The tumor suppressor p53 consists of four 393-residue chains, each of which has two natively unfolded (N-and C-terminal) and two folded (core and tetramerization) domains. Their structural organization is poorly characterized as the protein tends to aggregate, has defied crystallization, and is at the limits of NMR studies. We first stabilized the protein by mutation to make it more suitable for extended study and then acquired NMR spectra on full-length protein and various combinations of shorter domain constructs. The NMR spectrum ( 15 N, 1 H transverse relaxation optimized spectroscopy) of full-length p53 was close to that expected from the sum of the spectra of isolated individual domains. However, patterns of changes in chemical shifts revealed unexpected interactions between the core domains. We used the NMR data as constraints in docking algorithms and found a previously uncharacterized self-complementary surface for the association of core domains into dimers within the tetrameric complex. Binding to DNA requires about a 70°rotation to break those subunit interactions and form the known protein:protein interface in the p53-DNA complex. We verified the interactions by the effects of mutation on DNA binding. Spectroscopic, biophysical, and mutational data conspired to give a picture of the p53 tetramer as a dimer of loosely tethered core dimers of appropriate symmetry to be poised to bind target DNA. modular proteins ͉ NMR ͉ protein-protein interactions T he tumor suppressor p53 is a complex multifunctional protein that acts as a transcription factor in response to oncogenic and other stresses. It is at the centre of a multitude of networks in the cell, binds to DNA and a large number of protein signaling factors that modulate its activity, and is a subject to control by extensive posttranslational modification. Its activity is crucial in the prevention of cancer by inducing cell cycle arrest and apoptosis in response to oncogenic signals (1, 2). Each of its monomeric chains of 393 residues is comprised of several functional domains: the N-terminal domain (residues 1-93) comprises a transactivation domain (residues 1-60) (3) and a proline-rich regulatory domain (residues 64-92) (4), the DNAbinding core domain (CD) (residues 94-312) (5), the tetramerization domain (residues 324-355) (6), and the C-terminal negative regulatory domain (residues 360-393) (7). It undergoes a reversible equilibrium to form tetramers (8). The crystal structures of isolated human core domain bound to DNA (5), and of several oncogenic mutants, have been solved (9). The tetramerization domain is also well characterized by NMR (6, 10) and crystallography (11). The isolated N-terminal domain is natively unfolded (11, 12), apart from the formation of a nascent helix in the region 15-30 (11), which becomes fully helical when bound to Mdm2 (13) or in a membrane environment (14). The C-terminal negative regulatory domain of p53 is also unstructured (15, 16). The unstructured regions probably prevent the full-length protein being crystalli...

Potent and specific inhibitors of protein•protein interactions have significant potential both as therapeutic compounds and biological tools, yet discovery of such molecules remains a significant challenge. Whereas small molecules typically bind proteins in small, well-defined, deep clefts, proteins generally recognize each other through formation of large, heterogeneous complementary surfaces. Our laboratory has recently described a general solution, called protein grafting, for the identification of highly functional miniature proteins by stabilization of α-helical binding epitopes on a protein scaffold. In protein grafting, those residues that comprise a functional epitope are grafted onto the solvent-exposed α-helical face of the small yet stable protein avian pancreatic polypeptide (aPP). In this study, we use protein grafting in combination with molecular evolution by phage display to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. Furthermore, we show that grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold yields molecules capable of high affinity recognition of CBP KIX even in the absence of phosphorylation. Importantly, both classes of designed ligands exhibit high specificity for the target CBP KIX domain over carbonic anhydrase and calmodulin, two unrelated proteins that bind hydrophobic or α-helical molecules that might be encountered in vivo.

