Recognition of the disordered transactivation PNAS PLUS domain by the transcriptional adapter domains of CREB-binding protein

Alexander S. Kroisa,b, Josephine C. Ferreona,b,1, Maria A. Martinez-Yamouta,b, H. Jane Dysona, and Peter E. Wrighta,b,2

aDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; and bSkaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037

Contributed by Peter E. Wright, February 12, 2016 (sent for review January 8, 2016; reviewed by Mitsuhiko Ikura and Ishwar Radhakrishnan) An important component of the activity of p53 as a tumor suppres- binds to the C-terminal regulatory domain after sor is its interaction with the transcriptional coactivators cyclic-AMP acetylation at K382 by the CBP HAT domain in response to response element-binding protein (CREB)-binding protein (CBP) and DNA damage (26). The unphosphorylated p53TAD binds to the p300, which activate of p53-regulated stress response CBP/p300 domains with a broad range of affinities, with the genes and stabilize p53 against ubiquitin-mediated degradation. The strongest binding to TAZ2 (0.026 μM) (24, 25). It has been highest affinity interactions are between the intrinsically disordered hypothesized that each of the four TADs of the p53 tetramer N-terminal (TAD) of p53 and the TAZ1 and may bind to a separate domain of a single CBP/p300 molecule, TAZ2 domains of CBP/p300. The NMR spectra of simple binary resulting in an avidity effect that further stabilizes the p53– complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer CBP/p300 complex and enhances p53-mediated transcription from exchange broadening, but innovations in construct design and (24, 25). The p53TAD, which is disordered throughout its isotopic labeling have enabled us to obtain high-resolution struc- length in the absence of binding partners (27, 28), is bipartite tures using fusion proteins, uniformly labeled in the case of the and contains two activation subdomains, between residues 1–39 TAZ2–p53TAD fusion and segmentally labeled through transintein and 40–61, which function both synergistically and differen- – splicing for the TAZ1 p53TAD fusion. The p53TAD is bipartite, with tially in mediating p53 function (29–31). The activation sub- two interaction motifs, termed AD1 and AD2, which fold to form domains contain short amphipathic interaction motifs (32–34), short amphipathic helices upon binding to TAZ1 and TAZ2 whereas termed AD1 (residues 18–26) and AD2 (residues 44–54) (Fig. intervening regions of the p53TAD remain flexible. Both the AD1 1A), that have a weak propensity for transient helical secondary and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, structure in the unbound state (32–34) and are frequently ob- with AD2 making more extensive hydrophobic contacts consistent served to fold into stable amphipathic helices upon binding to with its greater contribution to the binding affinity. Binding of their partners (35–39). AD1 and AD2 is synergistic, and structural studies performed with

isolated motifs can be misleading. The present structures of the full- BIOPHYSICS AND length p53TAD complexes demonstrate the versatility of the inter- Significance COMPUTATIONAL BIOLOGY actions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the The tumor suppressor p53 regulates the cellular response to structural basis of the affinity changes that occur upon stress- genomic damage by recruiting the transcriptional related posttranslational modification. cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and its paralog p300 to activate stress response intrinsically disordered protein | binding motif | genes. We report NMR structures of the complexes formed transcriptional coactivator | intein | segmental labeling between the full-length, intrinsically disordered N-terminal transactivation domain of p53 and the transcriptional adapter zinc finger domains (TAZ1 and TAZ2) of CBP. Exchange broad- he tumor suppressor p53 plays a central role in the cellular ening of NMR spectra of the complexes was ameliorated by response to stress, functioning as an important signaling hub T using fusion proteins and segmental isotope labeling. The for the cellular response to various degrees of genomic damage structures show how the p53 transactivation domain uses bi- and instability (1, 2). p53 is a multidomain protein that contains partite binding motifs to recognize diverse partners, reveal the an N-terminal transactivation domain (TAD), a proline rich re- critical interactions required for high affinity binding, and gion, a core DNA-binding domain, a tetramerization domain, and provide insights into the mechanism by which phosphorylation a C-terminal regulatory domain (Fig. 1A). In unstressed cells, p53 enhances the ability of p53 to recruit CBP and p300. binds to the E3 ubiquitin ligase mouse double minute protein 2

