Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions.

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short ß-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREBTAD) construct. The CREBTAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR.

The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), which are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using trans-intein splicing, we have generated full-length p53 constructs in which the C-terminal region is isotopically labeled with 15N for NMR analysis, allowing us to obtain atomic-level information on the C-terminal domains in the context of the full-length protein. Resonances of NLS and CTD residues have narrow linewidths, showing that these regions are largely solvent-exposed and dynamically disordered, whereas resonances from the folded TET are broadened beyond detection. Two regions of the CTD, spanning residues 369-374 and 381-388 and with high lysine content, make dynamic and sequence-independent interactions with DNA in regions that flank the p53 recognition element. The population of DNA-bound states increases as the length of the flanking regions is extended up to approximately 20 base pairs on either side of the recognition element. Acetylation of K372, K373, and K382, using a construct of the transcriptional coactivator CBP containing the TAZ2 and acetyltransferase domains, inhibits interaction of the CTD with DNA. This work provides high-resolution insights into the behavior of the intrinsically disordered C-terminal regions of p53 within the full-length tetramer and the molecular basis by which the CTD mediates DNA binding and specificity.

Structural and dynamic studies of DNA recognition by NF-κB p50 RHR homodimer using methyl NMR spectroscopy.

Protein dynamics involving higher-energy sparsely populated conformational substates are frequently critical for protein function. This study describes the dynamics of the homodimer (p50)2 of the p50 Rel homology region (RHR) of the transcription factor NF-κB, using 13C relaxation dispersion experiments with specifically (13C, 1H)-labeled methyl groups of Ile (δ), Leu and Val. Free (p50)2 is highly dynamic in solution, showing µs-ms relaxation dispersion consistent with exchange between the ground state and higher energy substates. These fluctuations propagate from the DNA-binding loops through the core of the domain. The motions are damped in the presence of κB DNA, but the NMR spectra of the DNA complexes reveal multiple local conformations of the p50 RHR homodimer bound to certain κB DNA sequences. Varying the length and sequence of κB DNA revealed two factors that promote a single bound conformation for the complex: the length of the κB site in the duplex and a symmetrical sequence of guanine nucleotides at both ends of the recognition motif. The dynamic nature of the DNA-binding loops, together with the multiple bound conformations of p50 RHR with certain κB sites, is consistent with variations in the transcriptional activity of the p50 homodimer with different κB sequences.

Interactions of a Long Noncoding RNA with Domains of NF-κB and IκBα: Implications for the Inhibition of Non-Signal-Related Phosphorylation.

The transcription factor NF-κB is one of the central mediators of cellular signaling pathways. Under resting conditions, the canonical RelA-p50 (p65-p50) heterodimer NF-κB remains sequestered in the cytoplasm in complex with its inhibitor IκBα. Signal-mediated activation of NF-κB involves phosphorylation, ubiquitination and degradation of IκBα, and translocation of NF-κB to the nucleus. It was recently shown that a long noncoding RNA (termed NKILA) can modulate the NF-κB signaling circuit by interacting with the NF-κB-IκBα complex in the cytoplasm. In the current study, we investigated the interaction of RNA sequences derived from NKILA with domains of NF-κB and IκBα using NMR spectroscopy and native gel electrophoresis. Our results indicate that two RNA hairpin sequences interact with the DNA-binding domains of the Rel homology regions of RelA (p65) and p50 and that the same RNA sequences can affect the phosphorylation of the N-terminus of IκBα under low-salt conditions. We also observe that full-length RHR dimers (heterodimer of p65 and p50 and homodimer of p50) show a stronger interaction with the RNA hairpins than the individual domains of NF-κB. All of the interactions we observe between fragments of NKILA and domains of NF-κB are weak and nonspecific, consistent with the proposed function of the NKILA-NF-κB-IκBα interaction in protecting the NFκB-IκBα complex from aberrant activation of the NF-κB signaling pathway.

Role of Backbone Dynamics in Modulating the Interactions of Disordered Ligands with the TAZ1 Domain of the CREB-Binding Protein.

