one binding site for the E7(1-40) peptide per RbAB monomer and that the stoichiometry of the E7(1-40)– RbAB interaction is 1 : 1. Far-UV CD spectra of the complexes formed by binding of the RbAB domain to full-length E7, and to E7(1-40) and E7(40-98) peptides, revealed that formation of the complex does not induce significant structural changes in the secondary structure of the interacting proteins (data not shown). Figure 2C shows one representative binding curve per- formed at substoichiometric concentrations, and the residuals of the fit from which the K D value was calcu- lated. We tested the association of the RbAB domain with a 43-residue N-terminal fragment of the BPV1 E7 protein (BPVN), which does not contain an LxCxE motif. This interaction had marginal affinity, which was approximately 10 6 times lower than that of the full-length E7 protein (Table 1). Figure 2D summarizes all binding curves and shows the dynamic range of the assay, which allowed us to accurately determine subn- anomolar to micromolar dissociation constants. The E7(21-29) peptide, comprising the minimal LxCxE motif (DLYCYEQLN) [8], associated with RbAB with a K D of 4.7 ± 1.7 nm (Table 1), and the free energy of binding for this interaction was DG = A BCD Fig. 2. Interaction of different E7 fragments with the RbAB domain. (A) Scheme of HPV16 E7. The positions of conserved regions 1, 2 and 3 (CR1, CR2 and CR3) and the E7 fragments used in this study are shown; the LxCxE motif is underlined. Boxes denote the regions con- tained in each fragment: black, LxCxE motif; dark grey, CKII ⁄ PEST motif; light grey, CR1 helix-forming residues. Circles denote the position of FITC moieties. (B) Association of E7(1-40) and RbAB at 200 n M E7(1-40). (C) Association of E7(1-40) and the RbAB domain at 5 nM E7(1- 40). A fit to a 1 : 1 binding model and residuals are shown. The anisotropy value of the free peptide was 0.054 ± 0.001 and the anisotropy of the complex was 0.124 ± 0.001, indicating that no oligomerization occurred in this binding regime [76]. (D) Representative normalized binding curves for the different E7 fragments (symbols are as shown in panel A). Viral targeting of the retinoblastoma protein L. B. Chemes et al. 976 FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS )11.2 ± 0.2 kcalÆmol )1 . The E7(16-31) and E7(16-40) peptides, which contain the LxCxE motif plus addi- tional neighboring sequences from the CR2 region, and the E7(1-40) peptide, which comprises both CR1 and CR2, had the same affinity for the RbAB domain as the E7(21-29) peptide (Table 1). The full-length HPV16 E7 protein bound to the RbAB domain with a ten-fold increased affinity when compared with the E7(21-29) peptide (K D = 0.6 ± 0.3 nm), and the free energy of binding for the interaction between full- length E7 and the RbAB domain was DG = )12.4 ± 0.3 kcalÆmol )1 (Table 1). Therefore, our data show that the LxCxE motif contributes about 90% of the total binding energy for the HPV16 E7–RbAB interac- tion, providing quantitative support to previous results [47]. The CR1 region does not appear to contribute to RbAB binding within the context of an E7N mono- mer, as shown by the fact that the E7(16-40) and E7(1-40) peptides have the same affinity for the RbAB domain (Table 1). Finally, we showed that the E7 C-terminal domain contributes 1.0 ± 0.4 kcalÆmol )1 to the total free energy of binding, enhancing the affinity of the E7–RbAB complex by ten-fold. Previous semiquantitative assays have established that E7 proteins from HPV types highly associated with the development of cervical cancer (HPV16 and HPV18) bind to the full-length Rb protein more strongly than E7 proteins from HPV types associated with benign lesions (HPV11 and HPV6) [48]. In order to explore whether similar regions determine the