(MDM2), which mediates ubiquitination and degradation of p53 Author contributions: A.S.K., J.C.F., M.A.M.-Y., H.J.D., and P.E.W. designed research; A.S.K., (3–6). In response to stress, p53 can be phosphorylated at more than J.C.F., and M.A.M.-Y. performed research; A.S.K., J.C.F., M.A.M.-Y., H.J.D., and P.E.W. 20 sites, 9 of which are located within the TAD (7). These modi- analyzed data; and A.S.K., H.J.D., and P.E.W. wrote the paper. fications lower the affinity of the p53TAD for MDM2 and promote Reviewers: M.I., Ontario Cancer Institute–University of Toronto; and I.R., Northwestern binding to the transcriptional coactivator cyclic-AMP response el- University. ement-binding protein (CREB)-binding protein (CBP) and its The authors declare no conflict of interest. paralog p300 (8–13). Binding of p53 to CBP/p300 facilitates Data deposition: The atomic coordinates have been deposited in the , acetylation of the C-terminal domain of p53 and further inhibits www.pdb.org [PDB ID codes 5HPD (TAZ2–p53TAD), 5HP0 (TAZ2–p53AD2), and 5HOU (p53TAD–TAZ1)]. The NMR chemical shifts have been deposited in the BioMagResBank, its degradation (14, 15). The interaction between p53 and CBP/ www.bmrb.wisc.edu [accession nos. 30004 (TAZ2–p53TAD), 30003 (TAZ2–p53AD2), and p300 is also required for p53-mediated transcription and stabi- 30002 (p53TAD–TAZ1)]. – – lization of the p53 DNA interaction (16 18). Four domains of 1Present address: Department of Pharmacology, Baylor College of Medicine, Houston, CBP/p300 are involved in the interaction with the p53TAD: the TX 77030. folded transcriptional adapter zinc finger (TAZ) 1, KIX, and TAZ2 2To whom correspondence should be addressed. Email: [email protected]. domains, and the molten-globular nuclear coactivator-binding This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. domain (NCBD) (Fig. 1B) (16–25). In addition, the CBP/p300 1073/pnas.1602487113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1602487113 PNAS | Published online March 14, 2016 | E1853–E1862 Downloaded by guest on September 28, 2021 have determined solution structures of the full-length p53TAD bound to the TAZ1 and TAZ2 domains of CBP, as well as the structure of the isolated p53AD2 domain bound to TAZ2. Results Design of Protein Constructs. NMR structural studies of p53/CBP complexes are frequently hindered by conformational averaging and exchange broadening, making structural restraints difficult to obtain (25, 38, 53). Binding of the p53TAD to the TAZ1 (Kd 0.9 μM) and TAZ2 (Kd 0.026 μM) domains occurs in fast to intermediate exchange on the chemical shift timescale, resulting in weak or missing resonances as a result of exchange broadening (25). Additionally, the presence of secondary binding sites pre- vents the use of a large excess of one binding partner in an effort to drive the interaction into the slow-exchange regime (54). These difficulties have limited previous structural characteriza- tion of p53TAD:TAZ2 complexes to the isolated AD1 or AD2 subdomains, for which the resonances are less broadened be- cause they exchange on a faster timescale (25, 38, 39). To obtain insights into the molecular basis for recognition of TAZ1 and TAZ2 by the bipartite p53TAD, we attempted to determine NMR structures of p53(13–61) in a 1:1 complex with each of the TAZ domains. Because of exchange broadening and missing resonances, we were able to obtain only preliminary, low-resolution structures that revealed the location and N-to-C Fig. 1. Schematic diagrams showing the domain structure of (A) p53 and (B) orientation of the AD1 and AD2 helices but could not provide CBP. (C) Construct design for the TAZ2–p53TAD fusion protein. (D)Intein atomic details of the interactions with TAZ1 and TAZ2. To – ligation scheme and final construct for the p53TAD TAZ1 fusion protein. circumvent this problem, we created fusion proteins, in which the Blue colors denote the p53TAD, with the AD1 and AD2 consensus regions outlined in blue in A. Interaction and catalytic domains identified in CBP are two interacting partners are joined by a flexible linker. Fusion shown in green for the nuclear receptor interaction domain (NRID), the KIX proteins enforce close proximity of the components of the com- domain, the bromodomain (bromo), the Cys-His rich domain-2 (CH2), the plex, increasing the effective concentration and association rate histone acetyl transferase domain (HAT), the ZZ domain, and the nuclear and driving the binding process into the slow-exchange regime, coactivator binding domain (NCBD) and in red for TAZ1 and in yellow for thereby reducing line broadening and enhancing spectral quality TAZ2. Intein and other tag and linker sequences are shown in gray. (55, 56). On the basis of the preliminary structure of the non- tethered complex, we designed a fusion protein containing the TAZ2 domain of mouse CBP (residues 1764–1855) joined to the The transcriptional adapter zinc finger (TAZ) (40) domains of p53TAD (residues 2–61) by a six-residue Gly-Ser repeat sequence CBP and p300 frequently act as scaffolds for the binding-related (Fig. 1C). The improvement in the spectral quality of both the folding of the transactivation domains of numerous transcription TAZ2 and p53TAD spectra is illustrated in Fig. 2, with complete factors (41, 42). The TAZ1 and TAZ2 domains adopt similar spectra shown in Figs. S1 and S2. The backbone NH cross-peaks in α α –α folds that consist of four -helices ( 1 4) stabilized by binding to the spectrum of the free p53TAD are poorly dispersed (Fig. S1), three zinc atoms (43, 44). The largest structural differences be- consistent with the disordered nature of the free domain, but are α tween the two TAZ domains are the length of the 1 helix, which sharp and intense (cyan cross-peaks in Fig. 2 A and B and Fig. S1). α is extended in TAZ1, and the orientation of the 4 helix, which is The cross-peaks corresponding to the p53TAD in the TAZ2– rotated 180° between the two domains. Despite their topological p53TAD fusion protein are better dispersed (labeled black cross- similarity, TAZ1 and TAZ2 differ significantly in their amino peaks in Fig. 2 A and B and Fig. S1), showing that the p53TAD is acid sequence and are highly selective in their interactions, folded in the complex. In contrast, many cross-peaks are broad- preferentially binding different sets of transactivation domains. ened or missing from the spectrum of the complex obtained by Several structures of TAZ1 and TAZ2 complexes with tran- mixing equimolar amounts of p53TAD and TAZ2 (red cross- scriptional activation domains have been reported (45–52) and peaks in Fig. 2 A and B and Fig. S1), and those cross-peaks that have yielded insights into modes of binding: TAZ1 primarily are visible are frequently not shifted to the same extent as the binds long, extended activation domains in deep grooves whereas corresponding cross-peaks for the TAZ2–p53TAD fusion protein. TAZ2 binds tightly to discrete amphipathic helices through an Due to the presence of a weak (Kd 10 μM) secondary AD2-binding exposed hydrophobic surface (49, 50). site on TAZ2, the cross-peaks continue to shift beyond a 1:1 mole Whereas many intrinsically disordered transactivation domains ratio upon titration of p53TAD with TAZ2 (25, 54). Because the bind with high specificity to either TAZ1 or TAZ2, the p53TAD cross-peaks for a given residue in the various complexes shift in a binds to both TAZ domains, with differing affinities (24, 25). linear fashion, we infer that the fusion protein construct does not Although the isolated AD2 region of p53 binds with much higher cause a change in binding mode but stabilizes the complex with affinity than the isolated AD1, the AD2 and AD1 motifs syn- the p53 AD2 motif bound in its primary, high affinity site on ergize in the context of the full-length p53TAD to enhance the TAZ2. An increase in spectral quality is also observed for the binding affinity (25). Moreover, the p53TAD can bind simulta- heteronuclear single-quantum coherence (HSQC) spectra of neously to TAZ1 or TAZ2 (through AD2) and to MDM2 (through 15N-labeled TAZ2 in the fusion protein (black cross-peaks in Fig. AD1) to form a ternary complex that promotes polyubiquitination 2C), compared with in the 1:1 complex with unlabeled p53TAD and degradation of p53 (25). Structures have been reported for (magenta cross-peaks in Fig. 2C). The NMR spectra of the iso- complexes of the isolated AD1 and AD2 motifs bound to TAZ2 tope-labeled TAZ2–p53(2–61) fusion protein are of sufficient (38, 39), but no structural data are available for complexes of the quality for structure determination. full-length p53TAD or for p53 complexes with TAZ1. To gain in- To obtain additional evidence that our TAZ2–p53(2–61) fusion sights into the structural basis for CBP/p300 recruitment by p53, we protein represented an accurate binding model, we also determined

E1854 | www.pnas.org/cgi/doi/10.1073/pnas.1602487113 Krois et al. Downloaded by guest on September 28, 2021 labeled with isotope. The p53TAD–TAZ1 fusion was then pu- PNAS PLUS rified from the folded intein by-product, as described in Materials and Methods. The 1H-15N HSQC spectra of the intein-mediated fusion proteins selectively enriched with 15N in TAZ1 (Fig. 3A, black) or in the p53TAD (Fig. 3A, blue) are shown in Fig. 3A, super- imposed on the spectrum of the fully labeled nonintein fusion protein (Fig. 3A, orange). These spectra show clearly that the intein labeling method gives improved spectral quality and decreased spectral complexity. Fig. 3 B and C shows a comparison of regions of the 1H-15N HSQC spectrum of free 15N-labeled p53TAD(1–61) (cyan) with the corresponding regions of 15N-labeled p53TAD(1–61) in a 1:1 complex with unlabeled TAZ1 (red) and of the selectively labeled 15N p53TAD fused to unlabeled TAZ1 (black) (complete spectra are shown in Figs. S4 and S5).

Structure Determination. The structures of the TAZ2–p53(2–61), TAZ2–p53(37–61), and p53(1–61)–TAZ1 fusion proteins were determined using restraints derived from 15N-NOESY HSQC and 13C-NOESY HSQC spectra. NOEs between p53TAD and TAZ1 were identified using isotope-edited 13C-NOESY-HSQC spectra (58). Dihedral angle restraints were derived from 13Cα, 13 1 1 15 Cβ, Hα, HN and N chemical shifts using TALOS+ (59). Initial structures were calculated using CYANA with CANDID and were refined by restrained molecular dynamics, first in vacuo and finally using a generalized Born solvent model (60, 61). The NMR restraints and structure statistics are summarized in Table S1.

