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ISWI chromatin remodelers mobilize nucleosomes to control DNA accessibility. Complexes isolated to date pair one of six regulatory subunits with one of two highly similar ATPases. However, we find that each endogenously expressed ATPase co-purifies with every regulatory subunit, substantially increasing the diversity of ISWI complexes, and we additionally identify BAZ2B as a novel, seventh regulatory subunit. Through reconstitution of catalytically active human ISWI complexes, we demonstrate that the new interactions described here are stable and direct. Finally, we profile the nucleosome remodeling functions of the now expanded family of ISWI chromatin remodelers. By revealing the combinatorial nature of ISWI complexes, we provide a basis for better understanding ISWI function in normal settings and disease.

Bromodomain-containing protein 9 (BRD9), an epigenetic "reader" of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.

The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.

Deubiquitinases (DUBs) are proteolytic enzymes whose function is to oppose the process of the conjugation of ubiquitin to a specific substrate. This task is accomplished through an enzymatic cascade involving E1, E2, and E3 enzymes, which collectively produce a product that is either monoubiquitinated, or polyubiquitinated with multiple single ubiquitins or with ubiquitin chains. The resulting modifications may impact protein function or may lead to the degradation of the ubiquitinated species, so the removal of such modifications must be tightly regulated. On the basis of recent work featuring crystal structures and detailed biochemical or biophysical studies of DUBs, we will discuss here how posttranslational modifications, protein binding partners, and reactive oxygen species regulate their catalytic activity.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure–activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 μM, BRET IC50 = 0.41 μM, BRD4(1) IC50 = 13 μM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.

Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding. An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity. Phosphoactivation of DUBA represents an unprecedented mode of protease regulation and a clear link between two major cellular signal transduction systems: phosphorylation and ubiquitin modification.

The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have determined the crystal structure of a complex between the Bmi1/Ring1b RING–RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2–E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING–RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modelling of the interface between Bmi1/Ring1b–UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.

The chromosomal passenger complex (CPC) has been identified as a master regulator of mitosis. In particular, proper chromosome segregation and cytokinesis depend on the correct localization and function of the CPC. Within the complex, the kinase Aurora B associates with Incenp, Survivin, and Borealin. The stoichiometry of the complex as well as a complete understanding of how these four components interact with each other remains to be elucidated. Here, we identify a new domain of Borealin. We determined its structure using NMR spectroscopy and discovered a novel dimerization motif. Interestingly, we found that substitutions at Borealin T230, recently identified as an Mps1 phosphorylation site, can modulate the dimerization state of Borealin. Mutation of this single residue to alanine or valine impairs Aurora B activity during mitosis and causes chromosome segregation defects. This study reveals that Mps1 regulates the CPC through a novel Borealin domain.

The two major Aurora kinases carry out critical functions at distinct mitotic stages. Selective inhibitors of these kinases, as well as pan-Aurora inhibitors, show antitumor efficacy and are now under clinical investigation. However, the ATP-binding sites of Aurora A and Aurora B are virtually identical, and the structural basis for selective inhibition has therefore not been clear. We report here a class of bisanilinopyrimidine Aurora A inhibitors with excellent selectivity for Aurora A over Aurora B, both in enzymatic assays and in cellular phenotypic assays. Crystal structures of two of the inhibitors in complex with Aurora A implicate a single amino acid difference in Aurora B as responsible for poor inhibitory activity against this enzyme. Mutation of this residue in Aurora B (E161T) or Aurora A (T217E) is sufficient to swap the inhibition profile, suggesting that this difference might be exploited more generally to achieve high selectivity for Aurora A.

Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC50 < 100 nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in near stasis (92% TGI) in an HCT116 mouse xenograft model.

