The human orphan nuclear receptor tailless (TLX, NR2E1) is druggable.

Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.

Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity.

IscR from Escherichia coli is an unusual metalloregulator in that both apo and iron sulfur (Fe-S)-IscR regulate transcription and exhibit different DNA binding specificities. Here, we report structural and biochemical studies of IscR suggesting that remodeling of the protein-DNA interface upon Fe-S ligation broadens the DNA binding specificity of IscR from binding the type 2 motif only to both type 1 and type 2 motifs. Analysis of an apo-IscR variant with relaxed target-site discrimination identified a key residue in wild-type apo-IscR that, we propose, makes unfavorable interactions with a type 1 motif. Upon Fe-S binding, these interactions are apparently removed, thereby allowing holo-IscR to bind both type 1 and type 2 motifs. These data suggest a unique mechanism of ligand-mediated DNA site recognition, whereby metallocluster ligation relocates a protein-specificity determinant to expand DNA target-site selection, allowing a broader transcriptomic response by holo-IscR.

GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the ß-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the ß-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.

High-resolution conformation and backbone dynamics of a soluble aggregate of apomyoglobin119.

The structure and dynamics of soluble misfolded aggregates are poorly understood, despite their importance in protein science and disease. Water-soluble self-associated species that do not become insoluble over time are invaluable tools for high-resolution conformational studies aimed at dissecting the determinants of self-association. Here, we characterize the soluble model aggregate apomyoglobin(119) (apoMb(119)), generated upon truncating the residues corresponding to the C-terminal helix of sperm whale apomyoglobin. The secondary structure and backbone dynamics of apoMb(119), determined by multidimensional NMR at pH 6.0, reveal the presence of an N-terminal slow-tumbling core and a highly disordered flexible C-terminus displaying residual helicity and large-amplitude backbone motions on the picosecond-to-nanosecond timescale. The backbone of the apoMb(119) aggregate assumes progressively increased mobility as residues get further removed from the nonpolar core and closer to the more hydrophilic C-terminal end. This structural motif establishes a useful paradigm for the topology of soluble misfolded protein aggregates in aqueous solution in the absence of stabilizing additives. The partially helical and flexible C-terminus of apoMb(119)'s aggregate is in interesting contrast with the amyloid-related globulomers, which display dangling ends rich in ß-strand. Finally, we investigate how a molecular chaperone, the substrate-binding domain of DnaK, interferes with apoMb(119)'s aggregation.

Contribution of long-range interactions to the secondary structure of an unfolded globin.

This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an alpha-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable alpha-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins.

Secondary structure mapping of DnaK-bound protein fragments: chain helicity and local helix unwinding at the binding site.

Little is known about polypeptide conformation and folding in the presence of molecular chaperones participating in protein biosynthesis. In vitro studies on chaperone-substrate complexes have been mostly carried out with small peptide ligands. However, the technical challenges associated with either competing aggregation or spectroscopically unfavorable size and exchange rates have typically prevented analysis of larger substrates. Here, we report the high-resolution secondary structure of relatively large N-terminal protein fragments bound to the substrate-binding domain of the cotranslationally active chaperone DnaK. The all-alpha-helical protein apomyoglobin (apoMb), bearing the ubiquitous globin fold, has been chosen as a model substrate. On the basis of NMR secondary chemical shift analysis, we identify, for the first time, weak helical content (similar to that found in the chemically unfolded full-length protein) for the assigned residues of the chaperone-bound chain away from the chaperone binding sites. In contrast, we found that the residues corresponding to the strongest specific binding site for DnaK, examined via a short 13-mer apoMb peptide fragment matching the binding site sequence, display highly reduced helical content in their chaperone-bound form. Given that the free state of the peptide is weakly helical in isolation, we conclude that the substrate residues corresponding to the chaperone binding site undergo helix unwinding upon chaperone binding.

Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain.

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-beta. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-beta, however, formation of the original self-associated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-beta-bound state, which becomes folding-competent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently outcompetes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.

NMR spectroscopic filtration of polypeptides and proteins in complex mixtures.

Due to the inherent complexity of the natural biological environment, most studies on polypeptides, proteins and nucleic acids have so far been performed in vitro, away from physiologically relevant conditions. Nuclear magnetic resonance is an ideal technique to extend the in vitro analysis of simple model systems to the more complex biological context. This work shows how diffusion-based spectroscopic selection can be combined with isotopic labeling to tackle and optimize the NMR analysis of specific macromolecules in multicomponent mixtures. Typical media include cell-free systems containing overexpressed proteins, lysates and proteolytic mixtures. We present a few variants of diffusion-edited HSQC pulse sequences for the selective spectroscopic detection of protein and polypeptide resonances within complex mixtures containing undesired species of smaller molecular weight. Due to diffusion-based filtering, peak intensities of fast diffusing small molecules are attenuated more than peaks due to large molecules. The basic sequence, denoted as PFGSTE-HSQC, combines translational diffusion-ordering with two dimensional heteronuclear single quantum correlation spectroscopy. The GCSTE-HSQC and BPPSTE-HSQC sequences include bipolar gradients and are therefore suitable for both diffusion-based filtering and determination of diffusion coefficients of individual mixture components. Practical applications range from protein stability/folding investigations in physiologically relevant contexts to prescreening of tertiary fold and resonance assignments in structural genomics studies. A few applications of diffusion-edited HSQC to an E. coli cell lysate containing the (15)N-labeled B domain of streptococcal protein G (GB1), and to a (15)N-labeled N-acetylglycine/apomyoglobin mixture are presented. In addition, we provide specific guidelines for experimental setup and parameter optimization.