There is considerable interest in molecules that bind intra-or extracellular protein surfaces and inhibit protein-protein interactions. 1 Molecules with these properties have potential as validation tools or therapeutic leads for the vast array of proteins encoded by the human genome and can probe the functional relevance of molecular circuits that control the inner workings of the cell. Accurate interpretation of such chemical biology experiments, however, demands an exceedingly high and often elusive level of specificity, as the phenotypic readout will reflect the weighted integral of all cellular binding events, whether they involve the desired target or not. This caveat is especially important when closely related protein family members or paralogs are coexpressed and nonredundant. 2 Previously we reported a miniature protein design strategy in which the well-folded structure of the pancreatic-fold polypeptide aPP presents R-helical or PPII-helical recognition epitopes found on protein-protein interaction surfaces. [4][5][6] The miniature proteins designed in this way can recognize even shallow protein clefts with high affinity and specificity and inhibit protein-protein interactions. 4f One such miniature protein, PPBH3-1, binds the anti-apoptotic protein paralogs Bcl-2 and Bcl-X L with nanomolar affinity and a ∆∆G ) 1.2 kcal‚mol -1 preference for Bcl-X L in vitro. Moreover, PPBH3-1 competes effectively with a peptide comprising the BH3 domain of the pro-apoptotic Bcl-2 protein Bak. 4d Here we describe the evolution of PPBH3-1 into two new miniature proteins, PPBH3-5 and PPBH3-6, whose paralog specificity is reversed relative to PPBH3-1 ( Figure 1A). PPBH3-5 and PPBH3-6 bind Bcl-2 with nanomolar affinity and a ∆∆G ) 0.9-1.3 kcal‚mol -1 preference for Bcl-2 over Bcl-X L . PPBH3-5 and PPBH3-6 may have unique applications as early examples of nonnatural ligands that interact selectively with Bcl-2 proteins. 7,8 We began with a phage library of 5 × 10 8 PPBH3-1 variants whose sequences varied at six positions chosen to exploit subtle structural and electronic differences between the BH3-binding grooves of Bcl-X L and Bcl-2 ( Figure 1B). 9,10 These grooves are lined with remarkably similar side chains, with only three notable differences: Glu 136 in Bcl-X L is replaced by Arg 129 in Bcl-2, Ala 104 in Bcl-X L is replaced by Asp 111 in Bcl-2, and Leu 108 in Bcl-X L is replaced by Met 115 in Bcl-2. 11 The limited sequence changes within the BH3-binding grooves belie significant functional differences between the two proteins: Bcl-2 and Bcl-X L knock-out mice show dissimilar defects, 12 the proteins exhibit overlapping but distinct subcellular distributions, 13 and otherwise isogenic cell lines that overexpress Bcl-2 or Bcl-X L respond differently to chemotherapeutic agents. 14 We devised a positive/negative selection protocol to enrich the library with members that prefer Bcl-2 to Bcl-X L . This protocol selected members that bound GST-Bcl-2 1-205 (Bcl-2) with high affinity, eliminating those that also bound...

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We recently described a pair of ligands, PPKID4(P) (4(P)) and PPKID6(U) (6(U)), which present the alpha-helical functional epitope found on helix B of the CREB KID activation domain (KID(P)) on a pancreatic fold protein scaffold. 4(P) and 6(U) bind the natural target of KID(P), the KIX domain of the coactivator CBP, with equilibrium dissociation constants between 515 nM and 1.5 microM and compete effectively with KID(P) for binding to CBP KIX (KIX). Here we present a detailed investigation of the binding mode, orientation, and transcriptional activation potential of 4(P) and 6(U). Equilibrium binding experiments using a panel of well-characterized KIX variants support a model in which 4(P) binds KIX in a manner that closely resembles that of KID(P) but 6(U) binds an overlapping, yet distinct region of the protein. Equilibrium binding experiments using a judiciously chosen panel of 4(P) variants containing alanine or sarcosine substitutions along the putative alpha- or PPII helix of 4(P) support a model in which 4(P) folds into a pancreatic fold structure upon binding to KIX. Transcriptional activation assays performed in HEK293 cells using GAL4 DNA-binding domain fusion proteins indicate that 4(P) functions as a potent activator of p300/CBP-dependent transcription. Notably, 6(U) is a less potent transcriptional activator in this context than 4(P)despite the similarity of their affinities for CBP KIX. This final result suggests that thermodynamic affinity is an important, although not exclusive, criterion controlling the level of KIX-dependent transcriptional activation.