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

To address questions of protein stability, researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phage-display has proved to be a powerful tool for analyzing protein stability due to the large library size and the robustness of the phage particle to a variety of denaturing conditions. With the B1 domain of protein G (GB1) and a camelid heavy chain antibody as model systems, we are using phage-display libraries to experimentally address questions that have generally been addressed in silico, either through computational studies or statistical analysis of known protein structures. One effort has focused on identifying novel solutions to repacking the hydrophobic core of GB1, while maintaining stability comparable to the wild type protein. In a second study, a small set of substitutions in complimentarity-determining region 3 was found to stabilize the framework of the camelid antibody. Another major focus has been to obtain quantitative data on beta-sheet stability determinants. We have successfully adapted a phage-display method for quantitating affinities of protein variants (shotgun alanine scanning) to analysis of GB1 stability. Using this method, we have analyzed the energetic contributions of cross-strand side chain-side chain interactions. Finally, we discuss parameters to consider in using phage-display to discriminate subtle stability differences among fully folded variants. Overall, this method provides a fast approach for quantitatively addressing biophysical questions.

BAFF/BLyS, a member of the tumor necrosis family (TNF) superfamily of ligands, is a crucial survival factor for B cells. BAFF binds three receptors, TACI, BCMA, and BR3, with signaling through BR3 being essential for promoting B cell function. Typical TNF receptor (TNFR) family members bind their cognate ligands through interactions with two cysteine-rich domains (CRDs). However, the extracellular domain (ECD) of BR3 consists of only a partial CRD, with cysteine spacing distinct from other modules described previously. Herein, we report the solution structure of the BR3 ECD. A core region of only 19 residues adopts a stable structure in solution. The BR3 fold is analogous to the first half of a canonical TNFR CRD but is stabilized by an additional noncanonical disulfide bond. BAFF-binding determinants were identified by shotgun alanine-scanning mutagenesis of the BR3 ECD expressed on phage. Several of the key BAFF-binding residues are presented from a beta-turn that we have shown previously to be sufficient for ligand binding when transferred to a structured beta-hairpin scaffold [Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, D. M., Grewal, I. S., Cochran, A. G., Gordon, N. C., Yin, J., Starovasnik, M. A, and Dixit, V. M. (2002) Immunity 10, 515-524]. Outside of the turn, mutagenesis identifies additional hydrophobic contacts that enhance the BAFF-BR3 interaction. The crystal structure of the minimal hairpin peptide, bhpBR3, in complex with BAFF reveals intimate packing of the six-residue BR3 turn into a cavity on the ligand surface. Thus, BR3 binds BAFF through a highly focused interaction site, unprecedented in the TNFR family.

The turn-forming ability of a series of three-residue sequences was investigated by substituting them into a well-characterized beta-hairpin peptide. The starting scaffold, bhpW, is a disulfide-cyclized 10-residue peptide that folds into a stable beta-hairpin with two antiparallel strands connected by a two-residue reverse turn. Substitution of the central two residues with the three-residue test sequences leads to less stable hairpins, as judged by thiol-disulfide equilibrium measurements. However, analysis of NMR parameters indicated that each molecule retains a significant folded population, and that the type of turn adopted by the three-residue sequence is the same in all cases. The solution structure of a selected peptide with a PDG turn contained an antiparallel beta-hairpin with a 3:5 type I + G1 bulge turn. Analysis of the energetic contributions of individual turn residues in the series of peptides indicates that substitution effects have significant context dependence, limiting the predictive power of individual amino acid propensities for turn formation. The most stable and least stable sequences were also substituted into a more stable disulfide-cyclized scaffold and a linear beta-hairpin scaffold. The relative stabilities remained the same, suggesting that experimental measurements in the bhpW context are a useful way to evaluate turn stability for use in protein design projects. Moreover, these scaffolds are capable of displaying a diverse set of turns, which can be exploited for the mimicry of protein loops or for generating libraries of reverse turns.

We have developed polypeptide scaffolds that readily adopt aβ‒hairpin conformation (a pair of antiparallel strands connected by a turn) in solution. The study of such peptides allows us to understand the factors that govern stability and folding of these motifs in proteins, and permits mimicry of functionally important regions of proteins. Spectroscopic and biophysical methods have been used to characterize the conformational preferences and stability of these peptides, with a strong emphasis on using restraints generated from1H NMR spectroscopy to determine their three‒dimensional structure. By optimization of inter‒strand interactions, we have developed highly stable disulfide‒cyclized and linearβ‒hairpin peptides. In particular, tryptophan residues at non‒hydrogen bonded strand sites (NHB) are highly stabilizing. A variety of turn types have been presented from these scaffolds, suggesting that they might generally be useful in turn presentation. Interestingly,β‒hairpin peptides (containing a disulfide and a NHB tryptophan) have recently been discovered as antagonists of protein–protein interactions from naïve peptide libraries displayed on phage. Comparison of one suchβ‒hairpin peptide with anα‒helical peptide of very similar sequence provides further insight into the role that residue type and context play in determining polypeptide conformation.

