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Research has been conducted on the Escherichia coli TonS protein. The nature of the TonB interactions with the outer membrane has been investigated and the results indicate that the protein can interact with the outer membrane at the redundant sites prior to transducing energy to the outer membrane receptors.

A high affinity Streptavidin ligand was mined from a DNA-encoded library of non-peptidic oligimers and characterized structurally. [ABSTRACT FROM AUTHOR]

In spore forming microbes, germination protease (GPR) plays a key role in the initiation of the germination process. A critical step during germination is the degradation of small acid-soluble proteins (SASPs), which protect spore DNA from external stresses (UV, heat, low temperature, etc.). Inactive zymogen GPR can be activated by autoprocessing of the N-terminal pro-sequence domain. Activated GPR initiates the degradation of SASPs; however, the detailed mechanisms underlying the activation, catalysis, regulation, and substrate recognition of GPR remain elusive. In this study, we determined the crystal structure of GPR from Paenisporosarcina sp. TG-20 (PaGPR) in its inactive form at a resolution of 2.5 A. Structural analysis showed that the active site of PaGPR is sterically occluded by an inhibitory loop region (residues 202–216). The N-terminal region interacts directly with the self-inhibitory loop region, suggesting that the removal of the N-terminal pro-sequence induces conformational changes, which lead to the release of the self-inhibitory loop region from the active site. In addition, comparative sequence and structural analyses revealed that PaGPR contains two highly conserved Asp residues (D123 and D182) in the active site, similar to the putative aspartic acid protease GPR from Bacillus megaterium. The catalytic domain structure of PaGPR also shares similarities with the sequentially non-homologous proteins HycI and HybD. HycI and HybD are metal-loproteases that also contain two Asp (or Glu) residues in their active site, playing a role in metal binding. In summary, our results provide useful insights into the activation process of PaGPR and its active conformation. [ABSTRACT FROM AUTHOR]

Multifunctional pro-inflammatory cytokine CXCL8 is a small peptide of 8–10 kDa in size and it functions as a monomer or dimer. CXCL8 harbors 2 disulfide bonds for its stability. Although production of the CXCL8 protein in a large quantity in both mammalian and bacterial systems has been reported, the processes are complicated and lengthy. Here, we develop a new bacterial expression system for recombinant CXCL8 and simplify the purification system to yield a high amount of protein quickly. The purified CXCL8 protein from our new system develops a crystal structure that is identical to that produced through the mammalian expression system. Thus, we have established a simple and efficient recombinant CXCL8-producing system, which can be easily operated and is suitable to those requiring a large quantity of CXCL8. [ABSTRACT FROM AUTHOR]

The crystal structure of the heme containing, oxygen-sensing domain of the Escherichia coli direct oxygen sensor (Ec DosH) is found using heme Fe multiple-wavelength anomalous dispersion (MAD). Analysis provides insight into the possible mechanics of the signaling mechanism within the hemedomain following initiation due to O(sub 2) dissociation.

The Latin American plant Tabernaemontana longipes was studied in this work as a potential source of antiparasitic agents. The chloroform extract of T. longipes leaves was separated into several fractions, and tested for antitrypanosomal activity. One of the fractions displayed significant growth inhibitory activity against Trypanosoma brucei. The active principle in the fraction was isolated, purified, and characterized by NMR and mass spectrometry. The antitrypanosomal agent in the CHCl3 extract of T. longipes leaves is the pentacyclic triterpenoid bauerenol acetate. A metabolite profiling assay suggest that the triterpenoid influences cholesterol metabolism. The molecular target(s) of bauerenol and its acetate, like many other antiparasitic pentacyclic triterpenoids is/are unknown, but they present privileged structural scaffolds that can be explored for structure-based activity optimization studies using phenotypic assays. [ABSTRACT FROM AUTHOR]

Potent modulators of RNA function can be assembled in cellulo by using the cell as a reaction vessel and a disease-causing RNA as a catalyst. When designing small molecule effectors of function, a balance between permeability and potency must be struck. Low molecular weight compounds are more permeable whereas higher molecular weight compounds are more potent. The advantages of both types of compounds could be synergized if low molecular weight molecules could be transformed into potent, multivalent ligands by a reaction that is catalyzed by binding to a target in cells expressing a genetic defect. It was shown that this approach is indeed viable in cellulo. Small molecule modules with precisely positioned alkyne and azide moieties bind adjacent internal loops in r(CCUG)exp, the causative agent of myotonic dystrophy type 2 (DM2), and are transformed into oligomeric, potent inhibitors of DM2 RNA dysfunction by a Huisgen 1,3-dipolar cycloaddition reaction, a variant of click chemistry. [ABSTRACT FROM AUTHOR]