15 1 Structures of TAZ2 and TAZ1. The structures of the TAZ2 and Fig. 2. (A and B) Overlay of sections of the N- H HSQC spectra of free – – – – p53TAD (cyan), p53TAD with addition of one molar equivalent of TAZ2 (red), TAZ1 domains in the TAZ2 p53(2 61), TAZ2 p53(37 61), and and of the TAZ2–p53TAD fusion protein (black). Cross-peaks belonging to the TAZ2 portion of the (uniformly labeled) TAZ2–p53TAD fusion protein are labeled with a red asterisk. (C)Overlayof1H-15N HSQC spectra of free TAZ2 (green), TAZ2 with addition of one molar equivalent of p53TAD (magenta), and of the TAZ2–p53TAD fusion (black). Arrows indicate chemical shift changes for BIOPHYSICS AND individual cross-peaks. Complete spectra are shown in Figs. S1 and S2. COMPUTATIONAL BIOLOGY

the solution structure of a protein containing TAZ2 (residues 1764–1855) fused to only the AD2 region of the p53TAD (resi- dues 37–61) through a GSGSG linker. The 1H-15NHSQCspectra obtained with this construct were of sufficiently high quality (Fig. S3) to allow us to determine the solution structure of this complex. A uniformly labeled fusion protein with p53(13–61) tethered to the C terminus of TAZ1(340–439) gave similarly enhanced spectral quality compared with the complex formed between the isolated proteins. However, the TAZ1 and p53 resonances in the 1H-15N HSQC spectrum of this construct (orange cross-peaks in Fig. 3A) were not sufficiently well-resolved for detailed structural analysis. To increase resolution and enable the use of isotope- edited NMR techniques, we used a system of split inteins to prepare a selectively labeled fusion protein (57). The constructs used to obtain selectively labeled p53TAD–TAZ1 fusion pro- teins are shown in Fig. 1D. Preliminary structures calculated using the limited nuclear Overhauser effect (NOE) constraints available for a nontethered 1:1 TAZ1:p53(13–61) complex in- dicated that the C terminus of the p53TAD is located close to the N terminus of TAZ1. The intein-mediated fusion proteins were therefore designed to place the p53 sequence at the N terminus and the TAZ1 sequence at the C terminus of the fusion protein; p53(1–61) was fused to the N-terminal intein and TAZ1 to the C-terminal intein. To obtain a selectively labeled complex Fig. 3. (A) Overlay of the 1H-15N HSQC spectra of the uniformly labeled – – in the form of a fusion protein, one intein fusion (isotopically TAZ1 p53TAD (nonintein) fusion (orange), and the p53TAD TAZ1 intein labeled) was mixed with the other intein fusion (unlabeled). fusion construct segmentally labeled in the p53TAD portion (blue) and in the TAZ1 portion (black). (B and C) Overlay of HSQC spectra for free p53TAD Splicing occurs as the two portions of the split intein coalesce, (cyan), with one molar equivalent of TAZ1 (red) and of the p53-labeled producing a p53TAD–TAZ1 fusion protein with a GSCFNGT p53TAD–TAZ1 fusion protein (black). Arrows indicate chemical shift changes linker sequence and with either the p53 or the TAZ1 component for individual cross-peaks. Complete spectra are shown in Figs. S4 and S5.

Krois et al. PNAS | Published online March 14, 2016 | E1855 Downloaded by guest on September 28, 2021 nontethered complexes; we are therefore confident that the p53– TAZ1/2 interactions have not been altered by fusing the binding partners into a single polypeptide chain. Residues N-terminal to AD1 are unstructured and do not seem to be involved in the binding process, as their 1H-15N HSQC cross-peaks are minimally perturbed upon addition of the TAZ domains. Residues beyond the C-terminal end of the AD2 helix are also unstructured in the complex. The backbone conformation for residues between E28 and D42 of the p53TAD has a relatively large RMSD between struc- tures in both the TAZ1 and TAZ2 complexes (central blue region, Fig. 4 A and C). This disorder might be due to local conforma- tional flexibility or merely reflect the low density of intermolecular NOE restraints in this region. Heteronuclear {1H}-15N NOE ex- periments were performed to provide a direct measure of the backbone flexibility of 15N-labeled p53 in the p53TAD–TAZ1 fusion, the (untethered) TAZ2:p53(1–61) complex, and free p53(1–61) (Fig. 5). Except at the N- and C terminus, the NOE values for the complexes are substantially higher than for the free protein, consistent with the transition from the disordered free p53TAD to a more ordered state in the complexes. The NOE values for the p53TAD in both complexes are very similar at most positions, with two local maxima corresponding to the positions of the AD1 and AD2 helices. The dip in the NOE values for residues 28–42 confirms that the backbone between the AD1 and AD2 motifs is flexible on the ns timescale, even when the p53TAD is bound to the surface of TAZ1 or TAZ2.

Interactions Between the p53TAD and TAZ2. In the TAZ2–p53(2–61) and TAZ2–p53(37–61) constructs, residues 43–54 of the p53TAD adopt very similar backbone structures and the side chains make similar contacts with the TAZ2 surface (Fig. 4 A and B). The AD2 helix and two hydrophobic residues (M44, L45) that precede it bind to a hydrophobic patch, formed by the side chains of I1773, M1799, V1802, V1819, L1823, A1825, L1826, C1828, and Y1829, at the interface between the α1, α2,andα3 helices of TAZ2 (Fig. 6A). Binding of the AD2 helix is mediated by the side chains of Fig. 4. Backbone representation of ensembles of 20 solution structures I50, W53, and F54, with the phenylalanine ring docked into a (Left) and ribbon representation of lowest AMBER energy models (Right)of concave pocket on the surface of TAZ2. The M44 side chain of (A) TAZ2–p53TAD, (B) TAZ2–p53AD2, and (C) and p53TAD–TAZ1 fusion p53 packs against the side chains of A1825, C1828, and Y1829 on proteins. TAZ1 is colored pink, TAZ2 is colored gold, and p53 is colored blue, helix α3 whereas that of L45 participates in an extensive network with the AD1 helix shown in turquoise and the AD2 region in purple. The α fusion linkers between the p53 and TAZ domains are shown in gray. Zinc atoms of hydrophobic interactions involving residues located on the 1 and α3 helices of TAZ2 as well as side chains of p53 itself (M44, are shown as gray spheres. The TAZ1 and TAZ2 helices α1–α4 are labeled. I50, and W53). This extensive hydrophobic cluster probably plays

p53(1–61)–TAZ1 fusion constructs are well-defined (Fig. 4). Similar to the structures in the unligated state (43, 44), each TAZ domain consists of a bundle of four helices (α1 – α4) with zinc atoms bound in the interhelical loops. The backbone atom RMSDs between free TAZ2 (43) and TAZ2 fused to the p53TAD and p53AD2 are 1.13Å and 1.01Å respectively, with the largest differences being in the relative position of helix α1 in the free protein. There is also a substantial shift in the position of the α2–α3 loop of TAZ2, which forms part of the binding surface for the AD1 motif, in the TAZ2–p53(2–61) complex. The overall structure of TAZ1 in the TAZ1–p53TAD fusion resembles that of the free domain, with a backbone RMSD between free TAZ1 (44) and TAZ1 in the p53TAD–TAZ1 fusion of 1.57Å.

Structure and Dynamics of p53TAD in Complex with the TAZ Domains. In the absence of binding partners, the p53TAD is intrinsically disordered. Upon binding to TAZ2 and TAZ1, the AD1 and 1 15 AD2 regions fold into amphipathic helices (Fig. 4) that span Fig. 5. Per-residue plot of { H}– N heteronuclear NOEs measured at 600 – – MHz for 15N-labeled p53(1–61) in the free state (black), in the untethered residues Q16 L25 and P47 T55 in the complex with TAZ2 and – – – – – TAZ2:p53(1 61) complex (blue), and in the p53(1 61) TAZ1 fusion protein Q16 L25 and P47 W53 when bound to TAZ1. The AD1 and (red). The NOE values for backbone amides are shown as circles whereas AD2 helices in the fusion proteins are bound to the same surfaces NOEs for Trp indole Ne resonances are shown as triangles. The AD1 and AD2 and in the same orientation as in preliminary structures of the sequences are shown as boxes colored turquoise and purple, respectively.