Display of peptide libraries on filamentous phage has led to the identification of peptides of the form X 2-5 CX 2 GPXTWXCX 2-5 (where X is a variable residue) that bind to the extra-cellular portion of the erythropoietin receptor (EPO-R). These peptides adopt β-hairpin conformations when co-crystallized with EPO-R. Solution NMR studies reveal that the peptide is conformationally heterogeneous in the absence of receptor due to cis - trans isomerization about the Gly-Pro peptide bond. Replacement of the conserved threonine residue with glycine at the turn i + 3 position produces a stable β-hairpin conformation in solution, although this peptide no longer has activity in an EPO-R-dependent cell proliferation assay. A truncated form of the EPO-R-binding peptide (containing the i + 3 glycine residue) also forms a highly populated, monomeric β-hairpin. In contrast, phage-derived peptide antagonists of insulin-like growth factor binding protein 1 (IGFBP-1) have a high level of sequence identity with the truncated EPO-R peptide (eight of 12 residues) yet adopt a turn-α-helix conformation in solution. Peptides containing all possible pairwise amino acid substitutions between the EPO-R and IGFBP-1 peptides have been analyzed to assess the degree to which the non-conserved residues stabilize the hairpin or helix conformation. All four residues present in the original sequence are required for maximum population of either the β-hairpin or α-helix conformation, although some substitutions have a more dominant effect. The results demonstrate that, within a given sequence, the observed conformation can be dictated by a small subset of the residues (in this case four out of 12).

Polycomb repressive complex 1 (PRC1) is required for ubiquitination of histone H2A lysine 119, an epigenetic mark associated with repression of genes important in developmental regulation. The E3 ligase activity of PRC1 resides in the RING1A/B subunit when paired with one of six PCGF partners. The best known of these is the oncogene BMI1/PCGF4. We find that canonical PRC1 E3 ligases such as PCGF4-RING1B have intrinsically very low enzymatic activity compared with non-canonical PRC1 RING dimers. The structure of a high-activity variant in complex with E2 (PCGF5-RING1B-UbcH5c) reveals only subtle differences from an earlier PCGF4 complex structure. However, two charged residues present in the modelled interface with E2-conjugated ubiquitin prove critical: in BMI1/PCGF4, these residues form a salt bridge that may limit efficient ubiquitin transfer. The intrinsically low activity of the PCGF4-RING1B heterodimer is offset by a relatively favourable interaction with nucleosome substrates, resulting in an efficient site-specific monoubiquitination.

Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.

Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and, therefore, a prime therapeutic target for the development of antagonists for the treatment of cancer. As a first step toward this goal, phage display was used to generate peptides that bind to the receptor-binding domain (residues 8-109) of VEGF and compete with receptor [Fairbrother, W. J., Christinger, H. W., Cochran, A. G., Fuh, G., Keenan, C. J., Quan, C., Shriver, S. K., Tom, J. Y. K., Wells, J. A., and Cunningham, B. C. (1999) Biochemistry 38, 17754-17764]. The crystal structure of VEGF in complex with one of these peptides was solved and refined to a resolution of 1.9 A. The 20-mer peptide is unstructured in solution and adopts a largely extended conformation when bound to VEGF. Residues 3-8 form a beta-strand which pairs with strand beta6 of VEGF via six hydrogen bonds. The C-terminal four residues of the peptide point away from the growth factor, consistent with NMR data indicating that these residues are flexible in the complex in solution. In contrast, shortening the N-terminus of the peptide leads to decreased binding affinities. Truncation studies show that the peptide can be reduced to 14 residues with only moderate effect on binding affinity. However, because of the extended conformation and the scarcity of specific side-chain interactions with VEGF, the peptide is not a promising lead for small-molecule development. The interface between the peptide and VEGF contains a subset of the residues recognized by a neutralizing Fab fragment and overlaps partially with the binding site for the Flt-1 receptor. The location of the peptide-binding site and the hydrophilic character of the interactions with VEGF resemble more the binding mode of the Fab fragment than that of the receptor.