affinity for RbAB in E7 proteins from high-risk and low-risk HPV types, we used a competition assay to measure the association between the RbAB domain and the E7 proteins from HPV types 16, 18 and 11. We assembled a stoichiometric complex of RbAB and FITC-labeled HPV16 E7 or E7(16-31) and displaced labeled E7 with each of the different full-length pro- teins or N-terminal domains (Fig. 3A and Table 2). The HPV11 E7 protein associated with the RbAB domain 2.0 kcalÆmol )1 more weakly than the high-risk HPV16 and HPV18 type counterparts, providing quan- titative support to previous reports [48]. The N-termi- nal domain was the main contributor to the binding affinity of E7 from HPV11, HPV16 and HPV18 for the RbAB domain (Fig. 3B), pointing to a conserved mode of interaction. Phosphorylation of the conserved CKII sites within the E7 CR2 region increases affinity for RbAB The sequences C-terminal to the LxCxE motif in HPV16 E7 contain two serine residues, S31 and S32, which are phosphorylated in vitro and in vivo by CKII [36,37]. These serine residues are followed by a stretch of acidic amino acids that constitute an S ⁄ TxxD ⁄ E CKII consensus site. The PESTfind algorithm suggests Table 1. Determination of binding affinities for the E7–RbAB complex. The K D was calculated by fitting three to five independent binding curves to a 1 : 1 binding model, as described in the Materials and methods. Fragment K D (nM) DG a (kcalÆmol )1 ) E7 b 0.6 ± 0.3 )12.4 ± 0.3 E7(21-29) (LXCXE) b 4.7 ± 1.7 )11.2 ± 0.2 E7(16-31) (LXCXE) b 5.1 ± 1.3 )11.1 ± 0.3 E7(16-40) (CR2) b 6.5 ± 1.0 )11.0 ± 0.3 E7(16-40)PP (CR2PP) b 1.8 ± 0.4 )11.7 ± 0.1 E7(1-40) (E7N) b 3.0 ± 1.6 )11.4 ± 0.3 E7(1-20) (CR1) 19000 ± 2000 )6.3 ± 0.1 E7(40-98) (E7C) 2700 ± 600 )7.5 ± 0.1 BPV(1-43) (BPV-N) > 400 000 – a DG was calculated as DG = )RT *ln(K D ), with RT = 0.582 kcalÆmol )1 . b The stoichiometry for these complexes was deter- mined to be 1 : 1 by titrations performed at peptide concentrations at least 10 times greater than the determined K D . A B Fig. 3. The LxCxE motif is the main determinant of binding affinity in HPV-E7 proteins. (A) Competition experiments with full-length E7 proteins and a preformed complex of 5 n M RbAB and 5 nM FITC-HPV16-E7 protein. Competitor proteins were: BPV-Nter (s); HPV11-E7 ( ); HPV18-E7 ( ); and HPV16-E7 (d). (B) Comparison of DG values for different E7 full-length proteins (solid bars) and N-terminal domains (hatched bars). Data are from Table 2. L. B. Chemes et al. Viral targeting of the retinoblastoma protein FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS 977 that this site overlaps with a PEST degradation motif [49]. Figure 4A shows the sequence of the HPV16 E7 CR2 region, indicating the relative positions of the LxCxE motif, the phosphorylatable serine residues and the CKII ⁄ PEST region within CR2. Aligned below this sequence is a sequence logo created from the alignment of all 56 E7 proteins from genital HPV types (Fig. S3). The sequence logo clearly shows that serine residues are nearly as conserved as the LxCxE motif. Inspection of individual sequences revealed that all 56 E7 proteins present at least one CKII consensus site between positions 30 and 34. This region also contained a high proportion of negatively charged amino acids (D⁄ E), with 97% of sequences presenting a net charge that was equal to or lower than -6. The striking conservation of sequence features within the CR2 region of E7 underscores the impor- tance of this region for E7-mediated transformation. The CKII ⁄ PEST region of E7 and its phosphorylation have been postulated to play a role in the E7–Rb protein interaction. Here, we directly tested this hypothesis by comparing binding to the RbAB domain for E7(16-40) and for a synthetic E7(16-40) peptide phosphorylated at