One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded CAG repeats can cause Huntington's and other disease through translation of toxic proteins. Herein, a crystal structure of r[5′UUGGGC(CAG)3GUCC]2, a model of CAG expanded transcripts, refined to 1.65 Å resolution is disclosed that shows both anti-anti and syn-anti orientations for 1 × 1 nucleotide AA internal loops. Molecular dynamics (MD) simulations using AMBER force field in explicit solvent were run for over 500 ns on the model systems r(5′GCGCAGCGC)2 (MS1) and r(5′CCGCAGCGG)2 (MS2). In these MD simulations, both anti-anti and syn-anti AA base pairs appear to be stable. While anti-anti AA base pairs were dynamic and sampled multiple anti-anti conformations, no syn-anti ↔ anti-anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, an explicit solvent MD simulation over 800 ns was run on r[5′GGGC(CAG)3GUCC]2, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn-anti conformation), transformed to anti-anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn-anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1 × 1 nucleotide AA internal loops in r(CAG) repeats is anti-anti but can adopt syn-anti depending on the environment. These results are important to understand RNA dynamic-function relationships and to develop small molecules that target RNA dynamic ensembles. [ABSTRACT FROM AUTHOR]

Abstract: The PDZ domain of the shank protein interacts with numerous cell membrane receptors and cytosolic proteins via the loosely defined binding motif X-(Ser/Thr)-X-Φ-COOH (Φ represents hydrophobic residues) at the carboxyl terminus of its target protein. This enables shank to serve as a membrane-associated scaffold for the assembly of signaling complexes. As the list of proteins that bind to the shank PDZ domain grows, it is not immediately clear what structural element(s) mediate this domain’s target specificity or the plasticity required to bind its different targets. Here, we have determined the crystal structure of the shank1 PDZ in complex with the βPIX C-terminal pentapeptide (642–646, DETNL) at 2.3Å resolution and modeled shank1 PDZ binding to selected pentapeptide ligands. The resulting structures revealed a large hydrophobic pocket within the PDZ domain that can accommodate a variety of ligand residues at the P(0) position. A H-bond between His735 and Ser/Thr at the P(−2) position is invariant throughout the model structures. In addition, we identified multiple PDZ domain residues that are able to form H-bonds and salt bridges with an incoming target protein. Overall, our study provides a new level of understanding of the specificity and structural plasticity of the shank PDZ domain. [Copyright &y& Elsevier]

Interaction of the pattern recognition receptor, RAGE with key ligands such as advanced glycation end products (AGE), S100 proteins, amyloid β, and HMGB1 has been linked to diabetic complications, inflammatory and neurodegenerative disorders, and cancer. To help answer the question of how a single receptor can recognize and respond to a diverse set of ligands we have investigated the structure and binding properties of the first two extracellular domains of human RAGE, which are implicated in various ligand binding and subsequent signaling events. The 1.5-Å crystal structure reveals an elongated molecule with a large basic patch and a large hydrophobic patch, both highly conserved. Isothermal titration calorimetry (ITC) and deletion experiments indicate S100B recognition by RAGE is an entropically driven process involving hydrophobic interaction that is dependent on Ca2+ and on residues in the C'D loop (residues 54-67) of domain 1. In contrast, competition experiments using gel shift assays suggest that RAGE interaction with AGE is driven by the recognition of negative charges on AGE-proteins. We also demonstrate that RAGE can bind to dsDNA and dsRNA. These findings reveal versatile structural features of RAGE that help explain its ability to recognize of multiple ligands. [ABSTRACT FROM AUTHOR]

Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA-LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 Å Bragg spacing and structure determination. It was concluded that the improvement of crystal quality as indicated by the higher resolution of the new RMA-LC4 complex crystals was a consequence of the introduction of new lattice contacts at lower temperature. The lattice contacts corresponded to an increased number of interactions between high-entropy side chains that mitigate the lattice strain incurred upon cryocooling and accompanying mosaic spread increases. The thermodynamically unfavorable immobilization of high-entropy side chains used in lattice formation was compensated by an entropic increase in the bulk-solvent content owing to the greater solvent content of the crystal lattice. [ABSTRACT FROM AUTHOR]

Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca2+ ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca2+ binding curves were measured using atomic absorption spectroscopy. This method enables highly accurate measurements even for Ca2+ bound to polymerized protein. The binding curves for both skeletal and cardiac calsequestrin were complex, with binding increases that correlated with protein dimerization, tetramerization, and oligomerization. The Ca2+ binding capacities of skeletal and cardiac calsequestrin are directly compared for the first time, with ∼80 Ca2+ ions bound per skeletal calsequestrin and ∼60 Ca2+ ions per cardiac calsequestrin, as compared with net charges for these molecules of ∼80 and ∼69, respectively. Deleting the negatively charged and disordered C-terminal 27 amino acids of cardiac calsequestrin results in a 50% reduction of its calcium binding capacity and a loss of Ca2+-dependent tetramer formation. Based on the crystal structures of rabbit skeletal muscle calsequestrin and canine cardiac calsequestrin, Ca2+ binding capacity data, and previous light-scattering data, a mechanism of Ca2+ binding coupled with polymerization is proposed. [ABSTRACT FROM AUTHOR]