The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To gain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used nuclear magnetic resonance spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility observed for HIF-1α in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone dynamics in its various bound states. TAZ1 is more rigid in its CITED2-bound state than in its free state or in complex with HIF-1α, with increased rigidity observed not only in the CITED2 binding site but also in regions of TAZ1 that undergo conformational changes between the HIF-1α- and CITED2-bound structures. Taken together, these data suggest that backbone dynamics in TAZ1, as well as in the HIF-1α and CITED2 ligands, play a role in modulating the occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.

Structural Basis for Graded Inhibition of CREB:DNA Interactions by Multisite Phosphorylation.

Phosphorylation of the kinase inducible domain (KID) of the cyclic AMP response element binding transcription factor (CREB) regulates its function through several mechanisms. Transcriptional activation occurs following phosphorylation at serine 133, but multisite phosphorylation in a neighboring region termed the CK cassette, residues 108-117, results in inhibition of CREB-mediated transcription. A molecular-level understanding of the mechanism of these opposing reactions has been lacking, in part because of the difficulty of preparing multiply phosphorylated CREB in vitro. By substituting a single residue, we have generated an engineered mammalian CREB in which the CK cassette can be phosphorylated in vitro by casein kinases and have characterized its interactions with cyclic AMP response element DNA. Phosphorylation of the CK cassette promotes an intramolecular interaction between the KID domain and the site of DNA binding, the basic region of the C-terminal basic leucine zipper (bZip) domain. Competition between the phosphorylated KID domain and DNA for bZip binding results in a decreased affinity of CREB for DNA. The binding free energy calculated from the dissociation constant is directly proportional to the number of phosphate groups in the CK cassette, indicating that the DNA binding is regulated by a rheostat-like mechanism. The rheostat is modulated by variation of the concentration of cations such as Mg2+ and by alternative isoforms such as the natural CREB isoform that lacks residues 162-272. Multisite phosphorylation of CREB represents a versatile mechanism by which transcription can be tuned to meet the variable needs of the cell.

Structural basis for cooperative regulation of KIX-mediated transcription pathways by the HTLV-1 HBZ activation domain.

The human T cell leukemia virus I basic leucine zipper protein (HTLV-1 HBZ) maintains chronic viral infection and promotes leukemogenesis through poorly understood mechanisms involving interactions with the KIX domain of the transcriptional coactivator CBP and its paralog p300. The KIX domain binds regulatory proteins at the distinct MLL and c-Myb/pKID sites to form binary or ternary complexes. The intrinsically disordered N-terminal activation domain of HBZ (HBZ AD) deregulates cellular signaling pathways by competing directly with cellular and viral transcription factors for binding to the MLL site and by allosterically perturbing binding of the transactivation domain of the hematopoietic transcription factor c-Myb. Crystal structures of the ternary KIX:c-Myb:HBZ complex show that the HBZ AD recruits two KIX:c-Myb entities through tandem amphipathic motifs (L/V)(V/L)DGLL and folds into a long α-helix upon binding. Isothermal titration calorimetry reveals strong cooperativity in binding of the c-Myb activation domain to the KIX:HBZ complex and in binding of HBZ to the KIX:c-Myb complex. In addition, binding of KIX to the two HBZ (V/L)DGLL motifs is cooperative; the structures suggest that this cooperativity is achieved through propagation of the HBZ α-helix beyond the first binding motif. Our study suggests that the unique structural flexibility and the multiple interaction motifs of the intrinsically disordered HBZ AD are responsible for its potency in hijacking KIX-mediated transcription pathways. The KIX:c-Myb:HBZ complex provides an example of cooperative stabilization in a transcription factor:coactivator network and gives insights into potential mechanisms through which HBZ dysregulates hematopoietic transcriptional programs and promotes T cell proliferation.

Characterization of an Hsp90-Independent Interaction between Co-Chaperone p23 and Transcription Factor p53.