serine residues 31 and 32 [E7(16- 40)PP]. Phosphorylation increased the affinity four- fold (Table 1). The difference in free energy of binding of both peptides, DDG = )0.7 ± 0.3 kcalÆmol )1 , was significant across repeated assays. We further validated the data by carrying out competition experiments, where a stoichiometric complex of FITC-labeled E7(16-31) and the RbAB domain was titrated with increasing amounts of unlabeled E7(16-40) or E7(16- 40)PP peptides. Competition experiments (Fig. 4B,C) confirmed a positive contribution of phosphorylation to RbAB-binding affinity. The difference in free A BC Fig. 4. Phosphorylation of the E7 CR2 region increases the affinity for the RbAB domain. (A) Conservation of sequence features within E7 CR2. Upper panel: sequence of the HPV16 E7(16-40) peptide. The LxCxE motif is underlined, and the position of phosphoryl serine residues and the CKII ⁄ PEST consensus are marked. Lower panel: sequence logo of the CR2 region from genital E7 proteins. The height of the stack of letters at each position denotes the level of conservation (the maximum value is 4.32), while the relative proportions of each residue rep- resents the relative abundance. (B) Competition experiments with CR2 peptides and a preformed complex of 25 n M RbAB and the 25 nM FITC–E7(16-31) peptide. Competitor peptides were: E7(16-31) (d), E7(16-40) (.), E7(16-40)PP (s) and BPVN ()). (C) Comparison of DG val- ues for the E7(16-40) and E7(16-40)PP peptides with those for the E7(16-31) peptide. Data are from Table 1 and from panel B. Table 2. The LxCxE motif determines binding affinity in distantly related HPV E7 proteins. Fragment K D (nM) DG a (kcalÆmol )1 ) DDG b (kcalÆmol )1 ) Full-length protein HPV16 E7 2.4 ± 0.2 )11.6 ± 0.05 – HPV18 E7 7.8 ± 0.5 )10.9 ± 0.04 0.7 ± 0.06 HPV11 E7 108 ± 5 )9.3 ± 0.03 2.3 ± 0.06 N-terminal domain HPV16 E7 8.6 ± 1.3 )10.8 ± 0.09 – HPV18 E7 12.2 ± 0.8 )10.6 ± 0.04 0.2 ± 0.1 HPV11 E7 366 ± 25 )8.6 ± 0.04 2.2 ± 0.1 a DG = )RT*ln(K D ), with RT = 0.582 kcalÆmol )1 . b DDG was calcu- lated as DDG = DG ) DG E716 . Viral targeting of the retinoblastoma protein L. B. Chemes et al. 978 FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS energy of binding from competition experiments was DDG = )1.4 ± 0.2 kcalÆmol )1 , in agreement with the direct binding data. Our data demonstrated that phosphorylation of the CKII ⁄ PEST region contributes significantly to the RbAB–E7 interaction, enhancing the affinity by fourfold to 10-fold. Structural correlates of E7 phosphorylation at the CKII sites We have previously shown that E7N is an extended bona fide structural domain, with regions of dynamic residual secondary structure in solution. Far-UV CD analyses showed that HPV16 E7(1-40) displayed an extended PII structure, which was stabilized by phosphorylation of serine residues S31 and S32 [23]. We tested the E7 CR2 region for PII content by measuring the far-UV CD spectra of the E7(16-40) and the E7(16-40)PP peptides at 5 °C. Both peptides presented a CD spectrum charac- teristic for a disordered polypeptide with a positive band at 218 nm, which is characteristic of the PII conforma- tion (Fig. 5A). PII conformations are sensitive to tem- perature, with higher temperatures decreasing the intensity of the 218 and 198 nm peaks. Increasing the temperature to 85 °C decreased the intensity of both peaks for both peptides, characteristic for the disruption of the PII structure (Fig. 5A). The difference spectra (5– 85 °C) clearly showed the induction at 5 °C of the 218 nm peak (Fig. 5A, inset). The denaturant GdmCl is known to stabilize PII structures [50]. We have previ- ously shown that the stability of PII conformations can be estimated from GdmCl titrations, by validating changes in the CD spectra with NMR measurements of PII