Structural basis for subversion of cellular control mechanisms by the adenoviral E1A oncoprotein

Josephine C. Ferreon, Maria A. Martinez-Yamout, H. Jane Dyson, and Peter E. Wright1

Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Peter E. Wright, June 24, 2009 (sent for review May 9, 2009) The adenovirus early region 1A (E1A) oncoprotein mediates cell CR1 and CR2 function synergistically to enhance binding of E1A transformation by deregulating host cellular processes and acti- to pRb and efficiently disrupt the complex (17). vating viral expression by recruitment of cellular The transcriptional /acetyltransferase p300 was that include cyclic-AMP response element binding (CREB) binding originally identified through its specific interaction with E1A (5); (CBP)/p300 and the (pRb). While E1A also binds the close paralog CBP (4). Interactions between E1A is capable of independent interaction with CBP/p300 or pRb, E1A and CBP/p300 are central to its ability to transform cells simultaneous binding of both proteins is required for maximal and induce mitosis (2, 19). In addition, E1A represses CBP/ biological activity. To obtain insights into the mechanism by which p300-dependent transcriptional activity (20, 21) by binding to E1A hijacks the cellular machinery by competing with -bound CBP/p300 (22). By competing for binding to essential transcription factors for binding to CBP/p300, we have CBP/p300, E1A inhibits -mediated activation of p21 and determined the structure of the complex between the transcrip- other stress response and thereby suppresses cell cycle tional adaptor zinc finger-2 (TAZ2) domain of CBP and the con- arrest and apoptosis (23, 24). E1A has been variously reported served region-1 (CR1) domain of E1A. The E1A CR1 domain is to inhibit (21, 25) and to activate (26) the histone acetyltrans- unstructured in the free state and upon binding folds into a local ferase (HAT) activity of CBP/p300. helical structure mediated by an extensive network of intermolec- E1A utilizes its N-terminal region (residues 1–25) and residues ular hydrophobic contacts. By NMR titrations, we show that E1A within the CR1 region to bind CBP/p300 (13, 27–30), although BIOCHEMISTRY efficiently competes with the N-terminal of an additional weak interaction through CR3 has been reported p53 for binding to TAZ2 and that pRb interacts with E1A at 2 (31). The regions of CR1 that bind CBP/p300 and pRb appear independent sites located in CR1 and CR2. We show that pRb and to be distinct and nonoverlapping (28). The primary E1A binding the CBP TAZ2 domain can bind simultaneously to the CR1 site of site on CBP/p300 (Fig. 1B) appears to be the transcriptional E1A to form a ternary complex and propose a structural model for adaptor -2 (TAZ2) (or CH3) domain (4, 5) although the pRb:E1A:CBP complex on the basis of published x-ray data for interactions with the nuclear coactivator binding domain and the homologous binary complexes. These observations reveal the KIX domain have also been reported (32, 33). Simultaneous molecular basis by which E1A inhibits p53-mediated transcriptional interaction of both CBP/p300 and pRb is necessary for maximal activation and provide a rationale for the efficiency of cellular biological activity of E1A (34). transformation by the adenoviral E1A oncoprotein. To further our understanding of the mechanism by which E1A hijacks the cellular transcriptional machinery and deregulates CBP/p300 ͉ NMR ͉ protein structure ͉ transcriptional coactivator ͉ the cell cycle, we have undertaken an NMR structural analysis retinoblastoma protein of the interactions of E1A with CBP/p300 and pRb. In this paper, we report the solution structure of the CR1 region of E1A (residues 53–91) in complex with the TAZ2 domain of CBP and he adenovirus early region 1A (E1A) protein plays a central show that E1A competes efficiently with the transactivation Tfunction in viral infection by activating genes required for domain of p53 for binding. Most significantly, we show that E1A viral replication and by deregulating the host cell cycle to force is capable of forming a ternary complex with the CBP TAZ2 entry into S phase and repress differentiation (1–3). E1A domain and the pocket domain of pRb, and we present a deregulates the cell cycle through interactions with key cellular structural model of the ternary complex. Our experiments proteins, including the general transcriptional coactivators provide unique insights into the structural basis by which E1A cyclic-AMP response element binding (CREB) binding protein targets key cellular proteins to subvert cellular control mecha- (CBP) and p300 (4, 5) and the retinoblastoma protein (pRb) (6), nisms and provide an optimal environment for viral replication. which lead to epigenetic modifications and reprogramming of host cell transcriptional processes (7). Binding of E1A to pRb Results and Discussion displaces the E2F transcription factors, thereby relieving E2F Mapping the Interaction Sites of E1A with CBP. E1A binding has repression and resulting in premature S-phase entry and tran- been mapped previously to the TAZ2 domain of CBP/p300 (5). scriptional activation of E2F-regulated genes (8–10). Associa- Two regions of E1A are implicated in the interaction: the tion of E1A with CBP/p300 results in global hypoacetylation of N-terminal region (residues 1–25) and the CR1 region (residues K18 of histone H3 and may be linked to the ability of E1A to induce oncogenic transformation (11). E1A is a multifunctional protein (12) composed of 4 conserved regions (CR1–CR4, Fig. Author contributions: P.E.W. designed research; J.C.F. and M.A.M.-Y. performed research; 1A). E1A uses both CR1 and CR2 for interaction with pRb (13, M.A.M.-Y. contributed new reagents/analytic tools; J.C.F., H.J.D., and P.E.W. analyzed data; and J.C.F., H.J.D., and P.E.W. wrote the paper. 14). CR2 contains a characteristic pRb recognition motif, LX- Conflict of interest: The authors declare no conflict of interest. CXE, which binds with high affinity to the B cyclin fold domain Data deposition: The atomic coordinates reported in this paper have been deposited in the of the pRb pocket region (15, 16). The CR1 region of E1A binds , www.pdb.org (PDB ID code 2KJE). The resonance assignments reported at the interface of the A and B cyclin folds of pRb, in a site that in this paper have been deposited in the BioMagResBank (accession no. 16318). partially overlaps the binding site of E2F (17). From the struc- 1To whom correspondence should be addressed. E-mail: [email protected]. tural homology between E1A and E2F (18), a L/IXXLY/F motif This article contains supporting information online at www.pnas.org/cgi/content/full/ is implicated in the displacement of E2F from pRb by E1A (17). 0906770106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906770106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Domain organization in E1A and CBP. (A) Schematic diagram of E1A showing the 4 conserved regions (CR1–CR4). The sequences (residues 1–139) of representative E1A adenoviral serotypes are shown; conserved residues are indicated by colored shading. (B) Schematic diagram of CBP/p300 showing domain organization.