Peptides that inhibit binding of vascular endothelial growth factor (VEGF) to its receptors, KDR and Flt-1, have been produced using phage display. Libraries of short disulfide-constrained peptides yielded three distinct classes of peptides that bind to the receptor-binding domain of VEGF with micromolar affinities. The highest affinity peptide was also shown to antagonize VEGF-induced proliferation of primary human umbilical vascular endothelial cells. The peptides bind to a region of VEGF known to contain the contact surface for Flt-1 and the functional determinants for KDR binding. This suggests that the receptor-binding region of VEGF is a binding "hot spot" that is readily targeted by selected peptides and supports earlier assertions that phage-derived peptides frequently target protein-protein interaction sites. Such peptides may lead to the development of pharmacologically useful VEGF antagonists.

The recent rapid growth of protein sequence databases is outpacing the capacity of researchers to biochemically and structurally characterize new proteins. Accordingly, new methods for recognition of motifs and homologies in protein primary sequences may be useful in determining how these proteins might function. We have applied such a method, an iterative learning algorithm, to analyze possible coiled coil domains in histidine kinase receptors. The potential coiled coils have not yet been structurally characterized in any histidine kinase, and they appear outside previously noted kinase homology regions. The learning algorithm uses a combination of established sequence patterns in known coiled coil proteins and histidine kinase sequence data to learn to recognize efficiently this coiled coil-like motif in the histidine kinases. The common appearance of the structural motif in a functionally important part of the receptors suggests hypotheses for kinase regulation and signal transduction.

Wnt/beta-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/beta-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/beta-catenin signaling by Dkk1.

Survivin is a member of the IAP (inhibitor of apoptosis) protein family, defined in part by the presence of a zinc-binding baculoviral inhibitory repeat (BIR) domain. Most BIR domains bind short sequences beginning with alanine, and in this manner, they recognize and block the action of key targets in apoptotic pathways. However, Survivin binds only very weakly to typical IAP ligands. Unique features of Survivin are the long C-terminal helix following the BIR domain and a short segment (linking the helix and BIR domains) that mediates Survivin homodimerization. Despite this detailed knowledge of the structure of Survivin itself, there is a current lack of understanding about how Survivin recognizes cellular binding partners, and consequently, many questions about Survivin function remain unanswered. We determined two co-crystal structures of Survivin and a minimal binding fragment from the chromosomal passenger protein Borealin, a well validated functional interactor. The interaction between Survivin and Borealin involves extensive packing between the long C-terminal helix of Survivin and a long Borealin helix. Surprisingly, an additional important interaction occurs between the Survivin homodimerization interface and a short segment of Borealin. This segment both structurally mimics and displaces one Survivin monomer. The relevance of this unexpected interaction was tested by mutagenesis of two key Borealin residues. Mutant Borealin introduced into HeLa cells failed to localize properly during mitosis and also caused mislocalization of other chromosomal passenger proteins. This suggests that the mutant is dominant-negative and confirms the functional importance of the interaction surface identified in the crystal structures.