structure [51]. GdmCl increased the 218 nm band in the E7(16-40)PP peptide, but not in the E7(16-40) pep- tide (Fig. 5B), suggesting that the E7(16-40)PP peptide has a higher propensity for PII structure. The titration of the E7(16-40)PP peptide with GdmCl is shown in Fig. 5C, along with a fit of the data to a two-state coil- PII model. The calculated free energy for the coil-PII equilibrium in 0 m GdmCl is 1.7 ± 0.7 kcalÆmol )1 , which corresponds to 4.6 ± 6% of the PII population in the absence of denaturant. Although the model used is a crude estimate of the true conformational equilibria of the peptide, and the estimated parameters have high errors as a result of noise in the measurements, the GdmCl titration data clearly show that the E7(16-40)PP peptide is in equilibrium between coil and PII conforma- tions. Overall, our data indicate that both peptides from the HPV16 E7 CR2 region present residual PII structure in equilibrium with disordered conformations. GdmCl titrations strongly suggest that phosphorylation modu- lates the coil–PII equilibrium, increasing the PII propen- sity of the E7 CR2 region. The E7 C-terminal domain binds independently to RbAB The increased affinity of the full-length E7 protein compared with the E7 N-terminal domain suggested that additional regions within the E7 C-terminal domain contribute to association with the RbAB domain. In order to test for a direct interaction between E7C and RbAB, we performed a pull-down assay with recombinant purified proteins by forming a stoichiometric complex of His-tagged RbAB with E7 and E7C (Fig. 6A). Most of the full-length E7 protein (96%), and a fraction of the E7 C-terminal domain (23%), bound to RbAB at a concentration of 10 lm. These results confirmed a direct association of the RbAB domain with both E7 and E7C, and suggested that the E7C–RbAB interaction was weaker than the Fig. 5. Phosphorylation increases the PII content of the E7 CR2 region. (A) Far-UV CD spectra of the E7(16-40) (solid line) and the E7(16- 40)PP (dashed line) peptides, performed at 5 °C and 85 °C. Inset: difference spectra (5–85 °C) for the E7(16-40) (solid line) and the E7(16- 40)PP (dashed line) peptides. (B) CD spectra of E7(16-40) and E7(16-40)PP between 0 and 6 M GdmCl. Titration points graphed are [GdmCl] = 0, 1.2, 1.9, 2.4, 3.2, 3.7, 4.9 and 5.9 M. The curves corresponding to 0 and 5.9 M GdmCl are shown in bold. (C) GdmCl titration of the E7(16-40)PP peptide. Data were fit to a two-state coil-PII equilibrium (DG H2O E7(16-40)PP = 1.7 ± 0.7 kcalÆmol )1 ; m = 0.44 ± 0.22 kcal mol )1 ÆM )1 ). L. B. Chemes et al. Viral targeting of the retinoblastoma protein FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS 979 interaction of the full-length E7 protein with the RbAB domain. Direct titration showed that the E7C– RbAB complex had a dissociation constant of 2.7 ± 0.6 lm (Table 1, Fig. 6B). Titration with BPVN E7 yielded a dissociation constant of 400 lm or higher, supporting the specificity of the E7C–RbAB interac- tion. A peptide containing the CR2 region of E7 did not compete with E7C binding, indicating that E7C does not bind to the RbAB domain at the LxCxE- binding cleft (Table 1, Fig. 6B). The E7 CR1 region can form an alpha helix and binds independently to RbAB The CR1 region from E1A binds to the RbAB domain with micromolar affinity (K D =1lm) [43] at the interface between the A and B subdomains, which is also the binding site for E2F-TA (Fig. 1C,D). The fact that the E7 and AdE1A CR1 regions have similar functional properties [42] suggests that E7 CR1 might also bind to the RbAB domain at the E2F-TA-binding site. E1A CR1 and E2F-TA form a six-residue helix in the bound conformation (Fig. 1C, residues boxed in Fig. 7A) [16,18,43]. Four AdE1A residues that estab- lish intermolecular