Cancer-suppressing transcription factor p53 is regulated by a wide variety of cellular factors, including many chaperones. The DNA-binding domain (DBD) of p53 is known to interact with the chaperone Hsp90, but the role of other members of the chaperone network, including co-chaperones such as p23, is unknown. Using a combination of nuclear magnetic resonance (NMR) titration, isothermal titration calorimetry, fluorescence anisotropy, and native agarose gel electrophoresis, we have identified a direct interaction between the p53 DBD and Hsp90 co-chaperone p23 that occurs in the absence of Hsp90. The affinity is relatively weak and largely determined by electrostatic interactions between the acidic C-terminal disordered tail of p23 and the two DNA-binding regions of the p53 DBD. We show by NMR and native agarose gel electrophoresis that a p53-specific double-stranded DNA sequence competes successfully with p23 for binding to the p53 DBD. The Hsp90 independence of the interaction between p23 and p53 DBD, together with the competition of p23 versus DNA for p53, raises the intriguing possibility that p23, like other small charged proteins, may affect p53 in hitherto unknown ways.

Structural Basis for Interaction of the Tandem Zinc Finger Domains of Human Muscleblind with Cognate RNA from Human Cardiac Troponin T.

The human muscleblind-like proteins (MBNL) regulate tissue-specific splicing by targeting cardiac troponin T and other pre-mRNAs; aberrant targeting of CUG and CCUG repeat expansions frequently accompanies the neuromuscular disease myotonic dystrophy. We show, using biolayer interferometry (Octet) and NMR spectroscopy, that the zinc finger domains of MBNL isoform 1 (MBNL1) are necessary and sufficient for binding CGCU sequences within the pre-mRNA of human cardiac troponin T. Protein constructs containing zinc fingers 1 and 2 (zf12) and zinc fingers 3 and 4 (zf34) of MBNL1 each fold into a compact globular tandem zinc finger structure that participates in RNA binding. NMR spectra show that the stoichiometry of the interaction between zf12 or zf34 and the CGCU sequence is 1:1, and that the RNA is single-stranded in the complex. The individual zinc fingers within zf12 or zf34 are nonequivalent: the primary RNA binding surface is formed in each pair by the second zinc finger (zf2 or zf4), which interacts with the CGCU RNA sequence. The NMR structure of the complex between zf12 and a 15-base RNA of sequence 95GUCUCGCUUUUCCCC109, containing a single CGCU element, shows the single-stranded RNA wrapped around zf2 and extending to bind to the C-terminal helix. Bases C101, U102, and U103 make well-defined and highly ordered contacts with the protein, whereas neighboring bases are less well-ordered in the complex. Binding of the MBNL zinc fingers to cardiac troponin T pre-mRNA is specific and relatively simple, unlike the complex multiple dimer-trimer stoichiometries postulated in some previous studies.

Role of the CBP catalytic core in intramolecular SUMOylation and control of histone H3 acetylation.

The histone acetyl transferases CREB-binding protein (CBP) and its paralog p300 play a critical role in numerous cellular processes. Dysregulation of their catalytic activity is associated with several human diseases. Previous work has elucidated the regulatory mechanisms of p300 acetyltransferase activity, but it is not known whether CBP activity is controlled similarly. Here, we present the crystal structure of the CBP catalytic core encompassing the bromodomain (BRD), CH2 (comprising PHD and RING), HAT, and ZZ domains at 2.4-Å resolution. The BRD, PHD, and HAT domains form an integral structural unit to which the RING and ZZ domains are flexibly attached. The structure of the apo-CBP HAT domain is similar to that of acyl-CoA-bound p300 HAT complexes and shows that the acetyl-CoA binding site is stably formed in the absence of cofactor. The BRD, PHD, and ZZ domains interact with small ubiquitin-like modifier 1 (SUMO-1) and Ubc9, and function as an intramolecular E3 ligase for SUMOylation of the cell cycle regulatory domain 1 (CRD1) of CBP, which is located adjacent to the BRD. In vitro HAT assays suggest that the RING domain, the autoregulatory loop (AL) within the HAT domain, and the ZZ domain do not directly influence catalytic activity, whereas the BRD is essential for histone H3 acetylation in nucleosomal substrates. Several lysine residues in the intrinsically disordered AL are autoacetylated by the HAT domain. Upon autoacetylation, acetyl-K1596 (Ac-K1596) binds intramolecularly to the BRD, competing with histones for binding to the BRD and acting as a negative regulator that inhibits histone H3 acetylation.

Mapping the interactions of adenoviral E1A proteins with the p160 nuclear receptor coactivator binding domain of CBP.