42–80) (Fig. 1A) (28). It was necessary to truncate E1A for opposite face of TAZ2 are shifted and/or broadened by E1A(1– structural studies because the spectra of complexes formed with 36) binding (Fig. S1 C and D). Thus, although the N-terminal E1A constructs containing both binding regions were severely region of E1A clearly binds to a distinct surface of TAZ2, the broadened [supporting information (SI) Table S1]. Hetero- accompanying spectral broadening precludes determination of nuclear single quantum coherence (HSQC) titrations using the bound structure. truncated E1A constructs representing the isolated N-terminal and CR1 regions showed that the primary interaction site for Unbound E1A Is Intrinsically Disordered. Heteronuclear multidi- TAZ2 lies in CR1. Binding of E1A(1–36) is in intermediate mensional NMR spectra of the complex of E1A(53–91) with exchange and causes extensive broadening of both TAZ2 and TAZ2 were acquired, observing either isotope-labeled TAZ2 E1A cross peaks (Table S1, Fig. S1A), whereas binding of bound to unlabeled E1A or labeled E1A bound to unlabeled 1 13 15 E1A(53–91) is in slow exchange and gives rise to well dispersed TAZ2. Over 90% of the H, C, and N resonances were cross peaks with uniform line widths (Fig. 2). HSQC titrations assigned for both TAZ2 and E1A. The peaks in the HSQC were performed in which E1A(1–36) was titrated into the spectra of the free and bound TAZ2 domain are well dispersed 15N-TAZ2:E1A(53–91) complex. Cross peaks arising from (Fig. 2A), indicating that TAZ2 is well folded in both states; TAZ2 residues within the E1A(53–91) binding site are unaf- nevertheless, binding of E1A results in large chemical shift fected by E1A(1–36), while resonances from residues on the perturbations. More dramatic differences are observed between the HSQC spectra of free and bound E1A (Fig. 2B). E1A is largely unstructured in the free state as seen by the limited chemical shift dispersion in the 1H dimension; backbone reso- nances of both E1A(53–91) and E1A(1–139) show little devia- tion of chemical shifts from random coil values (Fig. S2 A and B). This result is consistent with functional studies where fully heat-denatured E1A injected into mammalian cells retains the ability to induce cellular transformation and other E1A functions (35). Upon interaction with TAZ2, there are large changes in the E1A chemical shifts (Fig. S2C) indicative of coupled folding and binding.