E1856 | www.pnas.org/cgi/doi/10.1073/pnas.1602487113 Krois et al. Downloaded by guest on September 28, 2021 PNAS PLUS

Fig. 6. Interaction between p53TAD and (A) TAZ2 and (B) TAZ1. (Top) Overview of the lowest AMBER energy structures, showing the positions of AD1 (turquoise) and AD2 (purple). (Bottom) Local side chain interactions for AD2 (purple) and AD1 (turquoise).

a major role in stabilization of the complex. The importance of TAZ2. These differences may explain why binding at this site hydrophobic interactions in this region has been previously dem- represents the “weaker” interaction of p53TAD with TAZ2. onstrated, as a W53Q/F54S mutant greatly diminishes AD2 binding to TAZ2 (24). The heteronuclear NOE for the indole NeHofW53 Interactions Between the p53TAD and TAZ1. Binding of p53TAD to is similar to the backbone amide NOEs in the AD2 helix (Fig. 5), TAZ1 is also mediated by hydrophobic interactions. Similar to its consistent with tight packing of theW53sidechaininthemolecular interaction with TAZ2, the AD2 helix binds at the interface of α α α interface. The regions of TAZ2 surrounding the α –α –α hydro- the 1, 2, and 3 helices of TAZ1. Residues N-terminal to the 1 2 3 α α phobic binding surface are strongly electropositive and acidic side AD2 helix pass through a surface groove between 1 and 2 and α chains on p53, namely D41, D42, and E51, make complementary wrap around 1; in the TAZ2 complex this part of the p53 chain α α electrostatic interactions (Fig. S6A). passes between the 1 and 3 helices. Residues L43, M44, L45, The p53 binding mode is very similar in the structures of the P47, I50, W53, and F54 of the AD2 region make hydrophobic TAZ2–p53(2–61) and TAZ2–p53(37–61) fusion proteins (Fig. contacts with L352, I353, Q356, L359, L360, M387, V390, S410, 7A), with a backbone RMSD of 0.85Å between the two struc- S411, I414, and V428 of TAZ1 (Fig. 6B). The F54 side chain α tures for p53 residues L43 to T55. The conformations of the reaches to the opposite side of 1 to make a hydrophobic contact with V428 at the beginning of α4. M40, L43 and L45 stack against

hydrophobic side chains of I50, W53, and F54 are almost iden- BIOPHYSICS AND α α α tical in the two structures while the preceding residues, M44 and 1 whereas M44 contacts 1 and the start of 2. I50 and W53 α α COMPUTATIONAL BIOLOGY L45, vary only slightly. The similarities in the mode of binding of protrude into the groove between 1 and 3. As in the complex the p53 AD2 region in the independently determined TAZ2– with TAZ2, the W53 side chain is tightly packed in the molecular – p53AD2 and TAZ2–p53TAD fusion protein structures provide interface and the heteronuclear NOE shows that its ps ns strong validation of our structural model. timescale motions are restricted (Fig. 5). The p53 structure be- In the absence of AD2, the isolated AD1 motif (residues 13– tween AD1 and AD2 is not well-defined, with increased back- 37) binds preferentially, but with much lower affinity than AD2 bone RMSD and decreased heteronuclear NOEs. Compared with the binding pocket of AD2 on TAZ2, the AD2 (24 μM vs. 32 nM), to the AD2 binding site at the TAZ2 α1–α2– binding site on TAZ1 presents a less continuous hydrophobic α3 interface; there is an even weaker (160 μM) secondary binding surface due to the presence of Q355, Q356, S410, S411, and site for the isolated AD1 in the vicinity of the α3 and α4 helices (54). In the context of the full-length p53TAD, AD1 occupies its Q413, which are all either in or proximal to the binding interface. secondary binding site on TAZ2, a deep hydrophobic groove Previous structures of TAZ1 in complexes with transactivation between the α3 and α4 helices and the α2–α3 loop, formed by residues T1812, L1823, I1824, A1825, F1843, I1847, and L1851 (Fig. 6A). The hydrophobic side chains of F19, L22, W23, L25, and L26 of p53TAD all project into this groove to make favor- able intermolecular contacts. The proximity of the K1850 side chain to the indole ring of W23 suggests the possibility for pi– cation interactions, which would be consistent with the NOE cross-peaks observed between these residues. Similar to W53, the ps–ns timescale dynamics of the W23 side chain are restricted in the TAZ2 complex (Fig. 5). Residues N-terminal to the AD1 helix are largely unstructured and lack long-range NOEs. The hydrophobic side chains of V31 and L32 of p53 contact L1851 and the aliphatic portion of K1832; however, the side chain confor- mations are not well defined and the interaction is likely transient Fig. 7. (A) Comparison of the AD2 region of p53 in the structures of the given the decreased heteronuclear NOE values for these p53 TAZ2–p53(2–61) (purple) and TAZ2–p53(37–61) (lavender) fusion proteins. residues (Fig. 5). The region of TAZ2 to which AD1 binds pre- The TAZ2 surface is shown in pale yellow. (B) Superposition of the structure of the TAZ2–p53(37–61) fusion protein (p53 AD2 region, purple; TAZ2 surface) and sents less exposed hydrophobic surface area than does the AD2 that of the complex between TAZ2 and an isolated p53AD1 peptide (brown) binding region, and there is less charge complementarity in the (2K8F, ref. 38). (C) Superposition of the structure of the TAZ2-p53(37-61) fusion AD1 binding site. This difference is mainly due to the presence of protein (p53 AD2 region, purple; TAZ2 surface) and that of the complex between several polar residues on the flexible loop between α2 and α3 of TAZ2 and an isolated p53AD2 peptide (red) (2MZD, ref. 39).