contacts with the RbAB domain (P41, L43, H44 and L49), and two residues that stabi- lize the helix by an intramolecular hydrogen bond (T42 and E45) [43], are conserved in E7 CR1 (E7 residues 6-10 and 15; Fig. 7A). Furthermore, the AGADIR algorithm [52] suggests that E7 residues 6 to 15 have local helical propensity (data not shown). We tested whether the E7 CR1 region could form an a-helix in solution by measuring the far-UV CD spec- trum of E7(1-20) in the presence of 1,1,1, trifluoroetha- nol (TFE), which is known to stabilize helical conformations in peptides [53]. The addition of 60% TFE induced an a-helix structure in E7(1-20) (Fig. 7B and inset). A fit of the TFE titration data to a two- state coil-helix model yielded a free energy for a-helix formation in 0% TFE of 1.3 ± 0.2 kcalÆmol )1 , corre- sponding to a residual a-helix population of 10 ± 4% in the absence of cosolvent. These results show that the E7 and E1A CR1 regions have similar conforma- tional properties. We tested for the association between E7 CR1 and the RbAB domain using three different approaches. First, we used nondenaturing PAGE and FITC-labeled E7 peptides to test for complex formation (Fig. 7C). As a positive control, we tested the association between FITC-labeled E7(1-40) and the RbAB domain, and as a test for the specificity of the interac- tion, we used ovalbumin in place of RbAB. Both E7(1-40) and E7(1-20) formed a complex with RbAB but not with the control protein ovalbumin, confirming the specificity of the interactions (Fig. 7C). A pull- down assay, similar to that performed with E7C, did not show significant interaction (data not shown), sug- gesting that the E7(1-20)–RbAB complex has a lower affinity than the E7C–RbAB complex. Fluorescence titration gives a dissociation constant of 19 ± 1 lm for the E7(1-20)–RbAB complex (Fig. 7D and Table 1). Titration with BPVN yielded a dissociation constant of 400 lm or higher. The 20-fold higher affin- ity for the E7(1-20)–RbAB complex supports the speci- ficity of the interaction. Peptides containing the CR2 region did not compete for the E7(1-20)–RbAB inter- action (Fig. 7C), which indicates that E7(1-20) does not bind RbAB at the LxCxE-binding site. A B Fig. 6. E7C binds independently to the RbAB domain. (A) Pull-down assay for the RbAB–E7C interaction. His-RbAB was incu- bated with E7 (lanes 3-4) or with E7C (lanes 7-8). Lanes 1-2 and 5-6: control experiments excluding His-RbAB. The labels to the left of the gel indicate the position of each protein. % E7: percentage of E7 or E7C protein in the bound (B) and unbound (U) fractions, as quantified by densitometry (see the Materials and methods). (B) Binding of E7C to the RbAB domain in solution. Titrations were per- formed at 1 l M FITC-E7C; the titrant was RbAB (d, K D = 4.8 ± 0.5 l M), RbAB-E7(16-40) (s, K D = 6.4 ± 0.9 lM). A control experiment was performed using 5 l M FITC-BPVN (4, K D > 400 lM). Viral targeting of the retinoblastoma protein L. B. Chemes et al. 980 FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS Discussion Despite its vast importance as the guardian of the cell cycle and its clinical relevance in human cancers, struc- tural and thermodynamic understanding of the mecha- nisms of action of the Rb protein is far behind that of p53, the keeper of the genome, mutated in most can- cers and targeted by the same DNA tumor viruses that target the Rb protein [44]. In this work, we set out to investigate the interaction mechanism of the RbAB pocket domain with one of the paradigmatic viral oncoproteins, HPV E7, which targets it for degrada- tion. Precise quantitative assessment of Rb protein interactions is fundamental for understanding viral- mediated subversion of cell cycle control and allows novel shared features of viral and cellular Rb protein