The X-ray crystal structure of the Escherichia coli (Ec) direct oxygen sensor heme domain (Ec DosH) has been solved to 1.8 A using Fe multiple-wavelength anomalous dispersion (MAD), and the positions of Met95 have been confirmed by selenomethionine (SeMet) MAD. Ec DosH is the sensing part of a larger two-domain sensing/signaling protein, in which the signaling domain has phosphodiesterase activity. The asymmetric unit of the crystal lattice contains a dimer comprised of two differently ligated heme domain monomers. Except for the heme ligands, the monomer heme domains are identical. In one monomer, the heme is ligated by molecular oxygen (O2), while in the other monomer, an endogenous Met95 with S→ Fe ligation replaces the exogenous O2 ligand. In both heme domains, the proximal ligand is His77. Analysis of these structures reveals sizable ligand-dependent conformational changes in the protein chain localized in the PG turn, the Gβ-strand, and the HI turn. These changes provide insight to the mechanism of signal propagation within the heme domain following initiation due to O2 dissociation. [ABSTRACT FROM AUTHOR]

Meprin α is a zinc metalloproteinase (metzincin) that has been implicated in multiple diseases, including fibrosis and cancers. It has proven difficult to find small molecules that are capable of selectively inhibiting meprin α, or its close relative meprin β, over numerous other metzincins which, if inhibited, would elicit unwanted effects. We recently identified possible molecular starting points for meprin α-specific inhibition through an HTS effort (see part I, preceding paper). Here, in part II, we report further efforts to optimize potency and selectivity. We hope that a hydroxamic acid meprin α inhibitor probe will help define the therapeutic potential for small molecule meprin α inhibition and spur further drug discovery efforts in the area of zinc metalloproteinase inhibition. [ABSTRACT FROM AUTHOR]

A correction to the article "A Toxic RNA Catalyzes the In Cellulo Synthesis of Its Own Inhibitor" is presented.

The ubiX gene of Colwellia psychrerythraea strain 34H encodes a 3-octaprenyl-4-hydroxybenzoate carboxylase (CpsUbiX, UniProtKB code: Q489U8) that is involved in the third step of the ubiquinone biosynthesis pathway and harbors a flavin mononucleotide (FMN) as a potential cofactor. Here, we report the crystal structures of two forms of CpsUbiX: an FMN-bound wild type form and an FMN-unbound V47S mutant form. CpsUbiX is a dodecameric enzyme, and each monomer possesses a typical Rossmann-fold structure. The FMN-binding domain of UbiX is composed of three neighboring subunits. The highly conserved Gly15, Ser41, Val47, and Tyr171 residues play important roles in FMN binding. Structural comparison of the FMN-bound wild type form with the FMN-free form reveals a significant conformational difference in the C-terminal loop region (comprising residues 170-176 and 195-206). Subsequent computational modeling and liposome binding assay both suggest that the conformational flexibility observed in the C-terminal loops plays an important role in substrate and lipid bindings. The crystal structures presented in this work provide structural framework and insights into the catalytic mechanism of CpsUbiX. [ABSTRACT FROM AUTHOR]

Three JNK isoforms, JNK1, JNK2, and JNK3 have been reported and unique biological function has been ascribed to each. It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit. To probe JNK isoform function we designed JNK2/3 inhibitors that have >30-fold selectivity over JNK1. Utilizing site-directed mutagenesis and x-ray crystallography we identified L144 in JNK3 as a key residue for selectivity. To test whether JNK2/3 selective inhibitors protect human dopaminergic neurons against neurotoxin-induced mitochondrial dysfunction, we monitored reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP). The results showed that JNK2/3 selective inhibitors protected against 6-hydroxydopamine-induced ROS generation and MMP depolarization. These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity. Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death. [ABSTRACT FROM AUTHOR]

Abstract: SAR and lead optimization studies for Rock inhibitors based on amino acid-derived quinazolines are described. Studies demonstrated that these amino acid derived quinazolinones were mainly pan-Rock (I & II) inhibitors. While selectivity against other kinases could be achieved, selectivity for most of these compounds against PKA was not achieved. This is distinct from Rock inhibitors based on non-amino acid derived quinazolinones, where high selectivity against PKA could be obtained.22 The inhibitors presented here in some cases possessed sub-nanomolar inhibition of Rock, nanomolar potency in ppMLC cell based assays, low to fair cytochrome P-450 inhibition, and good human microsomal stability. [Copyright &y& Elsevier]

Summary: The mitogen-activated kinases JNK1/2/3 are key enzymes in signaling modules that transduce and integrate extracellular stimuli into coordinated cellular response. Here, we report the discovery of irreversible inhibitors of JNK1/2/3. We describe two JNK3 cocrystal structures at 2.60 and 2.97 Å resolution that show the compounds form covalent bonds with a conserved cysteine residue. JNK-IN-8 is a selective JNK inhibitor that inhibits phosphorylation of c-Jun, a direct substrate of JNK, in cells exposed to submicromolar drug in a manner that depends on covalent modification of the conserved cysteine residue. Extensive biochemical, cellular, and pathway-based profiling establish the selectivity of JNK-IN-8 for JNK and suggests that the compound will be broadly useful as a pharmacological probe of JNK-dependent signal transduction. Potential lead compounds have also been identified for kinases, including IRAK1, PIK3C3, PIP4K2C, and PIP5K3. [ABSTRACT FROM AUTHOR]