Krois et al. PNAS | Published online March 14, 2016 | E1857 Downloaded by guest on September 28, 2021 domains have demonstrated that TAZ1 prefers to bind to - Here we report, to our knowledge, the first structures of the tively long intrinsically disordered regions containing multiple full-length p53TAD bound to the TAZ1 and TAZ2 domains of amphipathic motifs (45–50). By contrast, TAZ2 mostly interacts CBP/p300. The structure of the TAZ2 complex, which is likely to with short amphipathic helices. As such, isolated subdomains of be representative of the complex in vivo where intact AD1 and p53TAD may not represent an ‘ideal’ TAZ1 binding protein. AD2 motifs are required for efficient transactivation of the These differences may help explain the two orders of magnitude majority of p53-regulated genes (31, 63), differs significantly from lower Kd for binding of an AD2 peptide to TAZ2 compared with previously reported structures of the isolated AD1 and AD2 TAZ1 (0.055 μM vs. 4.9 μM) (25). peptides bound to the p300 TAZ2 domain. The isolated p53AD1 The AD1 motif of p53 binds in a deep hydrophobic pocket, and p53AD2 peptides bind preferentially to the same site on surrounded by positively charged side chains, formed by all four TAZ2, at the interface between the α1, α2,andα3 helices; how- of the TAZ1 helices. The side chains of F19, L22, and W23 in ever, AD2 binds with nearly 500-fold greater affinity than AD1 AD1 pack against L357, V358, L360, and L361 (α1), L391, M394, (0.055 μMvs.27μM) (25). Both motifs also bind with lower af- and T395 (α2), I415 and W418 (α3), L432 and A435 (α4). The finity to a common secondary site on the opposite surface of N-terminal region of AD1 makes contact with α4,andthe TAZ2, but again the strongest interaction is with AD2 (10 μM C-terminal region, starting with L26, begins to wrap around vs. 164 μM) (54). The affinity of the full-length TAD for TAZ2 the α1 helix of TAZ1 (Fig. 6B). L14 makes contact with W418, (0.026 μM) is increased only slightly over that of AD2 alone (24, although the structure in this region is poorly defined and the 25). On the basis of these observations, it is not surprising that our heteronuclear NOE data suggest that the interaction is likely to structure of the full-length p53TAD complex, with both the AD1 be transient (Fig. 5). Similarly, L32 binds in the groove between and AD2 motifs bound to different sites on TAZ2, differs from helices α1 and α4; as with L14, heteronuclear NOE data suggest that of the complex formed by AD1 alone (38). The structure this contact is transient. The side chain of F19, on the first turn shows the AD2 motif of the full-length p53TAD bound to the of the AD1 helix, is deeply buried in the p53:TAZ1 interface, hydrophobic surface that was previously identified through chem- closely packed against the plane of the indole ring of W418 and ical shift titrations as the primary binding site for isolated AD2 (54). contacting hydrophobic residues on helices α1, α3, and α4. The The very high affinity of AD2 for its primary site forces AD1 to side chain of W23 at the C-terminal end of the AD1 helix packs bind in its weak (164 μM) secondary binding site, where it con- 15 against L391, M394, and I415 on α2 and α3. The Ne resonance tributes very little to the overall binding free energy. of W23 has a much smaller heteronuclear NOE (0.39 ± 0.01) Comparison of our structures of the TAZ2–p53(2–61) and than the backbone NHs in the AD1 helix, suggesting significant TAZ2–p53(37–61) fusions with that of the TAZ2:AD1 complex side chain fluctuations on a picosecond to nanosecond timescale. of Feng et al. (38) suggests a molecular basis for the difference in This flexibility is fully consistent with AD1 being the weaker AD1 and AD2 affinities. Although the AD1 and AD2 helices binding subdomain of the bipartite p53TAD. In contrast to the bind in a similar location and orientation on TAZ2 (Fig. 7B), the TAZ2 complex, where L25 and L26 form an integral part of the AD2 motif makes significantly more hydrophobic contacts with molecular interface, the L25 side chain projects into solvent the surface of TAZ2. The side chains of W53 and F54 of AD2 where it makes minimal contact with TAZ1. L26 contacts the are deeply buried in the hydrophobic groove formed by the α1, α2, aliphatic portions of K365 and R368, two of the many positively and α3 helices and additional hydrophobic contacts are made by charged residues (the others being H364, K419, K433, K438, and M40, L41, and I50. In contrast, AD1 side chains are not well packed R439) that ring the AD1 binding pocket. in this pocket; only T18, F19, and L22 make hydrophobic contacts Although the AD2 motif of the p53TAD binds to both TAZ1 with TAZ2 while the W23, L25, and L26 side chains are on the and TAZ2 in a hydrophobic groove at the interface between the surface of the complex and are almost fully solvent exposed (38). α1, α2, and α3 helices (Fig. S7), the orientation of the AD2 helix An NMR structure of the complex between an isolated is opposite in the two structures. Although L45 and the hydro- p53(35–59) peptide and the p300 TAZ2 domain was recently phobic side chains on the AD2 helix adopt similar conformations reported (PDB accession code: 2MZD) (39), which differs sub- in the two complexes, the intermolecular interactions are differ- stantially from the structures determined in the present work. ent. For example, the W53 indole ring packs against hydrophobic The AD2 motif binds to the same hydrophobic surface on TAZ2 side chains in helix α3 of TAZ2, whereas it packs primarily against in both of the structures, but the orientation of the AD2 helix helix α1 in the TAZ1 complex. As a result of differences in the differs by ∼90° (Fig. 7C). The p53(35–59) peptide in structure orientation of helix α4 (Fig. S7A), the AD1 motif binds to dif- 2MZD uses the same hydrophobic residues (I50, W53, and F54) ferent surfaces of the TAZ 1 and TAZ2 domains. In TAZ2, the to bind to TAZ2; however, these side chains pack very differently α4 helix extends away from the core of the protein and creates a compared with our TAZ2–p53(2–61) and TAZ2–p53(37–61) hydrophobic groove between α3, α4, and the α2–α3 loop that structures. In structure 2MZD, the only residue fully buried in accommodates the AD1 helix. In contrast, the α4 helix of TAZ1 the hydrophobic groove of TAZ2 is I50 whereas W53 packs packs tightly against α1, creating an extensive hydrophobic sur- along the surface of helices α2 and α3, and F54 packs along the face, comprising side chains from the α1, α2, α3 and α4 helices, aliphatic portion of R1737 (CBP R1775) located on helix α1.By that binds the AD1 motif (Fig. S7C). contrast, in our TAZ2–p53(2–61) and TAZ2(37–61) structures, I50, W53, and F54 are all deeply buried in the hydrophobic in- Discussion terface between the AD2 helix and TAZ2. The hydrophobic The present work provides valuable insights into the molecular residues preceding the AD2 helix (M44 and L45, and to a lesser interactions through which p53 recruits CBP and p300 to activate extent L43) pack along the surface of the TAZ2 α3 helix in all of transcription of stress response genes and underscores the com- the structural models. The observed differences between 2MZD plex nature of molecular recognition by intrinsically disordered and the present structures are highly unlikely to be a conse- proteins containing multipartite binding motifs. Three structures quence of fusing the p53 peptides to the TAZ2 domain. Firstly, have been reported previously for complexes of the p53TAD with the orientation of the AD2 helix is identical in the structures of domains of CBP/p300: p53(13–61) bound to the CBP nuclear the TAZ2–p53(2–61) and TAZ2–p53(37–61) fusions (Fig. 7A), coactivator binding domain (NCBD) (62), p53(1–39) bound to the where the “linkers” between the two proteins are 52 and 16 p300 TAZ2 domain (38), and p53(35–59) bound to p300 TAZ2 residues in length, respectively. Secondly, the AD2 helix is bound (39). The NCBD:p53TAD complex includes both the AD1 and in a very similar position and orientation in the structures of the AD2 subdomains of p53TAD whereas the TAZ2 complexes two fusion constructs and in preliminary structures of the non- contain either the AD1 or AD2 motif. fused 1:1 p53(13–61):TAZ2 complex. We are therefore confident