Many viruses deregulate the cell and force transcription of viral genes by competing with cellular proteins for binding to the transcriptional co-activators CREB-binding protein (CBP) and p300. Through its interactions with CBP/p300 and the retinoblastoma protein, the adenovirus (AdV) early region 1A (E1A) oncoprotein hijacks the cell cycle and, in rodents, transforms the cell; the mechanistic and structural basis for these effects remain unclear. In this study we compare the affinity of protein constructs from the E1A proteins from two adenovirus serotypes, non-oncogenic AdV5 and highly oncogenic AdV12, for binding to the nuclear receptor coactivator binding domain (NCBD) of CBP. NMR spectra show that the E1A constructs from both serotypes are intrinsically disordered in the free state and that each contains three homologous binding sites for the NCBD, one in the N-terminal region and two within conserved region 1 (CR1) of E1A. The binding sites in CR1 correspond to the motifs that bind the retinoblastoma protein and the TAZ2 domain of CBP/p300. The E1A and NCBD peptides fold synergistically upon complex formation. Binding affinities determined from NMR titrations show that, although the overall affinities for AdV5 and AdV12 E1A are comparable, there are significant differences between the two E1A serotypes in the relative strength with which their constituent interaction motifs bind to the NCBD. The individual E1A interaction motifs were unable to compete effectively with p53 for binding to the NCBD and both the N-terminal region and CR1 region of E1A are required for efficient competition with p53.

Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein.

An important component of the activity of p53 as a tumor suppressor is its interaction with the transcriptional coactivators cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and p300, which activate transcription of p53-regulated stress response genes and stabilize p53 against ubiquitin-mediated degradation. The highest affinity interactions are between the intrinsically disordered N-terminal transactivation domain (TAD) of p53 and the TAZ1 and TAZ2 domains of CBP/p300. The NMR spectra of simple binary complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer from exchange broadening, but innovations in construct design and isotopic labeling have enabled us to obtain high-resolution structures using fusion proteins, uniformly labeled in the case of the TAZ2-p53TAD fusion and segmentally labeled through transintein splicing for the TAZ1-p53TAD fusion. The p53TAD is bipartite, with two interaction motifs, termed AD1 and AD2, which fold to form short amphipathic helices upon binding to TAZ1 and TAZ2 whereas intervening regions of the p53TAD remain flexible. Both the AD1 and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, with AD2 making more extensive hydrophobic contacts consistent with its greater contribution to the binding affinity. Binding of AD1 and AD2 is synergistic, and structural studies performed with isolated motifs can be misleading. The present structures of the full-length p53TAD complexes demonstrate the versatility of the interactions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the structural basis of the affinity changes that occur upon stress-related posttranslational modification.

The high-risk HPV16 E7 oncoprotein mediates interaction between the transcriptional coactivator CBP and the retinoblastoma protein pRb.

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.

Structural characterization of interactions between the double-stranded RNA-binding zinc finger protein JAZ and nucleic acids.

The interactions of the human double-stranded RNA-binding zinc finger protein JAZ with RNA or DNA were investigated using electrophoretic mobility-shift assays, isothermal calorimetry, and nuclear magnetic resonance spectroscopy. Consistent with previous reports, JAZ has very low affinity for duplex DNA or single-stranded RNA, but it binds preferentially to double-stranded RNA (dsRNA) with no detectable sequence specificity. The affinity of JAZ for dsRNA is unaffected by local structural features such as loops, overhangs, and bulges, provided a sufficient length of reasonably well-structured A-form RNA (about 18 bp for a single zinc finger) is present. Full-length JAZ contains four Cys2His2 zinc fingers (ZF1-4) and has the highest apparent affinity for dsRNA; two-finger constructs ZF12 and ZF23 have lower affinity, and ZF34 binds even more weakly. The fourth zinc finger, ZF4, has no measurable RNA-binding affinity. Single zinc finger constructs ZF1, ZF2, and ZF3 show evidence for multiple-site binding on the minimal RNA. Fitting of quantitative NMR titration and isothermal calorimetry data to a two-site binding model gave Kd1 ∼ 10 µM and Kd2 ∼ 100 µM. Models of JAZ-RNA complexes were generated using the high-ambiguity-driven biomolecular docking (HADDOCK) program. Single zinc fingers bind to the RNA backbone without sequence specificity, forming complexes with contacts between the RNA minor groove and residues in the N-terminal ß strands and between the major groove and residues in the helix-kink-helix motif. We propose that the non-sequence-specific interaction between the zinc fingers of JAZ with dsRNA is dependent only on the overall shape of the A-form RNA.