Structure of the TAZ2:E1A Complex. The structure of the complex of TAZ2 with E1A(53–91) was determined from distance and dihedral angle restraints derived from the NMR spectra. The structure statistics are summarized in Table 1. The structure of TAZ2 in the complex with E1A is very well defined by the NMR data (Fig. 3A) and is very similar to that of the free protein, with a backbone heavy atom (N, C␣,CЈ) root mean square deviation of 0.90 Å relative to the unbound state. The TAZ2 domain, which functions as a preformed helical scaffold for binding of disordered transcriptional activation domains (36), contains 4 ␣-helices (labeled ␣1Ϫ␣4) joined by 3 short loops that form binding sites for 3 zinc atoms (Fig. 3B). The fold is stabilized by an extensive hydrophobic core and by coordination of zinc (36). E1A binds in a deep hydrophobic groove in the surface of TAZ2, formed at the interfaces between helices ␣1/␣2 and ␣1/␣3, and buries Ϸ1400 Å2 of the TAZ2 surface (Fig. 3 B and C). The E1A backbone is generally well ordered between residues P54 Fig. 2. 1H-15N HSQC spectra of the TAZ2:E1A complex. (A) HSQC spectra of 15N TAZ2 free (black) and bound to equimolar E1A(53–91) (red). (B) HSQC and L80, except for a short loop from D68 to V70; residues at the spectra of 15N E1A(53–91) free (black) and bound to equimolar TAZ2 (red). N terminus [A53 and the 4 preceding residues (GSHM) that are Corresponding cross peaks from the free and bound forms are connected. an artifact from cleavage of the fusion protein] and at the C Asterisks denote cross peaks arising from E1A containing cis peptide bonds. terminus (L81–E91) do not interact strongly with TAZ2 and are

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906770106 Ferreon et al. Downloaded by guest on September 25, 2021 Table 1. NMR restraints and structural statistics groove formed between helix ␣1 of TAZ2 and the N terminus of ␣ ␣ NMR Restraints 2 and is oriented almost perpendicular to 1 (Fig. 3 B and C). Immediately beyond the helix, the aromatic ring of F66 projects Total NOE distance restraints 2,044 into a hydrophobic pocket formed at the interface between the Intraresidue (i, i) 472 ␣1, ␣2, and ␣3 helices of TAZ2. The side chains of A61, V62, I65, Sequential (i, i ϩ 1) 532 F66, and I78 of E1A form a hydrophobic cluster that packs Medium range (2 Յ ͉i Ϫ j͉ Յ 4) 636 against an extensive hydrophobic surface formed by the side Long range (͉i Ϫ j͉ Ͼ 4) 243 chains of I1773, C1776, M1799, V1802, L1823, A1825, and L1826 Intermolecular NOEs 161 at the ␣1/␣2/␣3 interface of TAZ2 (Fig. 3D). A second hydro- Total dihedral angle restraints 164 phobic cluster, formed by M71, A73, and V74 of E1A, packs ␾ 95 against V1802, P1818, and V1819 at the junction of helices ␣2 and ␺ 69 ␣3 of TAZ2. With the exception of V74 and I78, these hydro- Violation analysis phobic residues are strongly conserved among E1A serotypes Maximum distance violation (Å) 0.19 (37), consistent with their importance in mediating TAZ2 Maximum dihedral angle violation (°) 0 binding. Additional hydrophobic interactions are made by the Energies side chains of L80 and T82, located in a short helical turn at the Ϫ1 Ϫ Mean AMBER energy (kcal mol ) 5,492.42 C-terminal end of the E1A interaction interface. Although I78, Ϫ1 Mean restraint energy (kcal mol ) 3.56 L80, and T82 are not well conserved among E1A serotypes, they Deviations from idealized geometry appear to contribute to binding of AD5 E1A because constructs Bond lengths (Å) 0.0102 Ϯ 0.00007 Ϯ terminating at G77 interact more weakly with TAZ2 and give Bond angles (°) 2.65 0.022 exchange-broadened NMR spectra. The side chain of F83 of RMS deviations from mean structure* E1A is disordered and interacts only weakly with TAZ2; it is not Backbone atoms (N, C␣,CЈ) (Å) 0.55 conserved in the E1A sequence. All heavy atoms (Å) 0.96 The extensive intermolecular hydrophobic contacts are sup- Ramachandran plot* plemented by complementary charge interactions between Residues in most favorable regions (%) 88.9 acidic side chains on E1A and Arg and Lys residues on TAZ2 Residues in additionally allowed regions (%) 10.5 (Fig. 3E). E59 and E60 of E1A are in close proximity to K1798 Residues in generously allowed regions (%) 0.6 BIOCHEMISTRY Residues in disallowed regions (%) 0.0 and R1801 of TAZ2; acidic side chains are strongly conserved at both of these sites in all E1A serotypes (37). The more weakly *TAZ2 residues 1765–1851 and E1A residues 53–83. conserved E60 lies close to R1775 of TAZ2, and D79 forms a salt bridge with R1769 on the ␣1 helix of TAZ2. A small hydrophobic cluster, formed by the side chains of P67, V70, and L72, is disordered in the structure ensemble (Fig. 3A). A well-defined exposed on the surface of E1A (Fig. 3E). Because hydrophobic ␣-helix is formed by residues E59–I65, with shorter helical turns side chains are conserved at these positions (particularly 70 and between L72–V74 and L80–T82, and a compact loop between 72) in the majority of E1A serotypes, it is possible that this cluster F66–A73. The interaction of E1A with TAZ2 is mediated functions in recruitment of other proteins to the E1A:TAZ2 primarily through an extensive network of hydrophobic interac- complex. tions. The E1A helix (E59–I65) is bound in a deep hydrophobic The structure of a TAZ2 complex with the transcriptional