E1858 | www.pnas.org/cgi/doi/10.1073/pnas.1602487113 Krois et al. Downloaded by guest on September 28, 2021 that our structural models accurately represent the molecular in- T18 close to K438 and R439, S20 close to K418, and S33 close to PNAS PLUS terface between the CBP TAZ2 domain and the p53 AD2 motif. H362, K365, and R369. Although single site phosphorylation in We suggest two possible reasons for the observed differences this region leads to only a modest (∼twofold) increase in affinity, relative to 2MZD. The side chains that form the binding surface two- or three-site phosphorylation increases the TAZ1 binding for the AD2 motif are 100% identical in the CBP and p300 TAZ2 affinity by as much as 10-fold (12). T55, in the AD2 region of domains. However, the p300 TAZ2 construct used for structure p53, is proximal to K349. S37 and S46 are more distant from determination was mutated to replace four Cys residues (C1738A, basic residues in the family of NMR structures, but still contribute C1746A, C1789A, C1790A) by Ala (39) whereas the WT CBP to enhancement of the binding affinity upon phosphorylation (12, sequence, retaining all four Cys residues, was used in the present 13). It is of note that S46 is located at the N-cap position of the work. One of the mutated cysteines (C1738A in p300, C1776 in AD2 helix, and its phosphorylation may lead to substantial stabi- CBP) lies in the binding pocket for the AD2 helix, and it is pos- lization of the helical state (66). Similar electrostatic interactions sible that mutation of this residue influences the binding interface. are observed in the TAZ2–p53TAD structure (Fig. S6A). For Alternatively, the structure might represent an alternate binding p53AD1 bound to TAZ2, T18 and S20 are positioned close to conformation that is stabilized under the different buffer and so- K1850 and K1812 respectively. Within AD2, phosphorylation of lution conditions used to collect the p300 NMR data. S46 could serve to N-cap and stabilize the AD2 helix as well as promote interactions with the nearby side chain of R1769 whereas Comparison with Other p53 Transactivation Domain Structures. phosphorylation of T55 could stabilize interactions with K1798. Structures have now been reported for complexes of the p53 Although S15, S33, and S37 in the p53TAD do not make specific AD2 motif with several target proteins. The motif adopts a similar contacts with basic side chains of TAZ2 in the NMR structural structure, with helix between residues P47 and F54 and very similar ensemble, all are located close to electropositive regions of the conformations of the hydrophobic side chains, when bound to TAZ2 surface such that phosphorylation could enhance binding TAZ1, TAZ2 (this work), replication protein A (36), and the PH A through bulk electrostatic effects. Indeed, bulk electrostatics seem domain of the Tfb1 subunit of TFIIH (37) (Fig. 8 ). It thus seems to play a dominant role since the degree of enhancement of TAZ1 that the local amino acid sequence predisposes AD2, which is or TAZ2 binding upon phosphorylation of the p53TAD is de- disordered in the unbound state, toward a well conserved structure termined primarily by the number of phosphoryl groups and not when bound to its targets. A similar AD2 structure is observed in by their location in the p53 sequence (12). Similar to TAZ1, two- the complex with HMGB1, although the helix is somewhat shorter site and three-site phosphorylation of the AD1 region of the (64). Somewhat surprisingly, given the strong propensity of AD2 to p53TAD increases the TAZ2 binding affinity by up to 20-fold (12). fold into a helix, a doubly phosphorylated p53 AD2 motif binds in a more extended conformation to a highly electropositive region of Implications of the Bipartite Activation Domain. The bipartite p53 the PH domain of human TFIIH p62 (65). N-terminal activation domain interacts with its binding partners The AD1 region adopts a helical backbone conformation be- via two short amphipathic motifs that bind simultaneously to the tween T18 or F19 and L25 in its complexes with TAZ1, TAZ2, B target protein and fold into helical structure upon binding. In and MDM2 (this work and refs. 35 and 38) (Fig. 8 ). However, both the TAZ1 and TAZ2 complexes, the AD1 and AD2 motifs in contrast to hydrophobic side chains in AD2, the side chains of

bind to opposite surfaces of the target protein. Heteronuclear BIOPHYSICS AND F19 and W23 occupy different rotamers in the different AD1 NOE measurements reveal enhanced backbone flexibility of the COMPUTATIONAL BIOLOGY complexes, reflecting differences in the binding surfaces of the p53 residues between the motifs, and most of the side chains in target proteins. this region are disordered and make few or no contacts with the TAZ domains (Fig. 5). Despite the flexibility of the intervening Enhancement of CBP Binding by p53 Phosphorylation. Phosphoryla- sequence, binding is synergistic and the overall binding affinity of tion of the p53TAD has been shown to greatly enhance binding affinity for the TAZ domains (12, 13). The p53TAD can be the full-length p53TAD is two- to fivefold higher than for the AD2 phosphorylated at nine sites, many of which are well positioned to motif in isolation and up to 1,000-fold higher than for the isolated make favorable electrostatic interactions with TAZ1 and TAZ2. AD1 motif (25). Similar behavior was observed for the complex Phosphoryl groups can be accommodated at all of these sites of the p53TAD with the nuclear coactivator binding domain of without steric clashes with the TAZ domains. In the TAZ1 CBP (62). The exposed and flexible nature of the peptide be- complex, the AD1 motif binds in a region ringed with basic tween the AD1 and AD2 subdomains suggests the possibility of residues (Fig. S6B) with S15 positioned close to R439 and K433, posttranslational modification in this region while the bipartite p53TAD remains bound to TAZ2 or TAZ1. In addition to binding synergistically to a single target protein in a binary complex, the AD1 and AD2 interaction motifs of the p53 transactivation domain can also bind independently to two different target proteins to form a ternary complex. In the com- plexes with the TAZ2 and TAZ1 domains, the p53 AD2 motif occupies a more favorable binding pocket than does AD1 and makes the dominant contribution to the binding free energy (25). Because many of the residues between AD1 and AD2 remain flexible in the complex and contribute little to the binding inter- actions, ternary complexes can be formed with AD2 bound to TAZ1/2 and AD1 bound to a different target, such as MDM2, leading to cross talk between different signaling pathways (25). The ternary complex between p53, MDM2, and CBP/p300 seems Fig. 8. (A) Superposition of residues P47–F54 in the AD2 helix in complexes to play a central role in regulation of p53 stability, determining in with TAZ1 (pink, this work), TAZ2 (green, this work), replication protein A a phosphorylation-dependent manner whether p53 becomes [red (36)], and the Tfb1 subunit of TFIIH [blue (37)]. A schematic view of the backbone is shown, and the hydrophobic side chains are shown as sticks. polyubiquitinated and degraded or is activated to initiate tran- (B) Superposition of residues F19–L25 in the AD1 helix in complexes with scription of p53-regulated stress response genes (25, 67). The TAZ1 (pink, this work), TAZ2 (green, this work), TAZ2 [blue (38)], and MDM2 ability of multipartite systems to promiscuously interact with binding [red (35)]. The hydrophobic side chains are shown as sticks. partners has been shown with other CBP/p300 binding proteins