Amino acid structural propensities measured in "host-guest" model studies are often used in protein structure prediction or to choose appropriate residues in de novo protein design. While this concept has proven useful for helical structures, it is more difficult to apply successfully to beta-sheets. We have developed a cyclic beta-hairpin scaffold as a host for measurement of individual residue contributions to hairpin structural stability. Previously, we have characterized substitutions in non-backbone-hydrogen-bonded strand sites; relative stability differences measured in the cyclic host are highly predictive of changes in folding free energy for linear beta-hairpin peptides. Here, we examine the hydrogen-bonded strand positions of our host. Surprisingly, we find a large favorable contribution to stability from a valine (or isoleucine) substitution immediately preceding the C-terminal cysteine of the host peptide, but not at the cross-strand position of the host or in either strand of a folded linear beta-hairpin (trpzip peptide). Further substitutions in the peptides and NMR structural analysis indicate that the stabilizing effect of valine is general for CX(8)C cyclic hairpins and cannot be explained by particular side-chain-side-chain interactions. Instead, a localized decrease in twist of the peptide backbone on the N-terminal side of the cysteine allows the valine side chain to adopt a unique conformation that decreases the solvent accessibility of the peptide backbone. The conformation differs from the highly twisted (coiled) conformation of the trpzip hairpins and is more typical of conformations present in multistranded beta-sheets. This unexpected structural fine-tuning may explain why cyclic hairpins selected from phage-displayed libraries often have valine in the same position, preceding the C-terminal cysteine. It also emphasizes the diversity of structures accessible to beta-strands and the importance of considering not only "beta-propensity", but also hydrogen-bonding pattern and strand twist, when designing beta structures. Finally, we observe correlated, cooperative stabilization from side-chain substitutions on opposite faces of the hairpin. This suggests that cooperative folding in beta-hairpins and other small beta-structures is driven by cooperative strand-strand association.

Many biological processes are mediated by specific molecular recognition between proteins. However, the thermodynamic and structural ‘rules’ for such recognition are incompletely understood, as is the potential for inhibition by small molecules. Recent progress has included the discovery of small-molecule inhibitors for several targets important in cancer.

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.

Transmembrane signaling by bacterial chemotaxis receptors appears to require a conformational change within a receptor dimer. Dimers were engineered of the cytoplasmic domain of the Escherichia coli aspartate receptor that stimulated the kinase CheA in vitro. The folding free energy of the leucine-zipper dimerization domain was harnessed to twist the dimer interface of the receptor, which markedly affected the extent of CheA activation. Response to this twist was attenuated by modification of receptor regulatory sites, in the same manner as adaptation resets sensitivity to ligand in vivo. These results suggest that the normal allosteric activation of the chemotaxis receptor has been mimicked in a system that lacks both ligand-binding and transmembrane domains. The most stimulatory receptor dimer formed a species of tetrameric size.

Background and Objectives: The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. A longstanding question in this field is how a single lysine residue in the nucleosome is singled out for ubiquitin modification and why only one ubiquitin is added rather than a chain of ubiquitins. To better understand how Bmi1/Ring1b controls monoubiquitination of H2A, we structurally characterized the E2 and nucleosome interaction sites on Bmi1/Ring1b. Methods: X-ray crystallography was used to determine the structure of a complex between Bmi1/Ring1b and UbcH5c, its cognate E2 enzyme. Fluorescence-polarization assays were used to measure the binding of Bmi1/Ring1b to a synthetic DNA duplex. Site-directed mutagenesis was used to assess the importance of residues at the potential Bmi1/Ring1b-DNA interface, and in vitro ubiquitin ligase assays were used to measure the catalytic activity of mutant complexes. Molecular modeling studies were performed using HADDOCK v2.0. Results: We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Through site-directed mutagenesis we have been able to uncouple the E2 binding and the DNA binding activities of Bmi1/Ring1b, indicating that Bmi1/Ring1b uses distinct binding surfaces to recognize E2 and the nucleosomal substrate. Computational modeling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific mono-ubiquitination of H2A. Conclusions: Our data show that the direct interaction of the RING domains with nucleosomal DNA is crucial for the ubiquitin ligase activity of Bmi1/Ring1b. To our knowledge, this is the first example of a RING-domain E3 ligase binding directly to its substrate via the RING domain. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A160.

Protein–protein interactions are often attractive, but not straightforward, targets for disease therapy. Two strategies for identifying inhibitors of these interactions, peptide phage display and high-throughput screening, have recently shown new promise.

Aurora kinases are important mitotic regulators, and Aurora kinase inhibitors are under investigation as treatments for cancer. An ongoing debate in the field is which Aurora kinase is the better drug target. A new study (Girdler et al., 2008, in this issue of Chemistry & Biology) pushes the case forward for Aurora B.