Fig. 3. NMR structure of the TAZ2:E1A complex. (A) Ensemble of 20 lowest energy structures. The TAZ2 backbone is shown in blue, E1A is in coral, and the N and C termini of each protein are labeled. (B) Ribbon representation of the lowest-energy structure, colored as in A. Zinc atoms are shown as gray spheres. Helices ␣1–␣4 of TAZ2 are labeled. (C) Electrostatic potential mapped to the surface of TAZ2 showing basic residues in blue and acidic residues in red. The orientation is the same as in B. The backbone of the bound E1A is represented as a coral ribbon. (D) Close-up view showing E1A hydrophobic side chains (green, yellow for M71) that form the intermolecular interface. The surface formed by helices ␣1, ␣2, and ␣3 of TAZ2 is shown, together with a tube representation of the E1A backbone. (E) Electrostatic interactions in the TAZ2-E1A interface. The backbone of the bound E1A is represented as a coral ribbon, superimposed on the TAZ2 surface. Asp and Glu side chains of E1A are colored red, and Asn, Gln, and Ser side chains are shown in beige. The cluster of hydrophobic side chains exposed on the E1A surface is shown in green. The nitrogen atoms of Arg and Lys side chains of TAZ2 are colored blue. (F) Superposition of the complexes of TAZ2 (represented as a surface) with E1A (coral ribbon) and the STAT1 TAD (green ribbon). This figure and Figs. 4B and 5C were drawn using MOLMOL (52).

Ferreon et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 changes in TAZ2 chemical shifts caused by binding of p53(13– 61) map to the hydrophobic surface formed by the ␣1, ␣2, and ␣3 helices of TAZ2 (Fig. 4B). This surface partially overlaps with the E1A binding surface such that the p53 TAD and E1A would necessarily compete for binding to the TAZ2 domain of CBP/ p300.