interaction partners to be uncovered. We measured the contribution of the LxCxE motif of E7 to be 90% of the total binding free-energy, and showed that this motif is also the main determinant of binding for E7 proteins from three prototypical HPV types (Figs 2 and 3). The free energy of binding for full-length HPV16 E7 was 1.0 kcalÆmol )1 higher than that of the E7N domain, revealing that the E7C domain contributes a 10-fold increase in affinity through a dual-contact mode of interaction. Careful examination of conserved surface patches in the RbAB domain suggests a putative binding site for E7C, located in the RbA domain close to the AB cleft (Rb residues E492, F514, P515, K548 and H549; Fig. 1A,B, right). This site is nearly as conserved as the LxCxE cleft, the lysine-rich patch and the E2F-binding site [8], and a tumorigenic missense mutation, H549Y, has been described at this surface (Fig. 1A,B, asterisk) [3,54], which strongly suggests that this is an important functional surface in the RbAB domain for which cellular binding partners are likely to be described in the future [7]. Mutations in this region affect cell cycle regulation by E7 [41], suggesting that E7 may bind at this interaction site and displace Rb protein cellular targets. The viral transforming proteins AdE1A and SV40LT, in addition to nine cellular protein targets of Rb [17] [histone deacetylase (HDAC)1, HDAC2, tran- scriptional corepressor CtBP-interacting protein (CtIP), 95kDa retinoblastoma-associated protein (RBP95), ETS-related transcription factor 1 (Elf1), HMG Box transcription factor 1 (HBP1), kinetochore protein Hec1 (Hec1), RBP1 and replication factor C subunit 1 (RFC1)], present a putative serine-phosphorylation site following the LxCxE motif (Fig. 8). In addition, in vivo phosphorylation of the AdE1A, SV40-LT and HDAC sites has functional consequences [55–57]. In HPV E7, A B C D Fig. 7. The E7 CR1 region forms an a-helix and interacts with the RbAB domain. (A) Alignment of the E7 CR1 region with the E1A CR1 and E2F1-TA RbAB-binding sites. Bold: residues in AdE1A involved in complex formation with RbAB and conserved in E7. Asterisks: resi- dues of E1A and E2F1 involved in the RbAB-binding a-helix [16,43]. (B) TFE titration of the E7(1-20) peptide. Data were fit to a two- state helix-coil transition model (DG H2O = 1.3 ± 0.2 kcalÆmol )1 ; m = 25 ± 3 kcal mol )1 ÆM )1 ). Inset: difference spectrum (60–0% TFE) showing the conformation induced by TFE addition. (C) Interaction between E7(1-20) and the RbAB domain, determined using nondena- turing PAGE. Arrows mark the position of peptides ⁄ complexes: 1 = free peptide, 2 = E7(1-40)–RbAB complex, 3 = E7(1-20)–RbAB complex. (D) Interaction between E7(1-20) and RbAB in solution. Experiments were performed at 5 l M E7(1-20). The titrants were RbAB (d, K D =19±1lM; Table 1), RbAB–E7(16-40) ( , K D = 26 ± 1 l M) and RbAB–E7(1-40) (h, K D =30±2lM). A control exper- iment was performed using 5 l M FITC-BPVN (4, K D > 400 lM). L. B. Chemes et al. Viral targeting of the retinoblastoma protein FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS 981 phosphorylation is essential for S-phase re-entry of dif- ferentiating keratinocytes in organotypic raft models [39,40] and contributes to E7-mediated transformation [37]. Our results offer the first molecular insight into the functional role of E7 phosphorylation, by provid- ing direct evidence that phosphorylation of serines 31 and 32 of HPV16 E7 increases affinity for the RbAB domain (Fig. 4). The E7 region surrounding these resi- dues is natively unfolded [23] and presents a high den- sity of negative charge, which may interact with a conserved lysine-rich patch contiguous to the LxCxE cleft [41,58]. We showed that phosphorylation affects the local conformation of the