The CH2 domain of CBP/p300 is a novel zinc finger.

The transcriptional co-regulator CBP (CREB-binding protein) has a highly conserved cysteine/histidine-rich region (CH2) whose structure and function remain uncharacterized. Using nuclear magnetic resonance (NMR spectroscopy), sequence alignment, mass spectrometry, and mutagenesis, we show that the CH2 domain is not a canonical plant homeodomain (PHD) finger, as previously proposed, but binds an additional zinc atom through the region N-terminal to the putative PHD motif. The CH2 domain and the preceding bromodomain interact and mutually stabilize each other, implying a cooperative function. We tested the hypothesis that the bromodomain and the CH2 domain can interact with histones, but found that the CH2 does not participate in histone-recognition.

Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso.

Methylation of CpG dinucleotides in DNA is a common epigenetic modification in eukaryotes that plays a central role in maintenance of genome stability, gene silencing, genomic imprinting, development, and disease. Kaiso, a bifunctional Cys(2)His(2) zinc finger protein implicated in tumor-cell proliferation, binds to both methylated CpG (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) and represses transcription by recruiting chromatin remodeling corepression machinery to target genes. Here we report structures of the Kaiso zinc finger DNA-binding domain in complex with its nonmethylated, sequence-specific DNA target (KBS) and with a symmetrically methylated DNA sequence derived from the promoter region of E-cadherin. Recognition of specific bases in the major groove of the core KBS and mCpG sites is accomplished through both classical and methyl CH···O hydrogen-bonding interactions with residues in the first two zinc fingers, whereas residues in the C-terminal extension following the third zinc finger bind in the opposing minor groove and are required for high-affinity binding. The C-terminal region is disordered in the free protein and adopts an ordered structure upon binding to DNA. The structures of these Kaiso complexes provide insights into the mechanism by which a zinc finger protein can recognize mCpG sites as well as a specific, nonmethylated regulatory DNA sequence.

Kaiso uses all three zinc fingers and adjacent sequence motifs for high affinity binding to sequence-specific and methyl-CpG DNA targets.

Kaiso is a Cys(2)His(2) zinc finger (ZF) protein that mediates methyl-CpG-dependent and sequence-specific transcriptional repression. As a first step towards elucidating the structural and molecular basis for recognition of these disparate DNA sequences, the minimal binding region of Kaiso was identified and optimal DNA sequences for high-affinity interactions were characterized. Contrary to previous findings, Kaiso requires all three zinc fingers plus adjacent protein regions for DNA recognition. An N-terminal extension contributes to structural stability, while an extended C-terminal region augments DNA binding. Complexes formed between the optimized Kaiso construct and both DNA sequences are suitable for future structural evaluation.

Identification of endogenous ligands bound to bacterially expressed human and E. coli dihydrofolate reductase by 2D NMR.

Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed "humanized" mutant of ecDHFR switches binding specificity in the cell.

The client protein p53 adopts a molten globule-like state in the presence of Hsp90.

It is not currently known in what state (folded, unfolded or alternatively folded) client proteins interact with the chaperone Hsp90. We show that one client, the p53 DNA-binding domain, undergoes a structural change in the presence of Hsp90 to adopt a molten globule-like state. Addition of one- and two-domain constructs of Hsp90, as well as the full-length three-domain protein, to isotopically labeled p53 led to reduction in NMR signal intensity throughout p53, particularly in its central ß-sheet. This reduction seems to be associated with a change of structure of p53 without formation of a distinct complex with Hsp90. Fluorescence and hydrogen-exchange measurements support a loosening of the structure of p53 in the presence of Hsp90 and its domains. We propose that Hsp90 interacts with p53 by multiple transient interactions, forming a dynamic heterogeneous manifold of conformational states that resembles a molten globule.

Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.

The activity and stability of the tumor suppressor p53 are regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased level of dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and from Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1, residues 2066-2075; Cα2, residues 2081-2092; and Cα3, residues 2095-2105) with a hydrophobic groove into which p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.