Interaction of E1A with pRb. Interactions between the pRb pocket domain and 15N-labeled E1A peptides were examined using HSQC titrations (Fig. S4). As expected (16), the LXCXE motif in CR2 binds with high affinity to pRb; there is a large increase in dispersion of the HSQC spectrum of E1A(106–139) in the presence of pRb and exchange is slow on the chemical shift timescale (Fig. S4A). The affinity for CR1 is lower (14) and the HSQC spectrum of E1A(27–91) remains poorly dispersed in the presence of excess pRb; cross peaks corresponding to the pRb binding site (residues 39–51) are mostly broadened and those of adjacent residues (L32, S36, and A53) are shifted upon binding (Fig. S4B). Interactions were also observed with a pseudo- LXCXE motif (LAVQE, residues 72–76 of E1A) located in the TAZ2 binding region of CR1. HSQC cross peaks associated with these residues are broadened and/or shifted upon binding of pRb Fig. 4. E1A and the p53 transactivation domain compete for binding to to E1A(27–91) or E1A(53–91) (Fig. S4C). Binding of pRb to the TAZ2. (A) Region of the HSQC spectrum of 15N-labeled TAZ2 free (black); LAVQE motif in CR1 is weak and pRb is readily displaced by following addition of p53(13–61) at molar ratios of 1:0.25 (blue), 1:0.5 (or- TAZ2 (Fig. S4 D and E). ange), and 1:1 (red); in the presence of p53(13–61) and E1A(53–91) at 1:1:1 mol ratio (green); and in the 1:1 TAZ2:E1A binary complex (purple). The shifts Ternary Complex Formation by CBP/p300 (TAZ2), E1A, and pRb. While caused by p53 binding are highlighted by red arrows, and the shifts caused by our NMR experiments confirm formation of binary complexes addition of E1A to the p53:TAZ2 complex are indicated by green arrows. The between E1A and TAZ2 and between E1A and pRb, in vivo full HSQC spectra are shown in Fig. S3A.(B) Surface of TAZ2 colored according studies show that simultaneous interaction of CBP/p300 and pRb ⌬␦ to the weighted average amide chemical shift difference ( (N, H)av) between in a multimeric complex with E1A is necessary for efficient HSQC spectra of the free 15N-labeled TAZ2 domain and the 1:1 complex with p53(13–61). Residues with ⌬␦(N, H) Ͼ 0.2 ppm are colored red, those with oncogenic transformation (34, 42). To investigate ternary com- av plex formation, we performed HSQC titrations in which the pRb ⌬␦(N, H)av Ͼ 0.1 ppm are colored orange, and residues with ⌬␦(N, H)av Ͻ 0.1 ppm are white. The full histograms are shown in Fig. S3B. E1A is shown as a pocket domain was added to the E1A(27–91):TAZ2 complex. cyan ribbon. The cross peaks of residues V62, S69, M71, and A73 in the TAZ2 binding site of CR1 remain at their characteristic chemical shifts even after addition of a 3-fold excess of pRb (Fig. 5A, Fig. S5), activation domain of STAT1 has recently been reported (38). showing that pRb does not compete with TAZ2 for E1A binding. STAT1 also binds through an amphipathic helix, with comple- Upon titration with pRb, the A53 cross peak shifts in slow mentary electrostatic interactions between acidic side chains on exchange from its position in the spectrum of the TAZ2 binary the surface of the helix and arginine residues on helix ␣1 of complex to its position in the binary E1A(27–91):pRb complex TAZ2. Although STAT1 and E1A interact with a similar region (Fig. 5B) while the cross peaks of many residues in the region of the TAZ2 surface (Fig. 3F), they adopt very different 39–51 are broadened (Fig. S5), confirming binding of pRb to its structures upon binding and differ significantly in their inter- cognate site. Thus, despite the close proximity of the pRb and molecular interactions. In particular, the binding sites of the CBP/p300 binding sites in the E1A CR1 region, the NMR data amphipathic helices of STAT1 and E1A are different; the provide unequivocal evidence that both TAZ2 and the pRb STAT1 helix utilizes the hydrophobic groove between the ␣1 and pocket domain can bind simultaneously to form a ternary ␣3 helices of TAZ2 that is occupied by residues I78-T82 of E1A. TAZ2-E1A-pRb complex. Not surprisingly, significant line These results underscore the versatility of the TAZ2 domain for broadening accompanies formation of this large (Ϸ57 kDa) binding a diverse range of intrinsically disordered transcriptional multiprotein complex. activation domains and viral proteins. Structural Model of the Ternary Complex. We have used the x-ray E1A Competes with p53 for CBP/p300 (TAZ2) Interaction. E1A inhibits structures of the pRb pocket domain in complex with residues p53-dependent transcriptional activation and suppresses p53- 37–49 from the E1A CR1 region (17) and with the LXCXE motif induced cell cycle arrest and apoptosis by competing with p53 for from human papilloma virus (HPV) E7 (15), together with our binding to CBP/p300 (23, 24, 39, 40). The p53 transactivation NMR structure, to generate a model of the pRb:E1A:TAZ2 domain (TAD) interacts with 4 domains of CBP/p300 (TAZ1, ternary complex (Fig. 5C). Residues 37–49 of E1A bind in a deep KIX, TAZ2, and NCBD), with the highest affinity toward TAZ2 groove at the interface of the A and B cyclin fold domains of pRb (41). To test the hypothesis that E1A and p53 compete directly (17), while residues 53–91 in the C-terminal half of CR1 interact for TAZ2, p53(13–61) and E1A(53–91) were titrated into 15N- with the TAZ2 domain of CBP/p300. Residues 92–120 of E1A labeled TAZ2 and HSQC spectra were recorded. Addition of were modeled as a long, flexible linker between the TAZ2 increasing amounts of p53(13–61) results in shifting and broad- binding site in CR1 and the LXCXE motif (residues 122–126) in ening of cross peaks in the HSQC spectrum of TAZ2 (Fig. S3 A CR2. On the basis of the homology with HPV E7, the LXCXE and B); a region of this spectrum is shown in Fig. 4A. Addition motif of E1A is expected to bind in an extended ␤-like confor- of equimolar E1A to the TAZ:p53 complex causes all TAZ2 mation within a shallow hydrophobic groove on the B domain of cross peaks to shift to their characteristic positions in the HSQC pRb (15). spectrum of the binary TAZ2:E1A complex (Fig. 4A, Fig. S3C), The E1A CR1 and CR2 regions function synergistically to indicating that E1A displaces the p53 TAD from TAZ2. The displace E2F from pRb. It has been suggested that displacement