E7(16-40) fragment, increasing the PII content of this region and stabilizing an extended conformation that optimizes binding to the LxCxE cleft (Fig. 5). PII-coil transitions induced by phosphorylation in a similar natively unfolded PEST region can modulate the stability of a protein to intracellular degradation [51], which could also be the case for the E7 oncoprotein. The isolated E7 CR1 region is able to bind to the RbAB domain in vitro with measurable affinity, pos- sibly undergoing a coil-to-helix transition (Fig. 7). In the AdE1A protein, the 70-residue spacer between the CR1 and CR2 regions allows for the simulta- neous binding of both motifs at opposite sides of the same RbAB molecule (Fig. 1A,D) [59]. Our data clearly show that the E7(16-40) and E7(1-40) pep- tides have the same affinity for the RbAB domain (Table 1). This result implies that the HPV E7 CR1 region does not contribute to binding when CR2 is present, which is probably because of the short eight-residue spacer separating both binding motifs. In a complex between the Rb protein and the weak E7 dimer [29], the CR2 region of one E7 molecule may bind to the LxCxE cleft, while the CR1 region of the other E7 molecule binds to the E2F site of an RbAB monomer. This mode of interaction may cooperate in the displacement of E2F, as previously suggested [25,27]. Our results highlight the modular nature of E7 and its interaction with the RbAB domain (Fig. 8, top). It has long been recognized that AdE1A and SV40LT also present multiple interaction modules that bind to different Rb protein domains [21,22,59]. This is also a feature of prototypical Rb protein interacting part- ners, such as E2F1, HDAC, CtIP and EP300 interact- ing inhibitor of differentiation 1 (EID-1) (Fig. 8, bottom). The three secondary sites in E7 (E7C, CR1 and the CKII ⁄ PEST region) contribute far less than expected from their binding energy in isolation (this work), which suggests that their main role is to finely tune affinity and to target multiple interaction sur- faces of the RbAB domain. It will be interesting to investigate how the action of these modules is inte- grated with other known E7 interaction sites within Fig. 8. Interaction modules and affinities of viral and cellular Rb protein targets. Proteins and affinities reported are from: HPV-E7 [8,25,34,62] and, from this work, AdE1A [15,43,57], SV40LT [22,34,56], E2F1 [10,16,18], HDAC [14,34,55], CtIP [13,77] and EID-1 [9,78,79]. The interaction sites in each protein are marked as boxes. Linear motifs are marked in color: red (LxCxE motif), dark blue (CKII site), light blue [cyclin-dependent kinase phosphorylation (Cdk) site], orange (phosphorylatable serine residues), green (helix motif), violet (PENF motif) and yellow (FxxxV motif). Dark grey, interactions mediated by globular domains; light grey, interactions at unknown sites. Structural domains are indicated above each car- toon, and the Rb domains targeted, and the affinities, are indicated below each site. When known, the affinities of the full-length pro- teins and the effects of phosphorylation are indicated. Viral targeting of the retinoblastoma protein L. B. Chemes et al. 982 FEBS Journal 277 (2010) 973–988 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation Proc Natl Acad Sci USA 100, 2363–2368 Dick FA (2007) Structure-function analysis of the retinoblastoma tumor suppressor protein – is the whole a sum of its parts? Cell Div 2, 26 Lee C, Chang JH, Lee HS & Cho Y (2002) Structural basis for the recognition of the E2F transactivation domain by the retinoblastoma. .. height of each letter within a stack is proportional to its abundance Calculation of conservation scores for surface residues of the RbAB domain was carried out using an alignment of the RbAB domain from 46 vertebrate