Krois et al. PNAS | Published online March 14, 2016 | E1859 Downloaded by guest on September 28, 2021 and may represent a widely used mechanism by which proteins dominates binding of the p53TAD to the TAZ domains (24, 25), can use disparate motifs to regulate transcription (53, 68, 69). the AD2 and AD1 subdomains are both required for trans- The present work clearly illustrates the dangers of a re- activation of most p53 target genes and L22Q/W23S (human ductionist approach to characterize target binding by intrinsically numbering) mutant p53 is unable to induce cell cycle arrest or disordered proteins containing two or more interaction motifs. apoptosis in response to DNA damage in knock-in mice (31, 73). The isolated AD1 and AD2 regions of the p53 transactivation Activation of p21-like genes, which requires CBP/p300-mediated domain interact promiscuously with their targets (25, 53, 54). For acetylation of K382 in the C-terminal regulatory domain of p53, example, in the absence of AD2, the AD1 motif binds prefer- is severely impaired by deletion or mutation of the AD1 sub- entially in the AD2 binding site at the interface of the α1, α2, and domain (73, 74), suggesting that synergistic interactions involving α 3 helices, as was observed in the structure of the complex of both motifs are required for efficient recruitment of CBP/p300. TAZ2 with an AD1 peptide (38), and only weakly in a secondary Our results here demonstrate a structural basis for the impor- α α site between the 3 and 4 helices. When both motifs are present tance of AD1 in regulating the p53-mediated cellular stress re- in the same polypeptide chain, as in the present work, the AD2 sponse. In contrast to previous reports describing interactions of motif out-competes AD1 for binding in the preferred high af- TAZ2 with the isolated AD1 and AD2 subdomains (38, 39), the finity site, forcing AD1 to occupy its secondary binding site on current work reveals the molecular basis for binding of the full- TAZ2. Studies using isolated subdomains of disordered proteins length p53 transactivation domain to both TAZ1 and TAZ2, that contain bipartite or multipartite interaction motifs can po- providing insights into critical interactions required for activation tentially yield misleading results. of the p53-regulated damage response and showing how in- Although the AD2 subdomain of p53 dominates the interac- trinsically disordered proteins can recognize disparate binding tion with TAZ1 and TAZ2 and binds in a highly preferred site, partners. interactions with other targets can be more promiscuous. The p53 TAD forms an ensemble of conformational states in complex with Materials and Methods the KIX domain of CBP/p300, with each of the AD1 and AD2 Protein Production. The TAZ1 and TAZ2 domains of mouse CBP (residues 340– helices binding simultaneously in two distinct orientations and in 439 and 1764–1855, respectively) and p53TAD (residues 1–61 of human p53) two distinct site on the KIX surface (53). Similar promiscuous were expressed and purified as previously described (25). Fusion proteins interactions with both KIX sites have also been observed for the were expressed with the TAZ2 domain of mouse CBP (1764–1855) joined via bipartite transactivation domain of the FOXO3a transcription a (Gly-Ser)3 linker to the transactivation domain of p53. The design of the factor and are required for full transcriptional activity (68). fusion constructs was guided by knowledge of the binding surfaces derived from chemical shift titrations with free p53TAD constructs and by pre- liminary low-resolution NMR structures determined for the 1:1 TAZ2:p53(13– Competition with Other Transactivation Domains. p53 must compete 61) complex, which showed the location and orientation of the AD1 and with a multitude of cellular transcription factors for binding to AD2 helices. The linker length and site of fusion were optimized to give high- limiting amounts of CBP/p300 in the cell (41, 70, 71). Structures quality HSQC spectra, free of exchange broadening. A TAZ2-(GS)3–p53(2–61) have been determined for TAZ2 bound to the activation do- fusion gave high-quality NMR spectra with more uniform cross-peak in- mains of STAT1 (49), and C/EBP (51), both of which bind tensities. In contrast, a construct with a shorter linker between the C ter-

through helical structures to the same hydrophobic surface as the minus of TAZ2 and the AD1 motif (TAZ2-(GS)3–p53(13–61) resulted in exchange p53 AD2 motif. The adenovirus oncoprotein E1A and onco- broadening of resonances associated with the AD1 binding site. The TAZ2- – – protein E7 from high-risk strains of human papillomavirus also (GS)3 p53(2 61) fusion was therefore used for NMR structure analysis. Data bind to the same surface on TAZ2 as the AD2 motif of p53, but were also acquired for a TAZ2-GSGSG–p53AD2(37–61) fusion. The TAZ2–p53 with higher affinity (E1A, 3 nM; phosphorylated E7, 7 nM; p53, fusions were expressed in Escherichia coli BL21 (DE3) [DNAY] cells in M9 mini- – β 26 nM) (25, 52, 72). By binding with higher affinity to the same mal media for 12 16 h at 20 °C after induction with 0.8 mM isopropyl -D- thiogalactopyranoside (IPTG) and 150 μMZnSO4. Cells were lysed via sonication region of TAZ2, the viral proteins can out-compete p53 for in 20 mM Mes, pH 6.1, 40 mM NaCl, and 10 mM DTT. These constructs were binding to CBP/p300 and thereby inhibit activation of p53- purified via SP Sepharose on a gradient from 20 mM Mes, pH 6.1, 40 mM NaCl, mediated stress response or apoptotic pathways. 2 mM DTT to 20 mM Mes, pH 6.1, and 1 M NaCl. Further purification was Structures have been reported for TAZ1 bound to the acti- achieved via Sephacryl S100HR size exclusion in 20 mM Tris, pH 6.8, 100 mM vation domains of the hypoxia inducible factor 1α (HIF-1α) (45, NaCl, and 2 mM DTT. Purity was verified by SDS/PAGE and analytical HPLC, and 46), CITED2 (47, 48), STAT2 (49), and RelA (50), all of which molecular weights were verified by MALDI. Purified product was exchanged bind in the same hydrophobic groove as the p53TAD. Upon into 20 mM Tris, pH 6.8, 50 mM NaCl, 2 mM DTT, and 10% (vol/vol) D2Ofor binding to TAZ1, all of these activation domains form local el- NMR data collection. ements of amphipathic helical structure, but for the most part A segmentally labeled fusion protein with the C terminus of the p53TAD are located in different regions of the hydrophobic groove. Thus, joined to the N terminus of the TAZ1 domain was also constructed on – the p53 AD2 helix binds to the same surface as the C-terminal the basis of preliminary low-resolution structures of a 1:1 TAZ1:p53(13 61) complex. Segmentally labeled p53TAD–TAZ1 constructs were generated (αC) helix of HIF-1α but in a different orientation whereas the using the Nostoc punctiforme PCC73102 (Npu) DNA polymerase III (DnaE) AD1 motif binds in a groove that is occupied by the LPQL motif – α α intein system (75 77). p53TAD was expressed with an N-terminal H6GB1 tag and B helix of HIF-1 (45, 46). Although all of the above ac- and was fused to the N terminus of the N-split of the DnaE intein (Fig. 1D). Φ ΦΦ ΦΦ Φ Φ tivation domains contain XX or XX motifs, where The region between H6GB1 and p53(1–61) contained a tobacco etch virus is a bulky hydrophobic residue, they share little in common in (TEV) protease cleavage site. This construct was expressed in E. coli BL21 their interactions with TAZ1, which are clearly determined by (DE3) [DNAY] cells for 20 h at 16 °C in either isotopically enriched or un- the distribution of the amphipathic clusters within the peptide enriched media after induction with 1 mM IPTG. The TAZ1 domain of mouse sequence and by the nature of the flanking amino acids. CBP (residues 340–439) was expressed fused to the C terminus of the C-split of the DnaE intein. This construct had an H6GB1 tag N-terminal to the intein The p53-Mediated Stress Response Requires the Full-Length TAD. The fragment (Fig. 1D). This construct was expressed for 20 h at room temper- interaction between p53 and CBP/p300 is essential for activation ature after induction with 0.1 mM IPTG. Cells for both constructs were lysed of p53-mediated transcriptional programs in response to cellular via sonication in 20 mM Tris, pH 8.0, and 200 mM NaCl and purified using His60 Ni SuperflowTM Resin (Clontech) with elution in 20 mM Tris, pH 8.0, stress and genomic damage. High affinity binding is mediated 200 mM NaCl, and 150 mM imidazole. Intein ligation was performed at a primarily by interactions of the intrinsically disordered N-ter- ratio of 1:1.2 p53:TAZ1 in 20 mM Tris, pH 8.0, 200 mM NaCl, 0.5 mM tris(2- minal transactivation domain of p53 with the TAZ domains of carboxyethyl)phosphine (TCEP) for 1 h at room temperature before addition CBP/p300 and is enhanced by multisite phosphorylation of p53 of 1/200 molar equivalent of TEV protease. Intein ligation and TEV protease in response to cellular stress (12, 13). Although the AD2 motif cleavage reactions were carried out for an additional 12–15 h at 4 °C.