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906770106 Ferreon et al. Downloaded by guest on September 25, 2021 lates acetylation of pRb by CBP/p300. By forming a ternary complex with pRb and the TAZ2 domain, E1A brings pRb into close proximity with the neighboring HAT domain of CBP/p300 (Fig. 1B), thereby promoting acetylation at K873 and K874 of pRb (44). Acetylation at these sites hinders phosphorylation of pRb by cyclin-dependent kinases and enhances binding to the ubiquitin ligase MDM2, thereby regulating cell differentiation and promoting permanent cell cycle exit (44, 45).

Functional Significance of Intrinsic Disorder in E1A. NMR spectra of E1A(1–139) have limited dispersion and are characteristic of a largely unstructured protein (Fig. S2A). Chemical shifts are close to random coil values except for residues E13–D21, which have a weak propensity (Ϸ30% population) for helical structure (Fig. S2B). Thus, the NMR spectra are consistent with recent predictions that, except for CR3, E1A is intrinsically disordered (43). There are 4 known binding motifs located between residues 1 and 139 of E1A; residues 1–25 (13, 27–29) and residues 54–83 (this work) form binding sites for CBP/p300, while residues 37–51 (CR1) and 121–129 (CR2) function in pRb binding. The 4 binding motifs are capable of functioning either independently or synergistically to mediate interactions with cellular proteins (i.e., E1A can function as a ‘‘molecular hub’’) (43). In addition to CBP/p300 and pRb, the E1A domains interact with nearly 20 other cellular proteins to regulate cell cycling and suppress the cellular response to the adenoviral infection (2, 43, 46). The incorporation of these interaction motifs within an intrinsically disordered region confers upon E1A the flexibility to target BIOCHEMISTRY multiple cellular proteins and organize them combinatorially into higher-order complexes to efficiently disrupt cellular reg- ulatory pathways and reprogram . This ability to bind multiple cellular targets probably underlies the opposing effects of E1A in cellular apoptosis. E1A can prevent apoptosis by inhibiting the CBP/p300–p53 interaction (23) or induce Fig. 5. Ternary complex formation by CBP, E1A, and pRb. (A) Region of the 15 apoptosis by promoting formation of a pRb:MDM2:p53 multi- HSQC spectrum of N-labeled E1A(27–91) free (black), in the binary complex meric complex and initiating E1A-dependent CBP/p300 acety- with TAZ2 (1:1.5 mol ratio, red), and in the ternary complex with both TAZ2 and pRb (1:1.5:3 mol ratio, green). (B) Regions of the same spectrum showing lation of pRb (3, 44, 47). the cross peak of A53. As a reference, the spectrum of the binary E1A:pRb complex is superimposed in blue. Although the green and red cross peaks Materials and Methods become broader due to formation of a high molecular weight complex, it is Protein Expression and Purification. The TAZ2 domain of mouse CBP (residues clear that pRb does not displace TAZ2 and vice versa. The full HSQC spectra are 1764–1855) and the p53 transactivation domain (residues 13–61) were ex- shown in Fig. S5.(C) Structural model of the ternary pRb-E1A-TAZ2 complex. pressed and purified as described (36, 48). The pRb central pocket and ade- The model was generated using the crystal structure of the complex of pRb novirus serotype 5 (Ad5 and Ad6) E1A constructs were expressed in Escherichia with E1A (CR1, residues 37–49) (PDB entry 2R7G) (17), the NMR structure of the coli BL21 (DE3) cells; details are given in SI Text. For NMR titrations, refolded TAZ2-E1A(53–91) complex determined in this work, and the crystal structure TAZ2, the pRb pocket domain, p53(13–61), and the various E1A constructs of the HPV E7 peptide (DLYCYEQLN, homologous to residues 121–129 of E1A) were dialyzed into buffer (20 mM Tris, 50 mM NaCl, 1 mM DTT, pH 6.8, unless (in CR2) containing the LXCXE motif that interacts with pRb (PDB entry 1GUX) otherwise noted in SI Text). Protein concentration was determined from the (15). The flexible linker between residues 83 and 120 of E1A is indicated absorbance at 280 nm. For E1A(1–36) and E1A(53–91), which contain no schematically as a dotted line. The backbone structures of pRb, E1A, and TAZ2 tyrosine or tryptophan, protein concentration was estimated by BCA assay are represented as ribbons and are colored gray, coral, and blue, respectively. (Pierce) or from peak intensities observed in analytical HPLC, calibrated against known protein concentrations.