species, performed using ClustalW [70] and manual curation Sequences were obtained from PFAM and ENSEMBL [71,72] and from a Psi-Blast search of UNIPROT [73] using the human Rb protein as... Fundacion YPF C.S is a recipient of a University of Buenos Aires predoctoral fellowship I.E.S is the recipient of a CONICET postdoctoral Viral targeting of the retinoblastoma protein fellowship G.d.P.G is a career investigator from CONICET We acknowledge Diana Wetzler for assistance with CD data analysis and Guillermo Solovey for assistance with ProFit programming We thank Liliana Alonso for careful... is the dissociation constant and Po is the total peptide concentration The last term in the equation takes into account slight aggregation that may take place at higher 984 Data analysis We used TFE and GdmCl to stabilize a- helix and PII conformations respectively We assumed that PII populations (for TFE titrations) and a- helix populations (for GdmCl titrations) did not change during titrations, and. .. titrant concentrations, until saturation was achieved The stoichiometry was determined by extrapolation of two linear fits of the initial and final anisotropy signals For all reactions tested, full saturation was achieved at a 1 : 1 molar ratio of titrant, indicating a 1 : 1 stoichiometry, which validates the use of a 1 : 1 binding model (Figs 2B and S2) Binding curves were fit to a model considering a. .. retinoblastoma tumor suppressor Genes Dev 16, 3199–3212 McLaughlin-Drubin ME & Munger K (2009) The human papillomavirus E7 oncoprotein Virology 384, 335–344 Helt AM, Funk JO & Galloway DA (2002) Inactivation of both the retinoblastoma tumor suppressor and p21 by the human papillomavirus type 16 E7 oncoprotein is necessary to inhibit cell cycle arrest in human epithelial cells J Virol 76, 10559–10568 Pelka... routes for the prototypic viral oncoprotein: the amyloid-cancer connection Int J Cancer 125, 1902–1911 Uversky VN, Roman A, Oldfield CJ & Dunker AK (2006) Protein intrinsic disorder and human papillomaviruses: increased amount of disorder in E6 and E7 oncoproteins from high risk HPVs J Proteome Res 5, 1829–1842 Alonso LG, Garcia-Alai MM, Nadra AD, Lapena AN, Almeida FL, Gualfetti P & Prat-Gay GD (2002)... regions of the pRB pocket domain affect its inactivation by human papillomavirus E7 proteins J Virol 76, 6224–6234 Davies RC & Vousden KH (1992) Functional analysis of human papillomavirus type 16 E7 by complementation with adenovirus E 1A mutants J Gen Virol 73, 2135–2139 Liu X & Marmorstein R (2007) Structure of the retinoblastoma protein bound to adenovirus E 1A reveals the molecular basis for viral oncoprotein. .. identity was confirmed by western blots and by MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA) Peptide synthesis and labeling Peptides were synthesized at the W M Keck Facility (Yale University, New Haven, CT), and purified by RP-HPLC The phosphorylated E7( 16-40) peptide was obtained by incorporation of phosphoserine, instead of serine, in the synthesis The relative molecular mass of each peptide was confirmed... highlights the complex balance established between physiological and pathological interactions involving the Rb protein [7], and provides essential information towards a better understanding of the Rb network of interactions and the events that determine normal cell cycle regulation or the progression to cell transformation [66] Materials and methods Cloning and protein expression and purification The RbAB . Targeting mechanism of the retinoblastoma tumor suppressor by a prototypical viral oncoprotein Structural modularity, intrinsic disorder and phosphorylation. vast importance as the guardian of the cell cycle and its clinical relevance in human cancers, struc- tural and thermodynamic understanding of the mecha- nisms