E1860 | www.pnas.org/cgi/doi/10.1073/pnas.1602487113 Krois et al. Downloaded by guest on September 28, 2021 After completion of ligation and protease cleavage, the mixture was run TAZ1(14N) fusion protein (400 μM), and a sample of 300 μM 15N p53(1–61) PNAS PLUS over a 5-mL His60 Ni Superflow column (Clontech). The flow-through was with 1.2-fold excess of unlabeled TAZ2. purified via a 2 × 5-mL HiTrap Q HP Column (GE) on a linear gradient from 20 mM Tris, pH 7.5, 40 mM NaCl, 1 mM DTT to 20 mM Tris, pH 7.5, 1 M NaCl. Structure Calculations and Analyses. Initial structure generation and NOE The p53TAD–TAZ1 fusion eluted at ∼150 mM NaCl. Lastly, the fusion was assignment were performed through CYANA with CANDID (60), using a eluted from a 345-mL S100 size exclusion column with buffer containing subset of manually assigned, unambiguous NOE restraints. Distance re- 20 mM Tris, pH 8.0, 500 mM NaCl, and 1 mM DTT. Samples were exchanged straints were derived from NOE cross-peak volumes. Dihedral angle re- 13 13 1 1 15 into 20 mM Tris, pH 6.8, 50 mM NaCl, 1 mM DTT, and 5% D2O for NMR data straints were determined from Cα, Cβ, Hα, HN and N chemical shifts collection. The ligation reaction and purification were monitored by ana- using TALOS+ (59). Any nonglycine residues whose dihedral angles were lytical HPLC and SDS/PAGE, with ligation efficiency consistently above 90%. poorly defined in the TALOS+ output were restrained to negative phi an-

The molecular mass of the product was confirmed by mass spectroscopy. gles. Where possible, χ2 angles for leucine and isoleucine were derived from 13C chemical shifts (83, 84), and all leucine and isoleucine side chains were 1 15 15 15 15 NMR Experiments. H- N HSQC spectra for N p53(1–61), N TAZ1, 1:1 N restrained to sterically favorable regions of χ1,χ2 conformational space. Hy- p53TAD:14NTAZ1,1:115NTAZ1:14N p53TAD, and the p53TAD(15N)–TAZ1(14N) drogen bond restraints were used in the helices of TAZ2 and TAZ1 for initial and p53TAD(14N)–TAZ1(15N) fusion proteins were acquired with 100 μM pro- structure generation, but all hydrogen bond restraints (with the exception tein concentrations in 20 mM Tris, pH 6.8, 50 mM NaCl, 1 mM DTT, and 5% of the α1 and AD1 helices in TAZ2–p53TAD and α1 and α4 helices in TAZ2– 1 -15 15 15 14 D2O (NMR buffer). H N HSQC spectra for N TAZ2, 1:1 N p53TAD: N p53AD2) were removed before further refinement. Helical regions were TAZ2, 1:1 15N TAZ2:14N p53TAD, and the 15N TAZ2–p53TAD and 15N TAZ2– identified by comparison with previously determined TAZ2 and TAZ1 p53AD2 fusion proteins were acquired at 100-μM protein concentrations in structures and using dihedral angle restraints derived from TALOS+ (43, 44). NMR buffer. Hydrogen bond restraints for the α1 helix of TAZ2 and the p53AD1 helix in Spectra for resonance assignment and for restraint generation were ac- TAZ2–p53TAD were retained in the final structures to correct bends in the quired on Bruker Avance 900-MHz, DRX 800-MHz, DRX 600-MHz, and Avance helices that arose from the absence of long-range distance restraints in the 500-MHz spectrometers and were processed using NMRPipe (78) and N-terminal half of each helix. Hydrogen bond restraints were also main- α – NMRView5.0 (79). tained in 4 of TAZ2 in TAZ2 p53AD2 because of a similar lack of restraints – Backbone resonances for the p53TAD–TAZ1 construct were assigned using in the C-terminal half of the helix. Additionally, for the p53TAD TAZ1 fusion – HNCACB, HN(CO)CA, and 15N TOCSY spectra (80, 81) collected on segmen- protein, all distance restraints for residues 28 41 of p53 (region between tally labeled p53TAD(15N/13C)–TAZ1 and p53TAD–TAZ1(15N/13C) samples. AD1 and AD2) were loosened by 1 Å in an attempt to more realistically model These samples were also used to make side chain assignments using 3D the flexibility, indicated by the heteronuclear NOE, in this region. Sets of 200 HCCH-COSY and HCCH-TOCSY spectra (82). Backbone and side chain reso- initial structures were generated using CYANA and refined by restrained nance assignments for the TAZ2–p53TAD and TAZ2–p53AD2 constructs were molecular dynamics with AMBER12 (85, 86). The structures underwent 1,000 derived from 15N NOESY-HSQC, 13C NOESY-HSQC, and HNCACB experiments. steps of energy minimization with 20 ps of simulated annealing in vacuum or These assignments were validated by comparisons with assignments of TAZ2 in a generalized Born solvent model (61). The system was heated to 1,000 K complexes with multiple p53 constructs, including AD1(13–37), AD2(38–61), for the first 2 ps, kept at constant temperature for 4 ps, and then annealed – by cooling to 0 K over 12 ps. Force constants for NOE restraints were main- and p53(13 61). Assignments of these complexes were made through use of −1 -2 1 15 1 13 tained at 30 kcal·mol ·Å , and force constants for angle restraints were H- N and H- C HSQC titrations, HNCA and HNCO spectra, and HCCH and −1 −2 15 13 1,000 kcal·mol ·rad . Structures and restraints were initially refined using suc- CCHC COSY spectra, and through N NOESY-HSQC and C NOESY-HSQC cessive cycles of simulated annealing in vacuum. After final refinement using a experiments. These assignments were further validated by comparison with generalized Born solvent model, the 20 structures with the lowest AMBER en- previously published assignments of free TAZ2 (43), TAZ2 bound with STAT1 ergy were selected for analysis. Structure quality was assessed using PROCHECK-

(49), and bound chemical shifts of p53 with the NCBD and KIX domains of BIOPHYSICS AND NMR (87) and the Protein Structure Validation Software suite (88). Figures were CBP (53, 62). 15 prepared using the PyMOL Molecular Graphics System (Schrödinger, LLC). COMPUTATIONAL BIOLOGY Distance restraints were obtained from N NOESY-HSQC (τm = 100 ms) 13 and C NOESY-HSQC (τm = 120 ms) spectra. For the p53TAD–TAZ1 fusion 13 ACKNOWLEDGMENTS. We thank Gerard Kroon for expert assistance with protein, a C-filter-edit NOESY-HSQC spectrum (τm = 200 ms) was used to – NMR experiments, Benjamin Leach and Sulakshana Mukherjee for advice on derive intermolecular distance restraints (58). NMR data for the TAZ2 structure calculations, and Rebecca Berlow and Shanshan Lang for review of – p53TAD and TAZ2 p53AD2 constructs were collected at 32 °C, and data this manuscript. This work was supported by Grant CA096865 from the 1 15 for p53TAD–TAZ1 were acquired at 35 °C. { H}- N heteronuclear NOE data National Institutes of Health and by the Skaggs Institute for Chemical Biology. were acquired at 600 MHz and 35 °C on free 15Np53(1–61) (100 μM), p53(15N)– J.C.F. was supported by a Leukemia and Lymphoma Society Special Fellowship.

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