proceeds by a mechanism that involves initial high-affinity NMR Spectroscopy. Spectra were recorded using Bruker 600-, 800-, and 900- binding of CR2 to the B domain of pRb, thereby allowing CR1 MHz spectrometers. The complex formed by addition of E1A(53–91) to TAZ2 Ϸ to compete effectively with E2F for binding to the hydrophobic was concentrated to 1.5 mM in 10 mM d10-Tris in D2O (pH 6.4) or 90% interface between the A and B domains of pRb (17). The CR1 H2O/10% D2O (pH 6.8) buffer containing 1 mM DTT for NMR experiments. and CR2 binding sites on pRb are 30 Å apart (17). While CR1 Several samples with different isotope labeling patterns were used to reduce spectral complexity. Resonances were assigned using standard multidimen- and CR2 can bind independently to pRb, formation of a stable sional heteronuclear NMR experiments (49). Details are given in SI Text. ternary complex with CBP/p300 requires the presence of a long flexible spacer between CR1 and CR2 (34). The length require- Structure Calculations. Distance restraints were derived from 3D 13C- and 15N- ments can be understood from the structural model (Fig. 5C); edited nuclear Overhauser effect spectroscopy (NOESY)-HSQC and isotope- ␶ ϭ the CR1–CR2 linker must be long enough to allow simultaneous filtered and edited 3D NOESY-HSQC spectra with m 120 ms. Backbone dihedral binding of CR1 and CR2 in their cognate sites on pRb and to angle restraints were imposed in helical regions. Initial structures were generated accommodate binding of CBP/p300 without creating steric with the program CYANA (50) and refined by 2 cycles of restrained molecular Ͼ dynamics-simulated annealing, using the AMBER 8 software package (51). Details clashes. CR1 and CR2 are connected by a long ( 30 residues) of the structure calculations are given in SI Text, and the NOE restraints assigned intrinsically disordered linker (43) (Fig. S2B) in all sequenced manually and through the CANDID program are listed in Tables S2 and S3. The E1A serotypes (37). Our structural model of the ternary complex structure was visualized using the program MOLMOL (52) and the quality was provides insights into the molecular basis by which E1A stimu- checked using PROCHECK-NMR (53).

Ferreon et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. We thank Gerard Kroon for assistance with NMR experi- Salk Institute, La Jolla, CA) and Peiqing Sun (The Scripps Research Institute, La Jolla, ments; Mindy Landes, Spencer Tung, and Euvel Manlapaz for technical support; and CA), respectively. This work was supported by Grant CA96865 from the National Roberto De Guzman, Jonathan Wojciak, and Chul Won Lee for helpful discussions. Institutes of Health and by the Skaggs Institute for Chemical Biology. J.C.F. was The E1A cDNA and pRb plasmid were generous gifts from Drs. Marc Montminy (The supported by a Leukemia and Lymphoma Society Special Fellowship.

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